General question: Software vs. hardware

[Arvydas Matiukas]

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Hello list,

I am struggling to get a precisely circularly polarized beam. No matter how precisely I set fast axis of the quarter wave plate at 45 degrees  to the polarization of laser beam, the ellipticity is always <0.9 (Imin/Imax <0.9).

The laser beam is 588nm (5nm bandwidth) off OPO and pulse broadening fiber (from fs pulses of driving Mai Tai to ps pulses), diameter 4mm. Polarization is 1:70 that is further  improved to 1:600 by Glan polarizer. The zero order 588nm

 quarter wave plate  is aligned normal to the beam and rotated at 45 degrees (other angles only increase ellipticity).


Any suggestions what I may be missing are welcome.


Thanks,
Arvydas
************************

[Zdenek Svindrych-2]

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Hi Arvydas,
ellipticity of 0.9 may be well within your measurement error (at least it
is well within mine). Any optical surface that is not perfectly normal to
the beam will reflect different polarizations differently... On the other
hand, if you're after perfectly circular pol, and you're sure
detection/measurement is not the issue, you should be able to fin-tune the
beam properties with tilted glass plates (maybe).
Best, zdenek

On Mon, Mar 18, 2019 at 4:50 PM Arvydas Matiukas <[hidden email]>
wrote:

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> *****
>
> Hello list,
>
> I am struggling to get a precisely circularly polarized beam. No matter
> how precisely I set fast axis of the quarter wave plate at 45 degrees  to
> the polarization of laser beam, the ellipticity is always <0.9 (Imin/Imax
> <0.9).
>
> The laser beam is 588nm (5nm bandwidth) off OPO and pulse broadening fiber
> (from fs pulses of driving Mai Tai to ps pulses), diameter 4mm.
> Polarization is 1:70 that is further  improved to 1:600 by Glan polarizer.
> The zero order 588nm
>
>  quarter wave plate  is aligned normal to the beam and rotated at 45
> degrees (other angles only increase ellipticity).
>
>
> Any suggestions what I may be missing are welcome.
>
>
> Thanks,
> Arvydas
> ************************
>

--
--
Zdenek Svindrych, Ph.D.
Research Associate - Imaging Specialist
Department of Biochemistry and Cell Biology
Geisel School of Medicine at Dartmouth

[Benjamin Smith]

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Four thoughts come to mind:

1) 1:600 is pretty terrible performance for a Glan-laser polarizer.  They
are normally rated to around a 1:100,000 ratio, and even when paired with
an achromatic wave plate, we normally get a >1:12,000 extinction
coefficient.  This also gets to the second point.

2) Try an achromatic half-wave plate.   For a 5 nm bandwidth, a zero order
wave plate will have a 0.3% difference in retardation across the spectrum
due to dispersion, while an achromatic wave plate will have a 0.01%
difference.

3) Confirm the tuning of the laser.  If the OPO laser is tunable, the
tuning can drift over time, which may mean you are dealing with longer
wavelengths then you expect (which would give the result where 45° is still
elliptic).  The simplest solution to this issue would be to use a half-wave
plate.

4) Make sure there is nothing else in the path.  I was once helping a lab
trouble shoot their launch optics for a 2P system, and they were using a
non-polarizing beam splitter cube between the laser and the Pockels cell as
an initial attenuator.  The issue was that the Pockels cell had poor
dynamic range, and it turned out the cube was fairly birefringent.
Removing the cube fixed the problem.

Hope this helps,
   Ben Smith


On Mon, Mar 18, 2019 at 1:50 PM Arvydas Matiukas <[hidden email]>
wrote:

> *****
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> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hello list,
>
> I am struggling to get a precisely circularly polarized beam. No matter
> how precisely I set fast axis of the quarter wave plate at 45 degrees  to
> the polarization of laser beam, the ellipticity is always <0.9 (Imin/Imax
> <0.9).
>
> The laser beam is 588nm (5nm bandwidth) off OPO and pulse broadening fiber
> (from fs pulses of driving Mai Tai to ps pulses), diameter 4mm.
> Polarization is 1:70 that is further  improved to 1:600 by Glan polarizer.
> The zero order 588nm
>
>  quarter wave plate  is aligned normal to the beam and rotated at 45
> degrees (other angles only increase ellipticity).
>
>
> Any suggestions what I may be missing are welcome.
>
>
> Thanks,
> Arvydas
> ************************
>

--
Benjamin E. Smith, Ph. D.
Imaging Specialist, Vision Science
University of California, Berkeley
195 Life Sciences Addition
Berkeley, CA  94720-3200
Tel  [phone]
Fax [phone]
e-mail: [hidden email]
http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>

[Jeffrey Carmichael]

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 I agree that one needs to define the problem more carefully, and it is
like the screwdriver vs. wrench question. There are also other issues to
consider, one of them being photon economy.

I'm a bit biased and removed from the bench, originally coming from a lab
that relied heavily on widefield deconvolution data (the late Fred Fay's
lab at UMass Med. School) and having experience with earlier generations of
confocal microscopes. One of the common refrains was about the availability
of fluorescence photons. Much like the work of Agard and Sedat, the work of
Carrington, Fogarty, Fay et al. demonstrated the efficacy of a robust,
iterative deconvolution algorithm approach, using a minimization function
with a non-negativity constraint to resolve structures to 100-200nm.
Arriving at a best fit required providing certain inputs regarding
anticipated feature characteristics that an informed imaging scientist
would define and could also vary. Different variables would yield slightly
different results which could be used to help determine best fit with other
data. I think this level of engagement with and understanding of one's data
is important.

When one simply trusts either the computational technology or the imaging
technology, poor choices are made with little understanding.

Regarding the PSF needing to be at a higher resolution, this makes no sense
to me. The point of the PSF is to empirically model how light spreads in
your particular system, under the conditions you're using. Use a
sub-diffraction sized bead and image with the same parameters used to
acquire the data. A restorative deconvolution doesn't subtract anything. It
reassigns light to its purported origin. There should be constraints that
the total integrated optical density be the same before/after
deconvolution, else it's not really deconvolution but merely some sort of
filter.

Confocal by its very nature rejects something like 90-98% of available
fluorescence photons. That data is lost and irretrievable. This problem is
confounded by sample photobleaching. The relatively poor photon economy
means that in comparison to widefield, many more photons are emitted per
each photon detected, and fluorescence can be exhausted before the data is
even acquired.

Deconvolution -- if done properly -- not only can quantitatively reassign
fluorescence to it's point of origin, but it does this while collecting all
available photons in the case of widefield. This is why it's attractive as
an alternative - because it doesn't throw away data, and instead uses all
available fluorescence data throughout a volume to restore light back to
it's point of origin. As others have pointed out, one can also deconvolve
confocal and super resolution images.

This advantage is probably limited to those applications where relatively
large volumes are imaged and might otherwise be photobleached by confocal
laser excitation before being acquired, and yes there are forms of
structures that don't work well with deconvolution, and for those confocal
is preferable.

Time and expertise factor into whether this is practical, and for most,
confocal is the most practical.

*Jeff Carmichael*

*[hidden email] <[hidden email]>*

* <[hidden email]>*


On Mon, Mar 18, 2019 at 4:37 PM Benjamin Smith <[hidden email]>
wrote:

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> *****
>
> Not sure if this has been said, but this is basically like asking
> "screwdriver vs. wrench".  A laser scanning confocal microscope is not
> superior to a compound microscope, and the converse is also true, they are
> different tools for different tasks.  This is one of the key points I try
> to get across to students when I teach them about microscopy, we have
> macro, spinning disk, 2P, light sheet, stereo, confocal, STED, STORM, TEM,
> SEM, AFM, FIB-SEM, etc., etc, for a reason.  They all excel at tasks that
> other systems struggle with.
>
> Along these lines, here are two scenarios:
> Scenario 1) You want to get a kHz sample rate of a voltage dye in a
> cultured neuron.  In this case, a compound microscope with deconvolution
> (or likely even just a simple high-pass filter) is the clear winner as all
> the pixels in the frame are temporally correlated (as long as you have CCD
> or global shutter CMOS), and the frame rate will be much higher than with
> confocal, even with the most cutting edge technologies.
>
> Scenario 2) You want to measure the volume of densely packed nuclei using
> DAPI in a whole-mount sample.  Deconvolutions will quickly fall apart on
> this task simply because the deeper you go, the vast majority of the total
> signal is from out of focus light (much like trying to image a faint star
> right next to the sun).  This means that the amount of information you have
> about the sample plane itself becomes nearly non-existent.  Conversely,
> since confocal microscopes perform the deconvolution before light gets to
> the detector, you more or less eliminate this bottleneck caused by the
> dynamic range of the detector.
>
> Also, one quick point about deconvolutions.  Unless you measured the PSF in
> the sample (such as using TetraSpeck  beads) at a higher resolution than
> you acquired your image, you are not adding any information about the
> sample.  Rather, you are whittling away information you wish to discard
> (i.e. it is a lossy process, much like JPEG compression).  Along these
> lines, iterative blind deconvolutions are allowing a computer to guess what
> information should be removed.  Thus, just because the image looks better
> does not necessarily mean it is correct, otherwise STED, STORM, AFM, and
> cryo-EM would be obsolete.
>
> Just my own two cents,
>    Ben Smith
>
> On Mon, Mar 18, 2019 at 11:52 AM Feinstein, Timothy N <[hidden email]>
> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > Hi Alison,
> >
> > The signal to noise question strikes me as the key question WRT decon.
> > Most software packages default to a setting that seems unrealistic for
> > confocal imaging (ie SNR=20), and this leads to a lot of patterning
> > artifacts in deconvolved images.  At such high SNR any pixel of data is
> > treated as 'real', so background noise gets processed into weird
> moire-type
> > patterns.  This also happens when the algorithm doesn't quite converge
> and
> > you let it run for too many iterations (the default iteration #s tend to
> be
> > set high as well).  It's critical then to tell the software (when
> possible)
> > that your SNR is low, so it gets much more aggressive about detecting and
> > removing random noise (at the expense of losing details in the 1-3 pixel
> > range).  I would recommend folks take care if using any package that
> > doesn't let you correct the SNR.
> >
> > Honestly, when SNR is set properly I find that noisy images benefit a lot
> > more from decon than high-quality cover candidate-type pics.  In addition
> > to sharpening and Z blur removal the noise and background removal becomes
> > more dramatic (and beneficial for quantitation) the more background and
> > noise there is.  This comes up a lot when live imaging when we use
> resonant
> > scanning for speed and low photodamage, but have higher noise as a
> > trade-off.  I'd say that as long as you can image at 1.5x-2x Nyquist
> > resolution (and the extra time/expense is worth it), even super-noisy
> > images will benefit quite a bit.
> >
> > I understand that we're crossing fingers for the day when everyone has
> > GPU-enabled decon running seamlessly during acquisition.  For that to
> work
> > the algorighms need to automatically (accurately) estimate SNR.  That
> > doesn't seem like an insurmountable challenge, but I haven't seen it yet.
> >
> >
> > Best,
> >
> >
> > TF
> >
> > Timothy Feinstein, Ph.D.
> > Research Scientist
> > Department of Developmental Biology
> > University of Pittsburgh
> >
> >
> >
> >
> > On 3/18/19, 11:07 AM, "Confocal Microscopy List on behalf of Alison
> > North" <[hidden email] on behalf of
> > [hidden email]> wrote:
> >
> >
> >     Hi all,
> >
> >     So this is a very timely discussion because I have been discussing
> > with
> >     my staff whether there are data sets that should NOT be deconvolved,
> > and
> >     if so, how does one decide that?  I too attended Jim Pawley's
> > wonderful
> >     course (he was certainly a huge character as well as an incredibly
> >     fantastic microscopist, and he will certainly be remembered by all!),
> > so
> >     I have generally worked under the assumption that one should
> > deconvolve
> >     all confocal data.  But I am also very aware of the potential for
> >     artifacts if a data set isn't "good enough" for deconvolution.
> >     Obviously the ideal situation is to acquire an optimal data set -
> >     well-prepared sample, bright staining, Nyquist sampling etc. etc.,
> and
> > a
> >     high S:N ratio - and by sticking to these rules, our deconvolved
> >     DeltaVision images or confocal images of fixed samples have always
> >     looked great.  But nowadays we are faced with different scenarios,
> >     particularly when you are attempting to do very rapid imaging of
> live,
> >     weakly expressing cells, while attempting to minimize phototoxicity.
> > In
> >     that case you can end up with pretty lousy S:N ratios, because
> >     maintaining cell viability or imaging fast enough is more critical.
> >     For example, I have a lovely new iSIM in my lab, for which the
> initial
> >     resolution increase is achieved by the hardware, but the second step
> > in
> >     resolution increase is via deconvolution.   The whole point of this
> >     instrument is for rapid, super-resolution imaging - so we can't
> simply
> >     increase exposure times to improve S:N, turning up the laser power
> > will
> >     obviously kill the cells, and we can't increase the pixel size or
> > we'll
> >     lose the resolution.  And I assume a lot of the new types of
> >     super-resolution instrument out there must leave you facing the same
> >     issue, since live cell imaging invariably forces you to compromise
> >     somewhere within the imaging triangle (or hexagon, or whatever we've
> > got
> >     up to now!).
> >
> >     Therefore my question is, are there papers out there which have
> > compared
> >     deconvolution algorithms and looked at the potential for artifacts on
> >     really low S:N images, which we could use to advise our researchers
> on
> >     what is the minimum you can get away with before you really shouldn't
> > be
> >     deconvolving the data set at all? Also, are there papers showing the
> >     effect of undersampling in the z-axis on the resulting deconvolved
> >     images (as is often the case on our spinning disk system)?  I haven't
> >     managed to find any yet (though I confess I've been too busy with
> > other
> >     stuff to spend too many hours searching!), so if anybody could point
> > me
> >     to some good references I'd be most grateful.  I have spoken with
> >     several renowned microscopists about whether deconvolution is always
> a
> >     good idea under such circumstances, and the gut reaction appears to
> be
> >     no, but I could do with some hard and fast validation for teaching
> > purposes.
> >
> >     Many thanks in advance!
> >
> >     Alison
> >
> >
> >
> >     On 3/18/2019 9:56 AM, Feinstein, Timothy N wrote:
> >     > *****
> >     > To join, leave or search the confocal microscopy listserv, go to:
> >     >
> >
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> >
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> > and include the link in your posting.
> >     > *****
> >     >
> >     > Avi, I really agree with your point.  I feel that people to
> > deconvolve any time spatial information is critical, whether they're
> using
> > widefield, CLSM, spinning disc, or light sheet.  It's true that
> > deconvolving adds time and data volume and especially cost, but in trade
> > you get an image that is substantially sharper, with reduced noise and
> > background, and more quantitatively accurate*.
> >     >
> >     > Regarding whether to just go with a point scanning confocal, I
> don't
> > see it as a simple question of better or worse**.  A nuclear-cytoplasmic
> > translocation assay with monolayer cells works just as well on a
> widefield,
> > and (in my experience!) many types of biosensor assay work better with a
> > properly set up widefield.  The 16-bit depth of widefield images is nice
> > for quantitation, and modern sCMOS cameras have by far the best
> acquisition
> > speeds.  I don't know whether widefields still have a more linear
> > relationship between sample brightness and detected signal, but the last
> > time I checked that was still true.
> >     >
> >     > (*) Deconvolution is quantitatively useful as long as people make
> > sure to tell the software to preserve the original intensity values.  One
> > of my complaints about Hyvolution was that you could not do that, so I
> just
> > used the Huygens package that came with it.  I don't know whether
> Lightning
> > gives you that option...if not then caveat emptor.
> >     >
> >     > (**) My advice mostly applies to turnkey stuff that any lab can
> > implement, not exotic techniques available to folks with specialists or
> > engineers on hand.
> >     >
> >     > Best,
> >     >
> >     >
> >     > T
> >     > Timothy Feinstein, Ph.D. esearch Scientist
> >     > Department of Developmental Biology
> >     > University of Pittsburgh
> >     >
> >     >
> >     > On 3/18/19, 5:07 AM, "Confocal Microscopy List on behalf of Avi
> > Jacob" <[hidden email] on behalf of [hidden email]
> >
> > wrote:
> >     >
> >     >
> >     >      I'll point out, that you can, of course, deconvolve confocal
> > images too.
> >     >      So, while you can indeed get near confocal quality with
> > well-acquired wf
> >     >      data after deconvolution, you can also get near SR quality
> when
> >     >      deconvolving a well-acquired stack from a confocal. And then
> > you can also
> >     >      deconvolve a SR stack and get... well you get the idea! It's
> > like an arms
> >     >      race.
> >     >      I have the Hyvolution and had access for a couple of weeks to
> > the Lighting,
> >     >      and now confocal images look blurry to me.
> >     >      Avi
> >     >
> >     >      --
> >     >      Avi Jacob, Ph.D.
> >     >      Kanbar Light Microscopy Unit
> >     >      The Mina & Everard Goodman Faculty of Life Sciences
> >     >      Bar-Ilan University, Ramat-Gan 529002, Israel
> >     >
> >     >
> >     >
> >     >      On Sun, Mar 17, 2019 at 6:04 PM George McNamara <
> > [hidden email]>
> >     >      wrote:
> >     >
> >     >      > *****
> >     >      > To join, leave or search the confocal microscopy listserv,
> go
> > to:
> >     >      >
> >
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> >     >      > Post images on
> >
> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fwww.imgur.com-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3DMNM8i3iDaQuMA9LF766-252FyvCyp94jmie4IaC5qIEWclA-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DGy99yAWcbr6KHUA0kYKb4K-wOHL4iNGJsDecMfZ8X-M%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288894390&amp;sdata=2N%2BmlW5RE%2BAFuG%2FuM68FeI9LOfI1m2PlNu23S9eIQts%3D&amp;reserved=0=
> > and include the link in your posting.
> >     >      > *****
> >     >      >
> >     >      > Hi Mika,
> >     >      >
> >     >      > White et al 1987 (
> >
> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fjcb.rupress.org-252Fcontent-252F105-252F1-252F41.long-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3Da6oZCfeD7Gt4nYMS89PcklnveCdDLLkksafp3tCyld0-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3D67LyqPF2rBiBDSWJVZeG7TpJz1o4MRDf8ZyLysbeKx4%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288894390&amp;sdata=R3V0RHiTDpGkJUJyI8Q7SDJZjKdk3uDMgmBEPLrzcUw%3D&amp;reserved=0=
> > ) made a
> >     >      > compelling case for point scanning confocal microscopes:
> > collect just
> >     >      > the in focus light with instant gratification. The case has
> > not changed,
> >     >      > the hardware (especially data deluge side) has gotten a lot
> > better. I
> >     >      > note that both widefield detectors and PMT/APD/hybrid
> > detectors/others
> >     >      > have gotten a lot better in the 32 years from 1987! As have
> > the optics
> >     >      > and automation.
> >     >      >
> >     >      > Paul Goodwin 2014 (
> >
> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.ncbi.nlm.nih.gov-252Fpubmed-252F24974028-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3DGDve8BuxhLDAdBPKywRQoMTYV-252BqSk9aMcz2WrZCHPU8-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DPZ10eaR5B-jwZmf5y920Z41iJYUJcji_VsufABIeX84%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288894390&amp;sdata=dIZN7CiQOTIt24N0mwi0QgDFkcFcVScVNxaoDuS24HM%3D&amp;reserved=0=
> > ) made
> >     >      > a nice case for quantitative deconvolution microscopy (and
> on
> > very high
> >     >      > quality specimens, ~10% improvement in resolution compared
> to
> > simple
> >     >      > widefield), but historically slow.
> >     >      >
> >     >      > Now, with 'instant gratification' spatial deconvolution,
> > thanks to the
> >     >      > GPU revolution (NVidia RTX Titan~16 Teraflops [S.P.], 24 Gb
> > ram, $2500
> >     >      > ... not including the deconvolution software module price),
> > widefield,
> >     >      > spinning disk (and slightly exotic variants like iSIM, DMD
> > based, etc,
> >     >      > see also new THUNDER Imagers [see p.p.s.]), multiphoton (I'm
> > excited
> >     >      > about the price point of recent fiber lasers, which could
> > become much
> >     >      > better price if achieve 'economy of scale'), and of course,
> > point
> >     >      > scanning confocal microscopes.
> >     >      >
> >     >      > Spatial deconvolution (especially if someone $uccessfully
> > implements
> >     >      > joint spatial deconvolution and spectral unmixing, multiple
> > cameras -
> >     >      > for 4 cameras see Babcock 2018, mentions aiming for 8
> > cameras) helps
> >     >      > with Expansion Microscopy and/or DNA-PAINT, to go
> > super-resolution ...
> >     >      > really single molecule counting (and DNA-PAINT eliminates
> the
> > classic
> >     >      > issue of PALM/STORM/FPALM of not counting every molecule).
> > Sure,
> >     >      > DNA-PAINT (like STORM etc) have the issue of a whole lotta
> > images
> >     >      > acquired. Data deluge: who cares? Jerome & Price's 10th
> > commandment of
> >     >      > confocal imaging is: "10. Storage Media Is Essentially Free
> > and Infinite".
> >     >      >
> >     >      > More significantly, DNA-PAINT and related methods (single
> > molecule RNA
> >     >      > FISH, scRNAseq -> MERFISH = Moffitt 2018 as example, etc)
> > also enable
> >     >      > multiplex -- with single molecule counting -- to whatever
> > plex is needed
> >     >      > to answer the 'biological question(s)' being posed.
> >     >      >
> >     >      > All that said, the installed base of research grade point
> > scanning
> >     >      > confocal microscopes is large (5000+) and efficient at
> > acquiring high
> >     >      > quality images, to the point that user's sample preparation
> > (and
> >     >      > avoidance of purchasing stuff from 'Santa Crap' and similar
> > companies)
> >     >      > is much more limiting than the microscopes.
> >     >      >
> >     >      > George
> >     >      >
> >     >      > p.s. a couple of references not included in above:
> >     >      >
> >     >      > W. Gray (Jay) Jerome, Robert L. Price 2018... Basic Confocal
> > Microscopy
> >     >      > second edition
> >
> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Flink.springer.com-252Fbook-252F10.1007-25252F978-2D3-2D319-2D97454-2D5-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3D-252FMQlH4t81Pk9tNTHR8fsn1tQc0JAc2nc-252BnO2gdk60Us-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3D1JXaiNNf-bzSdjywfRJ8gowB9Yq0uGNulQMlGeSMxkE%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288894390&amp;sdata=3ua69ygadJRYIJltAT7qPoJzNd%2B16PWCCeNLQF13WIg%3D&amp;reserved=0=
> >     >      >
> >     >      > Expansion ... X10 protocol ... Truckenbrodt 2019 Nat Protoc,
> >     >      >
> >
> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.nature.com-252Farticles-252Fs41596-2D018-2D0117-2D3-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3DCF-252Fp9frimbrxZiMDwLWZjzuyoyQbCuKp4EcvaL3Wmmw-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DYeXbqWFX0mMxO3nVRt59pt6RJYlwIPBMZRePQbiF1yU%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288894390&amp;sdata=TEBfgj6Gg%2Bt02BMyFZCLb8%2Feb5nOyAJmnftouvPd1qY%3D&amp;reserved=0=
> >     >      >
> >     >      > DNA-PAINT acronym soup review ... Nieves 2018 Genes,
> >     >      >
> >
> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.mdpi.com-252F2073-2D4425-252F9-252F12-252F621-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3Diwx3Z1Pgr4a2SObO9F47jEtqlfsYQFk2R4gu0Qv1uJM-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DtJY5yunlGClYXKHrXuCDe5KU8D-qhc1ZS26_KuNdXpE%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288904398&amp;sdata=RUgIPB%2Fi0n6i28lOhKZAGSrlyirYALd0l75r5qhZCMk%3D&amp;reserved=0=
> >     >      >
> >     >      > Babcock 2018 (4 --> 8 cameras, single molecule localization
> > microscopy
> >     >      > with $1550 CMOS cameras) ...
> >     >      >
> >
> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.nature.com-252Farticles-252Fs41598-2D018-2D19981-2Dz-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3DwN-252FHP30wNUE0-252BJWeFJIJFsBk32ZhD4DfKi9gc3ZTz2c-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DTIL4NaeETsqkjycmtWw_CnoEWvrpY1Tkr_SAmz-QkRM%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288904398&amp;sdata=D26AicKRsRoIaCGZdaxaF5OKXfGuo8yS0zvSVgmoBwY%3D&amp;reserved=0=
> >     >      >
> >     >      > Moffitt et al 2018 ...
> >     >      >
> >
> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fscience.sciencemag.org-252Fcontent-252F362-252F6416-252Feaau5324.long-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DFR98V6ldS9Q38wI59kly9U8pCzp92Vzc1J6T8ydCU9w-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3D55vzrzqpm3n5Z7logfovDr0N73h1gF7gldtYUSkZ6c0%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288904398&amp;sdata=CQMKMRjzbJVLgZjtpCzzZSwaNvCbt72baFvqppyebSI%3D&amp;reserved=0=
> > and
> >     >      > commentary
> >
> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fscience.sciencemag.org-252Fcontent-252F362-252F6416-252F749-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3Do0B379uuh-252FrB6gS9n6lG-252BjAZeztiYHZVmICwX4ghKh8-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DXeBps6CWTMM197evmy1n1uC5qOcvU8mAN95bEPmieiQ%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288904398&amp;sdata=um%2F%2FPNffQLOyrTsQcJymSuR2XwRcVnNMwMgQ6qYy19A%3D&amp;reserved=0=
> >     >      >
> >     >      > ***
> >     >      >
> >     >      > Some resolution numbers:
> >     >      >
> >     >      > 1.4 NA objective lens ... 500 nm wavelength .. dxy = 0.61 *
> > wqavelength
> >     >      > / NA (I routinely drop the 0.61 from 0.61)
> >     >      >
> >     >      > 0.6 * 500 / 1.4 = 214 nm
> >     >      >
> >     >      > widefiel deconvolution (re: Goodwin 2014) ~10% better ...
> 193
> > nm (if
> >     >      > pixel size matched or interpolate optimally).
> >     >      >
> >     >      > point scanning confocal -- Zeiss has a nice PDF, "Zeiss 2008
> > Principles
> >     >      > - Confocal Laser Scanning Microscopy" (see fig 10) on
> confocal
> >     >      > resolution wrt 1 and smaller pinhole size, source of the
> > values below,
> >     >      >
> >     >      > 1 Airy unit: 0.51 * 500 / 1.4 = 182 nm.
> >     >      >
> >     >      > 0.5 Airy unit ... 0.44 * 500 / 1.4 = 157 nm ... ~0.25
> photons
> > throughput
> >     >      > (which doesn't matter if target is photostable).
> >     >      >
> >     >      > 0.2 Airy unit ... you can ask your Zeiss rep about AiryScan
> > (and
> >     >      > FastAiryScan).
> >     >      >
> >     >      > 0.1 Airy unit ... 0.37 * 500 / 1.4 = 132 nm ... ~0.10
> photons
> > throughput.
> >     >      >
> >     >      > Most modern point scanning confocal microscopes have a 405
> nm
> > laser, so
> >     >      > if using BV421 (and ignoring potential photobleaching for a
> > moment),
> >     >      >
> >     >      > 1 Airy unit: 0.51 * 421 / 1.4 = 153 nm.
> >     >      >
> >     >      > or in reflection mode, i.e. nanodiamond or nanogold,
> >     >      >
> >     >      > 1 Airy unit: 0.51 * 405 / 1.4 = 147 nm ... and reflection
> > implies no
> >     >      > photobleaching, so infinite number of photons (though also
> no
> > blinking,
> >     >      > so not usually eligible for precision localization) ...
> >     >      >
> >     >      > 0.1 Airy unit: 0.37 * 405 / 1.4 = 107 nm
> >     >      >
> >     >      > and not going completely exotic with NA (i.e. 1.65), if
> > perfectly
> >     >      > refractive index match with a fairly conventional 1.49 NA
> > lens, and
> >     >      > inreflectance:
> >     >      >
> >     >      > 0.1 Airy unit: 0.37 * 405 / 1.49 = 100.57 nm
> >     >      >
> >     >      > I think I'd rather invest a DNA-PAINT friendly rig than deal
> > with 157 to
> >     >      > 101 nm.
> >     >      >
> >     >      > DNA-PAINT makes resolution irrelevant, if you use it (and
> > don't run out
> >     >      > of disk space or time or money), since precision
> localization
> > is
> >     >      > resolution divided by square root of number of photons, ex.
> > 250 nm XY
> >     >      > resolution / sqrt(1,000,000) = 0.25 nm, and could increase
> > number of
> >     >      > photons per target further, but why bother?
> >     >      >
> >     >      > ***
> >     >      >
> >     >      > point scanning confocal microscopes are also great platforms
> > for
> >     >      > F-Techniques, such as FastFLIM (aka rapidFLIM, etc, much
> > faster than
> >     >      > classic TCSPC slow FLIM), FCS, FCCS, see Liu 2008,
> >     >      >
> >
> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.ncbi.nlm.nih.gov-252Fpubmed-252F18387308-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DZQTosrSoPFpCa8Y3IdEa9Xz-252B4RHC8JO4gBppHmzkazo-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DmeRUIZdF3FLQZGyok4cxyc4AP4yq2lXX41Vt8q9WMmw%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288904398&amp;sdata=XTEH306i5ez7vKq2cHKCVQlSphNiFIdnqIXmwnOmyNw%3D&amp;reserved=0=
> >     >      >
> >     >      > ***
> >     >      >
> >     >      > p.p.s. Disclosures I am ...
> >     >      >
> >     >      > 1. currently hosting a Leica THUNDER Imager tour event (ends
> > Monday
> >     >      > 3/18/2019 afternoon) ...  see pdf download page,
> >     >      >
> >     >      > THUNDER Technology Note
> >     >      > THUNDER Imagers: How Do They Really Work?
> >     >      >
> >     >      >
> >
> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.leica-2Dmicrosystems.com-252Fscience-2Dlab-252Fthunder-2Dtechnology-2Dnote-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DauRUORuEKAir87-252Bbw7RHyTR9IxxrLYl1g3bFBiRTXBM-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DLmFGYl_Icu6b9AAIYnHkoiH26bRZ688ODEc9I6k8yco%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288904398&amp;sdata=5ZGLmCZt85tKxH1CJYM%2Bvul5S4qXw9%2FOnyWG80m2QxM%3D&amp;reserved=0=
> >     >      >
> >     >      > 2. hosting Nikon confocal demos in May 2019.
> >     >      >
> >     >      > 3. aiming to co-host with ISS a FastFLIM (one day)
> > mini-symposium this
> >     >      > summer.
> >     >      >
> >     >      > 4. an unpaid advisor for Gary Brooker for FINCH/CINCH, re:
> >     >      >
> >
> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.ncbi.nlm.nih.gov-252Fpubmed-252F28261321-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DuVw0kiW1baZV0RJv-252Bk1ObKznhw51pemnCMehjHkUa2g-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DKC1HMkiDzpE2DLsuj1I2nHvopwFVMMc0GVecDV-fWkM%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288904398&amp;sdata=QC0LyEp9hbSKOgpV%2Fi%2B%2FABGDX4az02fRVdMZ4Ovufps%3D&amp;reserved=0=
> >     >      >
> >     >      >
> >     >      > On 3/17/2019 10:43 AM, Mika Ruonala wrote:
> >     >      > > *****
> >     >      > > To join, leave or search the confocal microscopy listserv,
> > go to:
> >     >      > >
> >
> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Flists.umn.edu-252Fcgi-2Dbin-252Fwa-253FA0-253Dconfocalmicroscopy-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DdHOCjeBda4sSZPf-252FB2-252B5Sv3Q8Qzcs708p4we8vHf-252FIg-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DqZ-tdRwhHG172zv4uHPeAJMNeIIHNxozhQudV-hF2kE%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288904398&amp;sdata=BnCAxorVJgPSFw70Kr%2B401vq4hMlZhvzdkivum9XGNE%3D&amp;reserved=0=
> >     >      > > Post images on
> >
> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fwww.imgur.com-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DcnhzoWOP4RwDR622fn447aQVxW8ZBI3D0utze67RiGg-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DYzF36y8eV4SG3E0LJEDu-9AMm3Nh05dwp_XKhB6BUyU%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288914407&amp;sdata=ec0HIKuOMVqlLU7knyQ1JdS1B6c1BMCIb%2FYsHIFdYbM%3D&amp;reserved=0=
> > and include the link in your
> >     >      > posting.
> >     >      > > *****
> >     >      > >
> >     >      > > Hi.
> >     >      > > There are software solutions that are able to create image
> > data from
> >     >      > wide-field and even microscope systems with seemingly
> similar
> > quality to
> >     >      > that obtained from confocal systems.
> >     >      > >
> >     >      > > The comparison of acquisition vs. software is essentially
> a
> > comparison
> >     >      > of image acquisition vs. image processing. While a software
> > solution is way
> >     >      > cheaper than a hardware solution if it is able to produce
> > image data with
> >     >      > equal quality why would anyone choose to invest to a
> confocal
> > anymore?
> >     >      > >
> >     >      > > I’m looking forward to a vidid discussion!
> >     >      > >
> >     >      > >> m
> >     >      >
> >     >
> >     >
> >     --
> >     Alison J. North, Ph.D.,
> >     Research Associate Professor and
> >     Senior Director of the Bio-Imaging Resource Center,
> >     The Rockefeller University,
> >     1230 York Avenue,
> >     New York,
> >     NY 10065.
> >     Tel: office    ++ 212 327 7488
> >     Tel: lab        ++ 212 327 7486
> >     Fax:            ++ 212 327 7489
> >
> >
> >
>
> --
> Benjamin E. Smith, Ph. D.
> Imaging Specialist, Vision Science
> University of California, Berkeley
> 195 Life Sciences Addition
> Berkeley, CA  94720-3200
> Tel  [phone]
> Fax [phone]
> e-mail: [hidden email]
> http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>
>
>

--
 <https://www.chroma.com/>CHROMA TECHNOLOGY CORP®
*an employee owned
company*
10 Imtec Lane, Bellows Falls, Vermont 05101 USA
800-824-7662 |
FAX: [phone]
www.chroma.com <https://www.chroma.com/> | 
[hidden email] <mailto:[hidden email]>                             

[Warren Zipfel]

*****
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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

I use a Berek compensator in the Ti:S beam and either a polarizer after the objective to verify the Berek settings for  X or Y linear polarization (for anisotropy imaging)  or a polarizer and 1/4 waveplate as an analyzer if I need R or L circular polarization.  The more complicated optical path on most setups makes the adjustability of a Berek necessary.

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Arvydas Matiukas
Sent: Monday, March 18, 2019 4:49 PM
To: [hidden email]
Subject: Circularly polarizing laser beam

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Hello list,

I am struggling to get a precisely circularly polarized beam. No matter how precisely I set fast axis of the quarter wave plate at 45 degrees  to the polarization of laser beam, the ellipticity is always <0.9 (Imin/Imax <0.9).

The laser beam is 588nm (5nm bandwidth) off OPO and pulse broadening fiber (from fs pulses of driving Mai Tai to ps pulses), diameter 4mm. Polarization is 1:70 that is further  improved to 1:600 by Glan polarizer. The zero order 588nm

 quarter wave plate  is aligned normal to the beam and rotated at 45 degrees (other angles only increase ellipticity).


Any suggestions what I may be missing are welcome.


Thanks,
Arvydas
************************

[George McNamara]

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Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Jeff et al,

I disagree with Jeff's statement:

     Confocal by its very nature rejects something like 90-98% of available fluorescence photons. That data is lost and irretrievable.

1. trivially, if it is not digitized, it is not data. If is from a
far-out of focus plane, deconvolution not going to reassign it very well.
2. Reducing to the simplest cases: one vs two sub-resolution features,
for simplicity, one or two 40 nm beads with some gap (or DNA origami),
at the coverglass, refractive index matched media (i.e. 1.4 NA objective
lens, R.I. 1.518 immersion oil and mounting medium). So: no out of focus
photons to reject, just at-focus-plane photons to collect, or not. With
confocal, choice of pinhole size (see my earlier email), which depending
on the gap size (and wavelength), may be resolvable. Sure, GaAsP or
(GaAsP)Hybrid detector has lower QE in the visible (~40%) than front
illuminated (~82% sCMOS) or back-illuminated (~95%, sCMOS, EMCCD, CCD)
... but confocal can have APD(s) with 80+% QE, so QE is a wash.
3a. Field of view and scanning:
   a. Camera based: at the mercy of whatever object lens magnification
and additional magnification in the instrument, readout (typically) some
number of entire rows, i.e. 25x2048 pixels, for sCMOS (sure, some CCDs
and EMCCDs have the acquisition area in the corner near the readout, so?).
   b. point scanning confocal: just scan the area of interest (and maybe
a few more pixels to give the GPU deconvlver a little more work). For
example, 25x25 pixels. Tweak the zoom as desired.
4. if 'change the game' a little ... reflectance (i.e. nanogold,
nannodiamond in reflectance), point scanning confocal is both trivial to
get just the in focus light, and effectively infinite number of photons
available, so shrink the pinhole, and shorter wavelength, as much as
desired; for widefield, good luck finding anyone's research
epi-illumination microscope to be clean enough and glare free enough for
this to work well (maybe some absolutely pristine light path darkfield
condenser and back of specimen might work ... good luck with that).

enjoy,
George
p.s. yes, I realize NIH does not (usually) give out grant money to
measure single vs pairs of sub-resolution beads, or nanogold etc.

On 3/18/2019 5:27 PM, Jeff Carmichael wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
>   I agree that one needs to define the problem more carefully, and it is
> like the screwdriver vs. wrench question. There are also other issues to
> consider, one of them being photon economy.
>
> I'm a bit biased and removed from the bench, originally coming from a lab
> that relied heavily on widefield deconvolution data (the late Fred Fay's
> lab at UMass Med. School) and having experience with earlier generations of
> confocal microscopes. One of the common refrains was about the availability
> of fluorescence photons. Much like the work of Agard and Sedat, the work of
> Carrington, Fogarty, Fay et al. demonstrated the efficacy of a robust,
> iterative deconvolution algorithm approach, using a minimization function
> with a non-negativity constraint to resolve structures to 100-200nm.
> Arriving at a best fit required providing certain inputs regarding
> anticipated feature characteristics that an informed imaging scientist
> would define and could also vary. Different variables would yield slightly
> different results which could be used to help determine best fit with other
> data. I think this level of engagement with and understanding of one's data
> is important.
>
> When one simply trusts either the computational technology or the imaging
> technology, poor choices are made with little understanding.
>
> Regarding the PSF needing to be at a higher resolution, this makes no sense
> to me. The point of the PSF is to empirically model how light spreads in
> your particular system, under the conditions you're using. Use a
> sub-diffraction sized bead and image with the same parameters used to
> acquire the data. A restorative deconvolution doesn't subtract anything. It
> reassigns light to its purported origin. There should be constraints that
> the total integrated optical density be the same before/after
> deconvolution, else it's not really deconvolution but merely some sort of
> filter.
>
> Confocal by its very nature rejects something like 90-98% of available
> fluorescence photons. That data is lost and irretrievable. This problem is
> confounded by sample photobleaching. The relatively poor photon economy
> means that in comparison to widefield, many more photons are emitted per
> each photon detected, and fluorescence can be exhausted before the data is
> even acquired.
>
> Deconvolution -- if done properly -- not only can quantitatively reassign
> fluorescence to it's point of origin, but it does this while collecting all
> available photons in the case of widefield. This is why it's attractive as
> an alternative - because it doesn't throw away data, and instead uses all
> available fluorescence data throughout a volume to restore light back to
> it's point of origin. As others have pointed out, one can also deconvolve
> confocal and super resolution images.
>
> This advantage is probably limited to those applications where relatively
> large volumes are imaged and might otherwise be photobleached by confocal
> laser excitation before being acquired, and yes there are forms of
> structures that don't work well with deconvolution, and for those confocal
> is preferable.
>
> Time and expertise factor into whether this is practical, and for most,
> confocal is the most practical.
>
> *Jeff Carmichael*
>
> *[hidden email] <[hidden email]>*
>
> * <[hidden email]>*
>
>
> On Mon, Mar 18, 2019 at 4:37 PM Benjamin Smith <[hidden email]>
> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> Not sure if this has been said, but this is basically like asking
>> "screwdriver vs. wrench".  A laser scanning confocal microscope is not
>> superior to a compound microscope, and the converse is also true, they are
>> different tools for different tasks.  This is one of the key points I try
>> to get across to students when I teach them about microscopy, we have
>> macro, spinning disk, 2P, light sheet, stereo, confocal, STED, STORM, TEM,
>> SEM, AFM, FIB-SEM, etc., etc, for a reason.  They all excel at tasks that
>> other systems struggle with.
>>
>> Along these lines, here are two scenarios:
>> Scenario 1) You want to get a kHz sample rate of a voltage dye in a
>> cultured neuron.  In this case, a compound microscope with deconvolution
>> (or likely even just a simple high-pass filter) is the clear winner as all
>> the pixels in the frame are temporally correlated (as long as you have CCD
>> or global shutter CMOS), and the frame rate will be much higher than with
>> confocal, even with the most cutting edge technologies.
>>
>> Scenario 2) You want to measure the volume of densely packed nuclei using
>> DAPI in a whole-mount sample.  Deconvolutions will quickly fall apart on
>> this task simply because the deeper you go, the vast majority of the total
>> signal is from out of focus light (much like trying to image a faint star
>> right next to the sun).  This means that the amount of information you have
>> about the sample plane itself becomes nearly non-existent.  Conversely,
>> since confocal microscopes perform the deconvolution before light gets to
>> the detector, you more or less eliminate this bottleneck caused by the
>> dynamic range of the detector.
>>
>> Also, one quick point about deconvolutions.  Unless you measured the PSF in
>> the sample (such as using TetraSpeck  beads) at a higher resolution than
>> you acquired your image, you are not adding any information about the
>> sample.  Rather, you are whittling away information you wish to discard
>> (i.e. it is a lossy process, much like JPEG compression).  Along these
>> lines, iterative blind deconvolutions are allowing a computer to guess what
>> information should be removed.  Thus, just because the image looks better
>> does not necessarily mean it is correct, otherwise STED, STORM, AFM, and
>> cryo-EM would be obsolete.
>>
>> Just my own two cents,
>>     Ben Smith
>>
>> On Mon, Mar 18, 2019 at 11:52 AM Feinstein, Timothy N <[hidden email]>
>> wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> Post images on http://www.imgur.com and include the link in your
>> posting.
>>> *****
>>>
>>> Hi Alison,
>>>
>>> The signal to noise question strikes me as the key question WRT decon.
>>> Most software packages default to a setting that seems unrealistic for
>>> confocal imaging (ie SNR=20), and this leads to a lot of patterning
>>> artifacts in deconvolved images.  At such high SNR any pixel of data is
>>> treated as 'real', so background noise gets processed into weird
>> moire-type
>>> patterns.  This also happens when the algorithm doesn't quite converge
>> and
>>> you let it run for too many iterations (the default iteration #s tend to
>> be
>>> set high as well).  It's critical then to tell the software (when
>> possible)
>>> that your SNR is low, so it gets much more aggressive about detecting and
>>> removing random noise (at the expense of losing details in the 1-3 pixel
>>> range).  I would recommend folks take care if using any package that
>>> doesn't let you correct the SNR.
>>>
>>> Honestly, when SNR is set properly I find that noisy images benefit a lot
>>> more from decon than high-quality cover candidate-type pics.  In addition
>>> to sharpening and Z blur removal the noise and background removal becomes
>>> more dramatic (and beneficial for quantitation) the more background and
>>> noise there is.  This comes up a lot when live imaging when we use
>> resonant
>>> scanning for speed and low photodamage, but have higher noise as a
>>> trade-off.  I'd say that as long as you can image at 1.5x-2x Nyquist
>>> resolution (and the extra time/expense is worth it), even super-noisy
>>> images will benefit quite a bit.
>>>
>>> I understand that we're crossing fingers for the day when everyone has
>>> GPU-enabled decon running seamlessly during acquisition.  For that to
>> work
>>> the algorighms need to automatically (accurately) estimate SNR.  That
>>> doesn't seem like an insurmountable challenge, but I haven't seen it yet.
>>>
>>>
>>> Best,
>>>
>>>
>>> TF
>>>
>>> Timothy Feinstein, Ph.D.
>>> Research Scientist
>>> Department of Developmental Biology
>>> University of Pittsburgh
>>>
>>>
>>>
>>>
>>> On 3/18/19, 11:07 AM, "Confocal Microscopy List on behalf of Alison
>>> North" <[hidden email] on behalf of
>>> [hidden email]> wrote:
>>>
>>>
>>>      Hi all,
>>>
>>>      So this is a very timely discussion because I have been discussing
>>> with
>>>      my staff whether there are data sets that should NOT be deconvolved,
>>> and
>>>      if so, how does one decide that?  I too attended Jim Pawley's
>>> wonderful
>>>      course (he was certainly a huge character as well as an incredibly
>>>      fantastic microscopist, and he will certainly be remembered by all!),
>>> so
>>>      I have generally worked under the assumption that one should
>>> deconvolve
>>>      all confocal data.  But I am also very aware of the potential for
>>>      artifacts if a data set isn't "good enough" for deconvolution.
>>>      Obviously the ideal situation is to acquire an optimal data set -
>>>      well-prepared sample, bright staining, Nyquist sampling etc. etc.,
>> and
>>> a
>>>      high S:N ratio - and by sticking to these rules, our deconvolved
>>>      DeltaVision images or confocal images of fixed samples have always
>>>      looked great.  But nowadays we are faced with different scenarios,
>>>      particularly when you are attempting to do very rapid imaging of
>> live,
>>>      weakly expressing cells, while attempting to minimize phototoxicity.
>>> In
>>>      that case you can end up with pretty lousy S:N ratios, because
>>>      maintaining cell viability or imaging fast enough is more critical.
>>>      For example, I have a lovely new iSIM in my lab, for which the
>> initial
>>>      resolution increase is achieved by the hardware, but the second step
>>> in
>>>      resolution increase is via deconvolution.   The whole point of this
>>>      instrument is for rapid, super-resolution imaging - so we can't
>> simply
>>>      increase exposure times to improve S:N, turning up the laser power
>>> will
>>>      obviously kill the cells, and we can't increase the pixel size or
>>> we'll
>>>      lose the resolution.  And I assume a lot of the new types of
>>>      super-resolution instrument out there must leave you facing the same
>>>      issue, since live cell imaging invariably forces you to compromise
>>>      somewhere within the imaging triangle (or hexagon, or whatever we've
>>> got
>>>      up to now!).
>>>
>>>      Therefore my question is, are there papers out there which have
>>> compared
>>>      deconvolution algorithms and looked at the potential for artifacts on
>>>      really low S:N images, which we could use to advise our researchers
>> on
>>>      what is the minimum you can get away with before you really shouldn't
>>> be
>>>      deconvolving the data set at all? Also, are there papers showing the
>>>      effect of undersampling in the z-axis on the resulting deconvolved
>>>      images (as is often the case on our spinning disk system)?  I haven't
>>>      managed to find any yet (though I confess I've been too busy with
>>> other
>>>      stuff to spend too many hours searching!), so if anybody could point
>>> me
>>>      to some good references I'd be most grateful.  I have spoken with
>>>      several renowned microscopists about whether deconvolution is always
>> a
>>>      good idea under such circumstances, and the gut reaction appears to
>> be
>>>      no, but I could do with some hard and fast validation for teaching
>>> purposes.
>>>
>>>      Many thanks in advance!
>>>
>>>      Alison
>>>
>>>
>>>
>>>      On 3/18/2019 9:56 AM, Feinstein, Timothy N wrote:
>>>      > *****
>>>      > To join, leave or search the confocal microscopy listserv, go to:
>>>      >
>>>
>> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttp-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DvfjahhKueherhfphxmr-Smw_FhB4QUlg-FsBATM1kl0%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288894390&amp;sdata=7V6L2MVDHIM%2BwC0pAgCi3pgsBuFzbd0XQQWFWD%2BTJfY%3D&amp;reserved=0=
>>>      > Post images on
>>>
>> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttp-3A__www.imgur.com%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DedOjY0NZEIhVq-zWzgNHHDaTrutHrPj1Rl6rcwE_B4M%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288894390&amp;sdata=1c1u00xUm3GBfY%2BbuasN%2FzjLdqR5THU38W7oITOW9k0%3D&amp;reserved=0=
>>> and include the link in your posting.
>>>      > *****
>>>      >
>>>      > Avi, I really agree with your point.  I feel that people to
>>> deconvolve any time spatial information is critical, whether they're
>> using
>>> widefield, CLSM, spinning disc, or light sheet.  It's true that
>>> deconvolving adds time and data volume and especially cost, but in trade
>>> you get an image that is substantially sharper, with reduced noise and
>>> background, and more quantitatively accurate*.
>>>      >
>>>      > Regarding whether to just go with a point scanning confocal, I
>> don't
>>> see it as a simple question of better or worse**.  A nuclear-cytoplasmic
>>> translocation assay with monolayer cells works just as well on a
>> widefield,
>>> and (in my experience!) many types of biosensor assay work better with a
>>> properly set up widefield.  The 16-bit depth of widefield images is nice
>>> for quantitation, and modern sCMOS cameras have by far the best
>> acquisition
>>> speeds.  I don't know whether widefields still have a more linear
>>> relationship between sample brightness and detected signal, but the last
>>> time I checked that was still true.
>>>      >
>>>      > (*) Deconvolution is quantitatively useful as long as people make
>>> sure to tell the software to preserve the original intensity values.  One
>>> of my complaints about Hyvolution was that you could not do that, so I
>> just
>>> used the Huygens package that came with it.  I don't know whether
>> Lightning
>>> gives you that option...if not then caveat emptor.
>>>      >
>>>      > (**) My advice mostly applies to turnkey stuff that any lab can
>>> implement, not exotic techniques available to folks with specialists or
>>> engineers on hand.
>>>      >
>>>      > Best,
>>>      >
>>>      >
>>>      > T
>>>      > Timothy Feinstein, Ph.D. esearch Scientist
>>>      > Department of Developmental Biology
>>>      > University of Pittsburgh
>>>      >
>>>      >
>>>      > On 3/18/19, 5:07 AM, "Confocal Microscopy List on behalf of Avi
>>> Jacob" <[hidden email] on behalf of [hidden email]
>>>
>>> wrote:
>>>      >
>>>      >
>>>      >      I'll point out, that you can, of course, deconvolve confocal
>>> images too.
>>>      >      So, while you can indeed get near confocal quality with
>>> well-acquired wf
>>>      >      data after deconvolution, you can also get near SR quality
>> when
>>>      >      deconvolving a well-acquired stack from a confocal. And then
>>> you can also
>>>      >      deconvolve a SR stack and get... well you get the idea! It's
>>> like an arms
>>>      >      race.
>>>      >      I have the Hyvolution and had access for a couple of weeks to
>>> the Lighting,
>>>      >      and now confocal images look blurry to me.
>>>      >      Avi
>>>      >
>>>      >      --
>>>      >      Avi Jacob, Ph.D.
>>>      >      Kanbar Light Microscopy Unit
>>>      >      The Mina & Everard Goodman Faculty of Life Sciences
>>>      >      Bar-Ilan University, Ramat-Gan 529002, Israel
>>>      >
>>>      >
>>>      >
>>>      >      On Sun, Mar 17, 2019 at 6:04 PM George McNamara <
>>> [hidden email]>
>>>      >      wrote:
>>>      >
>>>      >      > *****
>>>      >      > To join, leave or search the confocal microscopy listserv,
>> go
>>> to:
>>>      >      >
>>>
>> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Flists.umn.edu-252Fcgi-2Dbin-252Fwa-253FA0-253Dconfocalmicroscopy-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3DefIAtI3pbBSoyhJucB0DkDu4RXkFkgBZfGrO4PiXrfc-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3D9Uk5ZZzc8LP7V7Oj2aFSRbVluY0mpE-7XPLYdsyviMk%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288894390&amp;sdata=Jv2%2FvAnr1srE%2BjcBXMWe2mVTvO9rowobKYY65DWIRTo%3D&amp;reserved=0=
>>>      >      > Post images on
>>>
>> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fwww.imgur.com-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3DMNM8i3iDaQuMA9LF766-252FyvCyp94jmie4IaC5qIEWclA-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DGy99yAWcbr6KHUA0kYKb4K-wOHL4iNGJsDecMfZ8X-M%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288894390&amp;sdata=2N%2BmlW5RE%2BAFuG%2FuM68FeI9LOfI1m2PlNu23S9eIQts%3D&amp;reserved=0=
>>> and include the link in your posting.
>>>      >      > *****
>>>      >      >
>>>      >      > Hi Mika,
>>>      >      >
>>>      >      > White et al 1987 (
>>>
>> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fjcb.rupress.org-252Fcontent-252F105-252F1-252F41.long-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3Da6oZCfeD7Gt4nYMS89PcklnveCdDLLkksafp3tCyld0-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3D67LyqPF2rBiBDSWJVZeG7TpJz1o4MRDf8ZyLysbeKx4%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288894390&amp;sdata=R3V0RHiTDpGkJUJyI8Q7SDJZjKdk3uDMgmBEPLrzcUw%3D&amp;reserved=0=
>>> ) made a
>>>      >      > compelling case for point scanning confocal microscopes:
>>> collect just
>>>      >      > the in focus light with instant gratification. The case has
>>> not changed,
>>>      >      > the hardware (especially data deluge side) has gotten a lot
>>> better. I
>>>      >      > note that both widefield detectors and PMT/APD/hybrid
>>> detectors/others
>>>      >      > have gotten a lot better in the 32 years from 1987! As have
>>> the optics
>>>      >      > and automation.
>>>      >      >
>>>      >      > Paul Goodwin 2014 (
>>>
>> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.ncbi.nlm.nih.gov-252Fpubmed-252F24974028-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3DGDve8BuxhLDAdBPKywRQoMTYV-252BqSk9aMcz2WrZCHPU8-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DPZ10eaR5B-jwZmf5y920Z41iJYUJcji_VsufABIeX84%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288894390&amp;sdata=dIZN7CiQOTIt24N0mwi0QgDFkcFcVScVNxaoDuS24HM%3D&amp;reserved=0=
>>> ) made
>>>      >      > a nice case for quantitative deconvolution microscopy (and
>> on
>>> very high
>>>      >      > quality specimens, ~10% improvement in resolution compared
>> to
>>> simple
>>>      >      > widefield), but historically slow.
>>>      >      >
>>>      >      > Now, with 'instant gratification' spatial deconvolution,
>>> thanks to the
>>>      >      > GPU revolution (NVidia RTX Titan~16 Teraflops [S.P.], 24 Gb
>>> ram, $2500
>>>      >      > ... not including the deconvolution software module price),
>>> widefield,
>>>      >      > spinning disk (and slightly exotic variants like iSIM, DMD
>>> based, etc,
>>>      >      > see also new THUNDER Imagers [see p.p.s.]), multiphoton (I'm
>>> excited
>>>      >      > about the price point of recent fiber lasers, which could
>>> become much
>>>      >      > better price if achieve 'economy of scale'), and of course,
>>> point
>>>      >      > scanning confocal microscopes.
>>>      >      >
>>>      >      > Spatial deconvolution (especially if someone $uccessfully
>>> implements
>>>      >      > joint spatial deconvolution and spectral unmixing, multiple
>>> cameras -
>>>      >      > for 4 cameras see Babcock 2018, mentions aiming for 8
>>> cameras) helps
>>>      >      > with Expansion Microscopy and/or DNA-PAINT, to go
>>> super-resolution ...
>>>      >      > really single molecule counting (and DNA-PAINT eliminates
>> the
>>> classic
>>>      >      > issue of PALM/STORM/FPALM of not counting every molecule).
>>> Sure,
>>>      >      > DNA-PAINT (like STORM etc) have the issue of a whole lotta
>>> images
>>>      >      > acquired. Data deluge: who cares? Jerome & Price's 10th
>>> commandment of
>>>      >      > confocal imaging is: "10. Storage Media Is Essentially Free
>>> and Infinite".
>>>      >      >
>>>      >      > More significantly, DNA-PAINT and related methods (single
>>> molecule RNA
>>>      >      > FISH, scRNAseq -> MERFISH = Moffitt 2018 as example, etc)
>>> also enable
>>>      >      > multiplex -- with single molecule counting -- to whatever
>>> plex is needed
>>>      >      > to answer the 'biological question(s)' being posed.
>>>      >      >
>>>      >      > All that said, the installed base of research grade point
>>> scanning
>>>      >      > confocal microscopes is large (5000+) and efficient at
>>> acquiring high
>>>      >      > quality images, to the point that user's sample preparation
>>> (and
>>>      >      > avoidance of purchasing stuff from 'Santa Crap' and similar
>>> companies)
>>>      >      > is much more limiting than the microscopes.
>>>      >      >
>>>      >      > George
>>>      >      >
>>>      >      > p.s. a couple of references not included in above:
>>>      >      >
>>>      >      > W. Gray (Jay) Jerome, Robert L. Price 2018... Basic Confocal
>>> Microscopy
>>>      >      > second edition
>>>
>> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Flink.springer.com-252Fbook-252F10.1007-25252F978-2D3-2D319-2D97454-2D5-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3D-252FMQlH4t81Pk9tNTHR8fsn1tQc0JAc2nc-252BnO2gdk60Us-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3D1JXaiNNf-bzSdjywfRJ8gowB9Yq0uGNulQMlGeSMxkE%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288894390&amp;sdata=3ua69ygadJRYIJltAT7qPoJzNd%2B16PWCCeNLQF13WIg%3D&amp;reserved=0=
>>>      >      >
>>>      >      > Expansion ... X10 protocol ... Truckenbrodt 2019 Nat Protoc,
>>>      >      >
>>>
>> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.nature.com-252Farticles-252Fs41596-2D018-2D0117-2D3-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3DCF-252Fp9frimbrxZiMDwLWZjzuyoyQbCuKp4EcvaL3Wmmw-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DYeXbqWFX0mMxO3nVRt59pt6RJYlwIPBMZRePQbiF1yU%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288894390&amp;sdata=TEBfgj6Gg%2Bt02BMyFZCLb8%2Feb5nOyAJmnftouvPd1qY%3D&amp;reserved=0=
>>>      >      >
>>>      >      > DNA-PAINT acronym soup review ... Nieves 2018 Genes,
>>>      >      >
>>>
>> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.mdpi.com-252F2073-2D4425-252F9-252F12-252F621-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3Diwx3Z1Pgr4a2SObO9F47jEtqlfsYQFk2R4gu0Qv1uJM-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DtJY5yunlGClYXKHrXuCDe5KU8D-qhc1ZS26_KuNdXpE%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288904398&amp;sdata=RUgIPB%2Fi0n6i28lOhKZAGSrlyirYALd0l75r5qhZCMk%3D&amp;reserved=0=
>>>      >      >
>>>      >      > Babcock 2018 (4 --> 8 cameras, single molecule localization
>>> microscopy
>>>      >      > with $1550 CMOS cameras) ...
>>>      >      >
>>>
>> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.nature.com-252Farticles-252Fs41598-2D018-2D19981-2Dz-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3DwN-252FHP30wNUE0-252BJWeFJIJFsBk32ZhD4DfKi9gc3ZTz2c-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DTIL4NaeETsqkjycmtWw_CnoEWvrpY1Tkr_SAmz-QkRM%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288904398&amp;sdata=D26AicKRsRoIaCGZdaxaF5OKXfGuo8yS0zvSVgmoBwY%3D&amp;reserved=0=
>>>      >      >
>>>      >      > Moffitt et al 2018 ...
>>>      >      >
>>>
>> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fscience.sciencemag.org-252Fcontent-252F362-252F6416-252Feaau5324.long-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DFR98V6ldS9Q38wI59kly9U8pCzp92Vzc1J6T8ydCU9w-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3D55vzrzqpm3n5Z7logfovDr0N73h1gF7gldtYUSkZ6c0%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288904398&amp;sdata=CQMKMRjzbJVLgZjtpCzzZSwaNvCbt72baFvqppyebSI%3D&amp;reserved=0=
>>> and
>>>      >      > commentary
>>>
>> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fscience.sciencemag.org-252Fcontent-252F362-252F6416-252F749-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3Do0B379uuh-252FrB6gS9n6lG-252BjAZeztiYHZVmICwX4ghKh8-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DXeBps6CWTMM197evmy1n1uC5qOcvU8mAN95bEPmieiQ%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288904398&amp;sdata=um%2F%2FPNffQLOyrTsQcJymSuR2XwRcVnNMwMgQ6qYy19A%3D&amp;reserved=0=
>>>      >      >
>>>      >      > ***
>>>      >      >
>>>      >      > Some resolution numbers:
>>>      >      >
>>>      >      > 1.4 NA objective lens ... 500 nm wavelength .. dxy = 0.61 *
>>> wqavelength
>>>      >      > / NA (I routinely drop the 0.61 from 0.61)
>>>      >      >
>>>      >      > 0.6 * 500 / 1.4 = 214 nm
>>>      >      >
>>>      >      > widefiel deconvolution (re: Goodwin 2014) ~10% better ...
>> 193
>>> nm (if
>>>      >      > pixel size matched or interpolate optimally).
>>>      >      >
>>>      >      > point scanning confocal -- Zeiss has a nice PDF, "Zeiss 2008
>>> Principles
>>>      >      > - Confocal Laser Scanning Microscopy" (see fig 10) on
>> confocal
>>>      >      > resolution wrt 1 and smaller pinhole size, source of the
>>> values below,
>>>      >      >
>>>      >      > 1 Airy unit: 0.51 * 500 / 1.4 = 182 nm.
>>>      >      >
>>>      >      > 0.5 Airy unit ... 0.44 * 500 / 1.4 = 157 nm ... ~0.25
>> photons
>>> throughput
>>>      >      > (which doesn't matter if target is photostable).
>>>      >      >
>>>      >      > 0.2 Airy unit ... you can ask your Zeiss rep about AiryScan
>>> (and
>>>      >      > FastAiryScan).
>>>      >      >
>>>      >      > 0.1 Airy unit ... 0.37 * 500 / 1.4 = 132 nm ... ~0.10
>> photons
>>> throughput.
>>>      >      >
>>>      >      > Most modern point scanning confocal microscopes have a 405
>> nm
>>> laser, so
>>>      >      > if using BV421 (and ignoring potential photobleaching for a
>>> moment),
>>>      >      >
>>>      >      > 1 Airy unit: 0.51 * 421 / 1.4 = 153 nm.
>>>      >      >
>>>      >      > or in reflection mode, i.e. nanodiamond or nanogold,
>>>      >      >
>>>      >      > 1 Airy unit: 0.51 * 405 / 1.4 = 147 nm ... and reflection
>>> implies no
>>>      >      > photobleaching, so infinite number of photons (though also
>> no
>>> blinking,
>>>      >      > so not usually eligible for precision localization) ...
>>>      >      >
>>>      >      > 0.1 Airy unit: 0.37 * 405 / 1.4 = 107 nm
>>>      >      >
>>>      >      > and not going completely exotic with NA (i.e. 1.65), if
>>> perfectly
>>>      >      > refractive index match with a fairly conventional 1.49 NA
>>> lens, and
>>>      >      > inreflectance:
>>>      >      >
>>>      >      > 0.1 Airy unit: 0.37 * 405 / 1.49 = 100.57 nm
>>>      >      >
>>>      >      > I think I'd rather invest a DNA-PAINT friendly rig than deal
>>> with 157 to
>>>      >      > 101 nm.
>>>      >      >
>>>      >      > DNA-PAINT makes resolution irrelevant, if you use it (and
>>> don't run out
>>>      >      > of disk space or time or money), since precision
>> localization
>>> is
>>>      >      > resolution divided by square root of number of photons, ex.
>>> 250 nm XY
>>>      >      > resolution / sqrt(1,000,000) = 0.25 nm, and could increase
>>> number of
>>>      >      > photons per target further, but why bother?
>>>      >      >
>>>      >      > ***
>>>      >      >
>>>      >      > point scanning confocal microscopes are also great platforms
>>> for
>>>      >      > F-Techniques, such as FastFLIM (aka rapidFLIM, etc, much
>>> faster than
>>>      >      > classic TCSPC slow FLIM), FCS, FCCS, see Liu 2008,
>>>      >      >
>>>
>> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.ncbi.nlm.nih.gov-252Fpubmed-252F18387308-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DZQTosrSoPFpCa8Y3IdEa9Xz-252B4RHC8JO4gBppHmzkazo-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DmeRUIZdF3FLQZGyok4cxyc4AP4yq2lXX41Vt8q9WMmw%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288904398&amp;sdata=XTEH306i5ez7vKq2cHKCVQlSphNiFIdnqIXmwnOmyNw%3D&amp;reserved=0=
>>>      >      >
>>>      >      > ***
>>>      >      >
>>>      >      > p.p.s. Disclosures I am ...
>>>      >      >
>>>      >      > 1. currently hosting a Leica THUNDER Imager tour event (ends
>>> Monday
>>>      >      > 3/18/2019 afternoon) ...  see pdf download page,
>>>      >      >
>>>      >      > THUNDER Technology Note
>>>      >      > THUNDER Imagers: How Do They Really Work?
>>>      >      >
>>>      >      >
>>>
>> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.leica-2Dmicrosystems.com-252Fscience-2Dlab-252Fthunder-2Dtechnology-2Dnote-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DauRUORuEKAir87-252Bbw7RHyTR9IxxrLYl1g3bFBiRTXBM-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DLmFGYl_Icu6b9AAIYnHkoiH26bRZ688ODEc9I6k8yco%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288904398&amp;sdata=5ZGLmCZt85tKxH1CJYM%2Bvul5S4qXw9%2FOnyWG80m2QxM%3D&amp;reserved=0=
>>>      >      >
>>>      >      > 2. hosting Nikon confocal demos in May 2019.
>>>      >      >
>>>      >      > 3. aiming to co-host with ISS a FastFLIM (one day)
>>> mini-symposium this
>>>      >      > summer.
>>>      >      >
>>>      >      > 4. an unpaid advisor for Gary Brooker for FINCH/CINCH, re:
>>>      >      >
>>>
>> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.ncbi.nlm.nih.gov-252Fpubmed-252F28261321-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DuVw0kiW1baZV0RJv-252Bk1ObKznhw51pemnCMehjHkUa2g-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DKC1HMkiDzpE2DLsuj1I2nHvopwFVMMc0GVecDV-fWkM%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288904398&amp;sdata=QC0LyEp9hbSKOgpV%2Fi%2B%2FABGDX4az02fRVdMZ4Ovufps%3D&amp;reserved=0=
>>>      >      >
>>>      >      >
>>>      >      > On 3/17/2019 10:43 AM, Mika Ruonala wrote:
>>>      >      > > *****
>>>      >      > > To join, leave or search the confocal microscopy listserv,
>>> go to:
>>>      >      > >
>>>
>> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Flists.umn.edu-252Fcgi-2Dbin-252Fwa-253FA0-253Dconfocalmicroscopy-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DdHOCjeBda4sSZPf-252FB2-252B5Sv3Q8Qzcs708p4we8vHf-252FIg-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DqZ-tdRwhHG172zv4uHPeAJMNeIIHNxozhQudV-hF2kE%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288904398&amp;sdata=BnCAxorVJgPSFw70Kr%2B401vq4hMlZhvzdkivum9XGNE%3D&amp;reserved=0=
>>>      >      > > Post images on
>>>
>> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fwww.imgur.com-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DcnhzoWOP4RwDR622fn447aQVxW8ZBI3D0utze67RiGg-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DYzF36y8eV4SG3E0LJEDu-9AMm3Nh05dwp_XKhB6BUyU%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288914407&amp;sdata=ec0HIKuOMVqlLU7knyQ1JdS1B6c1BMCIb%2FYsHIFdYbM%3D&amp;reserved=0=
>>> and include the link in your
>>>      >      > posting.
>>>      >      > > *****
>>>      >      > >
>>>      >      > > Hi.
>>>      >      > > There are software solutions that are able to create image
>>> data from
>>>      >      > wide-field and even microscope systems with seemingly
>> similar
>>> quality to
>>>      >      > that obtained from confocal systems.
>>>      >      > >
>>>      >      > > The comparison of acquisition vs. software is essentially
>> a
>>> comparison
>>>      >      > of image acquisition vs. image processing. While a software
>>> solution is way
>>>      >      > cheaper than a hardware solution if it is able to produce
>>> image data with
>>>      >      > equal quality why would anyone choose to invest to a
>> confocal
>>> anymore?
>>>      >      > >
>>>      >      > > I’m looking forward to a vidid discussion!
>>>      >      > >
>>>      >      > >> m
>>>      >      >
>>>      >
>>>      >
>>>      --
>>>      Alison J. North, Ph.D.,
>>>      Research Associate Professor and
>>>      Senior Director of the Bio-Imaging Resource Center,
>>>      The Rockefeller University,
>>>      1230 York Avenue,
>>>      New York,
>>>      NY 10065.
>>>      Tel: office    ++ 212 327 7488
>>>      Tel: lab        ++ 212 327 7486
>>>      Fax:            ++ 212 327 7489
>>>
>>>
>>>
>> --
>> Benjamin E. Smith, Ph. D.
>> Imaging Specialist, Vision Science
>> University of California, Berkeley
>> 195 Life Sciences Addition
>> Berkeley, CA  94720-3200
>> Tel  [phone]
>> Fax [phone]
>> e-mail: [hidden email]
>> http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>
>>
>>

[Lutz Schaefer]

*****
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Post images on http://www.imgur.com and include the link in your posting.
*****

Dear Gabor,
resistance to explain details may indicate a lack of substance. I had a look
at the "Leica Microsystems Thunder Technology Note" document PDF. As
explained it becomes clear that "Thunder Technology" can't be much different
than a simple nearest neighbor de-blurring. Perhaps it is not implemented as
first described in Castleman's textbook "Digital Image Processing" (1979),
but the described additive 2D model suggests an approach that is very close
to this. Using Wavelets to simulate out of focus information does not
significantly change the idea of subtracting questionably, approximate
out-of-focus information. In my opinion a subtractive 2D model should never
even be brought in context with a rigorous 3D forward model and
deconvolution in general. There is always a lot of marketing BS in these
notes lacking scientific evidence of the consistency of claims made.

Regards
Lutz

-----Original Message-----
From: Csúcs Gábor
Sent: Monday, March 18, 2019 3:56 PM
To: [hidden email]
Subject: Re: General question: Software vs. hardware

*****
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*****

Connected to the same thread I would like your opinion about the new Leica
davalopment (they call it "computational clearing" and sell it under the
name "Thunder".
They are very resistant to tell any mathematical details about it (even in a
recently published white paper). Based on some rudimentaqry explanation it
seems (really my subjective interpretation)that they do a wavelet
conversion, filter out the large spatial components (claiming that those
correspond to the out-of-focus background) and then transfer back the data.
With this they claim to have the same effect as structured illumination
(e.g. ApoTome) or a spinning disk (though this is a clear overstatement).
Any thoughts from the community?

Greetings Gabor

[Jeffrey Carmichael]

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*****

 George,

True, if it's not acquired it's not data, but that's semantics because
those same photons would be acquired as data in widefield. The point is
that a small fraction of the light that is acquired with widefield
illumination is acquired in a typical confocal configuration using the same
objective lens, so I guess I should have said that more data of the same
sample is acquired with widefield.....which is simply stating the obvious
when you look at a convolved, widefield, blurry image.

To clarify the rejection of 90 - 98% estimate, this means relative to
widefield collection. Even widefield fails to collect the large majority
because it's only collecting a cone out of a 3D sphere of emitted
fluorescence, influenced somewhat by the polarity of the fluorophores which
generally are randomly distributed.

Partly because of the geometry, most of the out-of-focus light from any
particular object acquired in widefield is within a couple microns of the
focal plane, not at a large distance. Much of the light from a large
distance from the focal plane is dispersed outside the collection angles.
Good deconvolution algorithms take this into account.

Regarding the bead example [*one or two 40 nm beads with some gap (or DNA
origami), at the coverglass*] this doesn't strike me as very representative
of most real-world biological specimens, which tend towards many structures
in or on a cell (and not in an ideally perpendicular plane), surrounded by
many cells, with lots of fluorescence from different focal planes. However,
even in this case, deconvolution could well provide the same resolution.

Even with no additional out-of-focus fluorescent objects to muddle the
situation, the widefield collection will collect far more fluorescence.You
simply image a cube (image stack) even though the beads are on one plane,
acquiring z planes above and below, just like you do when acquiring a PSF
with a bead. Now, voila - lots of out-of-focus fluorescence (real
data).....all of which can be used to fuel the deconvolution to describe
the size and shape and separation of the objects with ever-increasing
accuracy.

Regarding your earlier email George, I am always impressed by your
encyclopedic knowledge and deep understanding of imaging, and I can't
compete, nor do I wish to :)

Jeff

*Jeff Carmichael*

*Director of Marketing*

*[hidden email] <[hidden email]> | 802-428-2528*

* <[hidden email]>*


On Mon, Mar 18, 2019 at 8:14 PM George McNamara <[hidden email]>
wrote:

> Hi Jeff et al,
>
> I disagree with Jeff's statement:
>
>      Confocal by its very nature rejects something like 90-98% of
> available fluorescence photons. That data is lost and irretrievable.
>
> 1. trivially, if it is not digitized, it is not data. If is from a
> far-out of focus plane, deconvolution not going to reassign it very well.
> 2. Reducing to the simplest cases: one vs two sub-resolution features,
> for simplicity, one or two 40 nm beads with some gap (or DNA origami),
> at the coverglass, refractive index matched media (i.e. 1.4 NA objective
> lens, R.I. 1.518 immersion oil and mounting medium). So: no out of focus
> photons to reject, just at-focus-plane photons to collect, or not. With
> confocal, choice of pinhole size (see my earlier email), which depending
> on the gap size (and wavelength), may be resolvable. Sure, GaAsP or
> (GaAsP)Hybrid detector has lower QE in the visible (~40%) than front
> illuminated (~82% sCMOS) or back-illuminated (~95%, sCMOS, EMCCD, CCD)
> ... but confocal can have APD(s) with 80+% QE, so QE is a wash.
> 3a. Field of view and scanning:
>    a. Camera based: at the mercy of whatever object lens magnification
> and additional magnification in the instrument, readout (typically) some
> number of entire rows, i.e. 25x2048 pixels, for sCMOS (sure, some CCDs
> and EMCCDs have the acquisition area in the corner near the readout, so?).
>    b. point scanning confocal: just scan the area of interest (and maybe
> a few more pixels to give the GPU deconvlver a little more work). For
> example, 25x25 pixels. Tweak the zoom as desired.
> 4. if 'change the game' a little ... reflectance (i.e. nanogold,
> nannodiamond in reflectance), point scanning confocal is both trivial to
> get just the in focus light, and effectively infinite number of photons
> available, so shrink the pinhole, and shorter wavelength, as much as
> desired; for widefield, good luck finding anyone's research
> epi-illumination microscope to be clean enough and glare free enough for
> this to work well (maybe some absolutely pristine light path darkfield
> condenser and back of specimen might work ... good luck with that).
>
> enjoy,
> George
> p.s. yes, I realize NIH does not (usually) give out grant money to
> measure single vs pairs of sub-resolution beads, or nanogold etc.
>
> On 3/18/2019 5:27 PM, Jeff Carmichael wrote:
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> >   I agree that one needs to define the problem more carefully, and it is
> > like the screwdriver vs. wrench question. There are also other issues to
> > consider, one of them being photon economy.
> >
> > I'm a bit biased and removed from the bench, originally coming from a lab
> > that relied heavily on widefield deconvolution data (the late Fred Fay's
> > lab at UMass Med. School) and having experience with earlier generations
> of
> > confocal microscopes. One of the common refrains was about the
> availability
> > of fluorescence photons. Much like the work of Agard and Sedat, the work
> of
> > Carrington, Fogarty, Fay et al. demonstrated the efficacy of a robust,
> > iterative deconvolution algorithm approach, using a minimization function
> > with a non-negativity constraint to resolve structures to 100-200nm.
> > Arriving at a best fit required providing certain inputs regarding
> > anticipated feature characteristics that an informed imaging scientist
> > would define and could also vary. Different variables would yield
> slightly
> > different results which could be used to help determine best fit with
> other
> > data. I think this level of engagement with and understanding of one's
> data
> > is important.
> >
> > When one simply trusts either the computational technology or the imaging
> > technology, poor choices are made with little understanding.
> >
> > Regarding the PSF needing to be at a higher resolution, this makes no
> sense
> > to me. The point of the PSF is to empirically model how light spreads in
> > your particular system, under the conditions you're using. Use a
> > sub-diffraction sized bead and image with the same parameters used to
> > acquire the data. A restorative deconvolution doesn't subtract anything.
> It
> > reassigns light to its purported origin. There should be constraints that
> > the total integrated optical density be the same before/after
> > deconvolution, else it's not really deconvolution but merely some sort of
> > filter.
> >
> > Confocal by its very nature rejects something like 90-98% of available
> > fluorescence photons. That data is lost and irretrievable. This problem
> is
> > confounded by sample photobleaching. The relatively poor photon economy
> > means that in comparison to widefield, many more photons are emitted per
> > each photon detected, and fluorescence can be exhausted before the data
> is
> > even acquired.
> >
> > Deconvolution -- if done properly -- not only can quantitatively reassign
> > fluorescence to it's point of origin, but it does this while collecting
> all
> > available photons in the case of widefield. This is why it's attractive
> as
> > an alternative - because it doesn't throw away data, and instead uses all
> > available fluorescence data throughout a volume to restore light back to
> > it's point of origin. As others have pointed out, one can also deconvolve
> > confocal and super resolution images.
> >
> > This advantage is probably limited to those applications where relatively
> > large volumes are imaged and might otherwise be photobleached by confocal
> > laser excitation before being acquired, and yes there are forms of
> > structures that don't work well with deconvolution, and for those
> confocal
> > is preferable.
> >
> > Time and expertise factor into whether this is practical, and for most,
> > confocal is the most practical.
> >
> > *Jeff Carmichael*
> >
> > *[hidden email] <[hidden email]>*
> >
> > * <[hidden email]>*
> >
> >
> > On Mon, Mar 18, 2019 at 4:37 PM Benjamin Smith <
> [hidden email]>
> > wrote:
> >
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> Post images on http://www.imgur.com and include the link in your
> posting.
> >> *****
> >>
> >> Not sure if this has been said, but this is basically like asking
> >> "screwdriver vs. wrench".  A laser scanning confocal microscope is not
> >> superior to a compound microscope, and the converse is also true, they
> are
> >> different tools for different tasks.  This is one of the key points I
> try
> >> to get across to students when I teach them about microscopy, we have
> >> macro, spinning disk, 2P, light sheet, stereo, confocal, STED, STORM,
> TEM,
> >> SEM, AFM, FIB-SEM, etc., etc, for a reason.  They all excel at tasks
> that
> >> other systems struggle with.
> >>
> >> Along these lines, here are two scenarios:
> >> Scenario 1) You want to get a kHz sample rate of a voltage dye in a
> >> cultured neuron.  In this case, a compound microscope with deconvolution
> >> (or likely even just a simple high-pass filter) is the clear winner as
> all
> >> the pixels in the frame are temporally correlated (as long as you have
> CCD
> >> or global shutter CMOS), and the frame rate will be much higher than
> with
> >> confocal, even with the most cutting edge technologies.
> >>
> >> Scenario 2) You want to measure the volume of densely packed nuclei
> using
> >> DAPI in a whole-mount sample.  Deconvolutions will quickly fall apart on
> >> this task simply because the deeper you go, the vast majority of the
> total
> >> signal is from out of focus light (much like trying to image a faint
> star
> >> right next to the sun).  This means that the amount of information you
> have
> >> about the sample plane itself becomes nearly non-existent.  Conversely,
> >> since confocal microscopes perform the deconvolution before light gets
> to
> >> the detector, you more or less eliminate this bottleneck caused by the
> >> dynamic range of the detector.
> >>
> >> Also, one quick point about deconvolutions.  Unless you measured the
> PSF in
> >> the sample (such as using TetraSpeck  beads) at a higher resolution than
> >> you acquired your image, you are not adding any information about the
> >> sample.  Rather, you are whittling away information you wish to discard
> >> (i.e. it is a lossy process, much like JPEG compression).  Along these
> >> lines, iterative blind deconvolutions are allowing a computer to guess
> what
> >> information should be removed.  Thus, just because the image looks
> better
> >> does not necessarily mean it is correct, otherwise STED, STORM, AFM, and
> >> cryo-EM would be obsolete.
> >>
> >> Just my own two cents,
> >>     Ben Smith
> >>
> >> On Mon, Mar 18, 2019 at 11:52 AM Feinstein, Timothy N <[hidden email]>
> >> wrote:
> >>
> >>> *****
> >>> To join, leave or search the confocal microscopy listserv, go to:
> >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >>> Post images on http://www.imgur.com and include the link in your
> >> posting.
> >>> *****
> >>>
> >>> Hi Alison,
> >>>
> >>> The signal to noise question strikes me as the key question WRT decon.
> >>> Most software packages default to a setting that seems unrealistic for
> >>> confocal imaging (ie SNR=20), and this leads to a lot of patterning
> >>> artifacts in deconvolved images.  At such high SNR any pixel of data is
> >>> treated as 'real', so background noise gets processed into weird
> >> moire-type
> >>> patterns.  This also happens when the algorithm doesn't quite converge
> >> and
> >>> you let it run for too many iterations (the default iteration #s tend
> to
> >> be
> >>> set high as well).  It's critical then to tell the software (when
> >> possible)
> >>> that your SNR is low, so it gets much more aggressive about detecting
> and
> >>> removing random noise (at the expense of losing details in the 1-3
> pixel
> >>> range).  I would recommend folks take care if using any package that
> >>> doesn't let you correct the SNR.
> >>>
> >>> Honestly, when SNR is set properly I find that noisy images benefit a
> lot
> >>> more from decon than high-quality cover candidate-type pics.  In
> addition
> >>> to sharpening and Z blur removal the noise and background removal
> becomes
> >>> more dramatic (and beneficial for quantitation) the more background and
> >>> noise there is.  This comes up a lot when live imaging when we use
> >> resonant
> >>> scanning for speed and low photodamage, but have higher noise as a
> >>> trade-off.  I'd say that as long as you can image at 1.5x-2x Nyquist
> >>> resolution (and the extra time/expense is worth it), even super-noisy
> >>> images will benefit quite a bit.
> >>>
> >>> I understand that we're crossing fingers for the day when everyone has
> >>> GPU-enabled decon running seamlessly during acquisition.  For that to
> >> work
> >>> the algorighms need to automatically (accurately) estimate SNR.  That
> >>> doesn't seem like an insurmountable challenge, but I haven't seen it
> yet.
> >>>
> >>>
> >>> Best,
> >>>
> >>>
> >>> TF
> >>>
> >>> Timothy Feinstein, Ph.D.
> >>> Research Scientist
> >>> Department of Developmental Biology
> >>> University of Pittsburgh
> >>>
> >>>
> >>>
> >>>
> >>> On 3/18/19, 11:07 AM, "Confocal Microscopy List on behalf of Alison
> >>> North" <[hidden email] on behalf of
> >>> [hidden email]> wrote:
> >>>
> >>>
> >>>      Hi all,
> >>>
> >>>      So this is a very timely discussion because I have been discussing
> >>> with
> >>>      my staff whether there are data sets that should NOT be
> deconvolved,
> >>> and
> >>>      if so, how does one decide that?  I too attended Jim Pawley's
> >>> wonderful
> >>>      course (he was certainly a huge character as well as an incredibly
> >>>      fantastic microscopist, and he will certainly be remembered by
> all!),
> >>> so
> >>>      I have generally worked under the assumption that one should
> >>> deconvolve
> >>>      all confocal data.  But I am also very aware of the potential for
> >>>      artifacts if a data set isn't "good enough" for deconvolution.
> >>>      Obviously the ideal situation is to acquire an optimal data set -
> >>>      well-prepared sample, bright staining, Nyquist sampling etc. etc.,
> >> and
> >>> a
> >>>      high S:N ratio - and by sticking to these rules, our deconvolved
> >>>      DeltaVision images or confocal images of fixed samples have always
> >>>      looked great.  But nowadays we are faced with different scenarios,
> >>>      particularly when you are attempting to do very rapid imaging of
> >> live,
> >>>      weakly expressing cells, while attempting to minimize
> phototoxicity.
> >>> In
> >>>      that case you can end up with pretty lousy S:N ratios, because
> >>>      maintaining cell viability or imaging fast enough is more
> critical.
> >>>      For example, I have a lovely new iSIM in my lab, for which the
> >> initial
> >>>      resolution increase is achieved by the hardware, but the second
> step
> >>> in
> >>>      resolution increase is via deconvolution.   The whole point of
> this
> >>>      instrument is for rapid, super-resolution imaging - so we can't
> >> simply
> >>>      increase exposure times to improve S:N, turning up the laser power
> >>> will
> >>>      obviously kill the cells, and we can't increase the pixel size or
> >>> we'll
> >>>      lose the resolution.  And I assume a lot of the new types of
> >>>      super-resolution instrument out there must leave you facing the
> same
> >>>      issue, since live cell imaging invariably forces you to compromise
> >>>      somewhere within the imaging triangle (or hexagon, or whatever
> we've
> >>> got
> >>>      up to now!).
> >>>
> >>>      Therefore my question is, are there papers out there which have
> >>> compared
> >>>      deconvolution algorithms and looked at the potential for
> artifacts on
> >>>      really low S:N images, which we could use to advise our
> researchers
> >> on
> >>>      what is the minimum you can get away with before you really
> shouldn't
> >>> be
> >>>      deconvolving the data set at all? Also, are there papers showing
> the
> >>>      effect of undersampling in the z-axis on the resulting deconvolved
> >>>      images (as is often the case on our spinning disk system)?  I
> haven't
> >>>      managed to find any yet (though I confess I've been too busy with
> >>> other
> >>>      stuff to spend too many hours searching!), so if anybody could
> point
> >>> me
> >>>      to some good references I'd be most grateful.  I have spoken with
> >>>      several renowned microscopists about whether deconvolution is
> always
> >> a
> >>>      good idea under such circumstances, and the gut reaction appears
> to
> >> be
> >>>      no, but I could do with some hard and fast validation for teaching
> >>> purposes.
> >>>
> >>>      Many thanks in advance!
> >>>
> >>>      Alison
> >>>
> >>>
> >>>
> >>>      On 3/18/2019 9:56 AM, Feinstein, Timothy N wrote:
> >>>      > *****
> >>>      > To join, leave or search the confocal microscopy listserv, go
> to:
> >>>      >
> >>>
> >>
> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttp-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DvfjahhKueherhfphxmr-Smw_FhB4QUlg-FsBATM1kl0%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288894390&amp;sdata=7V6L2MVDHIM%2BwC0pAgCi3pgsBuFzbd0XQQWFWD%2BTJfY%3D&amp;reserved=0=
> >>>      > Post images on
> >>>
> >>
> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttp-3A__www.imgur.com%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DedOjY0NZEIhVq-zWzgNHHDaTrutHrPj1Rl6rcwE_B4M%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288894390&amp;sdata=1c1u00xUm3GBfY%2BbuasN%2FzjLdqR5THU38W7oITOW9k0%3D&amp;reserved=0=
> >>> and include the link in your posting.
> >>>      > *****
> >>>      >
> >>>      > Avi, I really agree with your point.  I feel that people to
> >>> deconvolve any time spatial information is critical, whether they're
> >> using
> >>> widefield, CLSM, spinning disc, or light sheet.  It's true that
> >>> deconvolving adds time and data volume and especially cost, but in
> trade
> >>> you get an image that is substantially sharper, with reduced noise and
> >>> background, and more quantitatively accurate*.
> >>>      >
> >>>      > Regarding whether to just go with a point scanning confocal, I
> >> don't
> >>> see it as a simple question of better or worse**.  A
> nuclear-cytoplasmic
> >>> translocation assay with monolayer cells works just as well on a
> >> widefield,
> >>> and (in my experience!) many types of biosensor assay work better with
> a
> >>> properly set up widefield.  The 16-bit depth of widefield images is
> nice
> >>> for quantitation, and modern sCMOS cameras have by far the best
> >> acquisition
> >>> speeds.  I don't know whether widefields still have a more linear
> >>> relationship between sample brightness and detected signal, but the
> last
> >>> time I checked that was still true.
> >>>      >
> >>>      > (*) Deconvolution is quantitatively useful as long as people
> make
> >>> sure to tell the software to preserve the original intensity values.
> One
> >>> of my complaints about Hyvolution was that you could not do that, so I
> >> just
> >>> used the Huygens package that came with it.  I don't know whether
> >> Lightning
> >>> gives you that option...if not then caveat emptor.
> >>>      >
> >>>      > (**) My advice mostly applies to turnkey stuff that any lab can
> >>> implement, not exotic techniques available to folks with specialists or
> >>> engineers on hand.
> >>>      >
> >>>      > Best,
> >>>      >
> >>>      >
> >>>      > T
> >>>      > Timothy Feinstein, Ph.D. esearch Scientist
> >>>      > Department of Developmental Biology
> >>>      > University of Pittsburgh
> >>>      >
> >>>      >
> >>>      > On 3/18/19, 5:07 AM, "Confocal Microscopy List on behalf of Avi
> >>> Jacob" <[hidden email] on behalf of
> [hidden email]
> >>>
> >>> wrote:
> >>>      >
> >>>      >
> >>>      >      I'll point out, that you can, of course, deconvolve
> confocal
> >>> images too.
> >>>      >      So, while you can indeed get near confocal quality with
> >>> well-acquired wf
> >>>      >      data after deconvolution, you can also get near SR quality
> >> when
> >>>      >      deconvolving a well-acquired stack from a confocal. And
> then
> >>> you can also
> >>>      >      deconvolve a SR stack and get... well you get the idea!
> It's
> >>> like an arms
> >>>      >      race.
> >>>      >      I have the Hyvolution and had access for a couple of weeks
> to
> >>> the Lighting,
> >>>      >      and now confocal images look blurry to me.
> >>>      >      Avi
> >>>      >
> >>>      >      --
> >>>      >      Avi Jacob, Ph.D.
> >>>      >      Kanbar Light Microscopy Unit
> >>>      >      The Mina & Everard Goodman Faculty of Life Sciences
> >>>      >      Bar-Ilan University, Ramat-Gan 529002, Israel
> >>>      >
> >>>      >
> >>>      >
> >>>      >      On Sun, Mar 17, 2019 at 6:04 PM George McNamara <
> >>> [hidden email]>
> >>>      >      wrote:
> >>>      >
> >>>      >      > *****
> >>>      >      > To join, leave or search the confocal microscopy
> listserv,
> >> go
> >>> to:
> >>>      >      >
> >>>
> >>
> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Flists.umn.edu-252Fcgi-2Dbin-252Fwa-253FA0-253Dconfocalmicroscopy-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3DefIAtI3pbBSoyhJucB0DkDu4RXkFkgBZfGrO4PiXrfc-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3D9Uk5ZZzc8LP7V7Oj2aFSRbVluY0mpE-7XPLYdsyviMk%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288894390&amp;sdata=Jv2%2FvAnr1srE%2BjcBXMWe2mVTvO9rowobKYY65DWIRTo%3D&amp;reserved=0=
> >>>      >      > Post images on
> >>>
> >>
> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fwww.imgur.com-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3DMNM8i3iDaQuMA9LF766-252FyvCyp94jmie4IaC5qIEWclA-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DGy99yAWcbr6KHUA0kYKb4K-wOHL4iNGJsDecMfZ8X-M%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288894390&amp;sdata=2N%2BmlW5RE%2BAFuG%2FuM68FeI9LOfI1m2PlNu23S9eIQts%3D&amp;reserved=0=
> >>> and include the link in your posting.
> >>>      >      > *****
> >>>      >      >
> >>>      >      > Hi Mika,
> >>>      >      >
> >>>      >      > White et al 1987 (
> >>>
> >>
> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fjcb.rupress.org-252Fcontent-252F105-252F1-252F41.long-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3Da6oZCfeD7Gt4nYMS89PcklnveCdDLLkksafp3tCyld0-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3D67LyqPF2rBiBDSWJVZeG7TpJz1o4MRDf8ZyLysbeKx4%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288894390&amp;sdata=R3V0RHiTDpGkJUJyI8Q7SDJZjKdk3uDMgmBEPLrzcUw%3D&amp;reserved=0=
> >>> ) made a
> >>>      >      > compelling case for point scanning confocal microscopes:
> >>> collect just
> >>>      >      > the in focus light with instant gratification. The case
> has
> >>> not changed,
> >>>      >      > the hardware (especially data deluge side) has gotten a
> lot
> >>> better. I
> >>>      >      > note that both widefield detectors and PMT/APD/hybrid
> >>> detectors/others
> >>>      >      > have gotten a lot better in the 32 years from 1987! As
> have
> >>> the optics
> >>>      >      > and automation.
> >>>      >      >
> >>>      >      > Paul Goodwin 2014 (
> >>>
> >>
> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.ncbi.nlm.nih.gov-252Fpubmed-252F24974028-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3DGDve8BuxhLDAdBPKywRQoMTYV-252BqSk9aMcz2WrZCHPU8-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DPZ10eaR5B-jwZmf5y920Z41iJYUJcji_VsufABIeX84%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288894390&amp;sdata=dIZN7CiQOTIt24N0mwi0QgDFkcFcVScVNxaoDuS24HM%3D&amp;reserved=0=
> >>> ) made
> >>>      >      > a nice case for quantitative deconvolution microscopy
> (and
> >> on
> >>> very high
> >>>      >      > quality specimens, ~10% improvement in resolution
> compared
> >> to
> >>> simple
> >>>      >      > widefield), but historically slow.
> >>>      >      >
> >>>      >      > Now, with 'instant gratification' spatial deconvolution,
> >>> thanks to the
> >>>      >      > GPU revolution (NVidia RTX Titan~16 Teraflops [S.P.], 24
> Gb
> >>> ram, $2500
> >>>      >      > ... not including the deconvolution software module
> price),
> >>> widefield,
> >>>      >      > spinning disk (and slightly exotic variants like iSIM,
> DMD
> >>> based, etc,
> >>>      >      > see also new THUNDER Imagers [see p.p.s.]), multiphoton
> (I'm
> >>> excited
> >>>      >      > about the price point of recent fiber lasers, which could
> >>> become much
> >>>      >      > better price if achieve 'economy of scale'), and of
> course,
> >>> point
> >>>      >      > scanning confocal microscopes.
> >>>      >      >
> >>>      >      > Spatial deconvolution (especially if someone $uccessfully
> >>> implements
> >>>      >      > joint spatial deconvolution and spectral unmixing,
> multiple
> >>> cameras -
> >>>      >      > for 4 cameras see Babcock 2018, mentions aiming for 8
> >>> cameras) helps
> >>>      >      > with Expansion Microscopy and/or DNA-PAINT, to go
> >>> super-resolution ...
> >>>      >      > really single molecule counting (and DNA-PAINT eliminates
> >> the
> >>> classic
> >>>      >      > issue of PALM/STORM/FPALM of not counting every
> molecule).
> >>> Sure,
> >>>      >      > DNA-PAINT (like STORM etc) have the issue of a whole
> lotta
> >>> images
> >>>      >      > acquired. Data deluge: who cares? Jerome & Price's 10th
> >>> commandment of
> >>>      >      > confocal imaging is: "10. Storage Media Is Essentially
> Free
> >>> and Infinite".
> >>>      >      >
> >>>      >      > More significantly, DNA-PAINT and related methods (single
> >>> molecule RNA
> >>>      >      > FISH, scRNAseq -> MERFISH = Moffitt 2018 as example, etc)
> >>> also enable
> >>>      >      > multiplex -- with single molecule counting -- to whatever
> >>> plex is needed
> >>>      >      > to answer the 'biological question(s)' being posed.
> >>>      >      >
> >>>      >      > All that said, the installed base of research grade point
> >>> scanning
> >>>      >      > confocal microscopes is large (5000+) and efficient at
> >>> acquiring high
> >>>      >      > quality images, to the point that user's sample
> preparation
> >>> (and
> >>>      >      > avoidance of purchasing stuff from 'Santa Crap' and
> similar
> >>> companies)
> >>>      >      > is much more limiting than the microscopes.
> >>>      >      >
> >>>      >      > George
> >>>      >      >
> >>>      >      > p.s. a couple of references not included in above:
> >>>      >      >
> >>>      >      > W. Gray (Jay) Jerome, Robert L. Price 2018... Basic
> Confocal
> >>> Microscopy
> >>>      >      > second edition
> >>>
> >>
> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Flink.springer.com-252Fbook-252F10.1007-25252F978-2D3-2D319-2D97454-2D5-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3D-252FMQlH4t81Pk9tNTHR8fsn1tQc0JAc2nc-252BnO2gdk60Us-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3D1JXaiNNf-bzSdjywfRJ8gowB9Yq0uGNulQMlGeSMxkE%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288894390&amp;sdata=3ua69ygadJRYIJltAT7qPoJzNd%2B16PWCCeNLQF13WIg%3D&amp;reserved=0=
> >>>      >      >
> >>>      >      > Expansion ... X10 protocol ... Truckenbrodt 2019 Nat
> Protoc,
> >>>      >      >
> >>>
> >>
> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.nature.com-252Farticles-252Fs41596-2D018-2D0117-2D3-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3DCF-252Fp9frimbrxZiMDwLWZjzuyoyQbCuKp4EcvaL3Wmmw-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DYeXbqWFX0mMxO3nVRt59pt6RJYlwIPBMZRePQbiF1yU%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288894390&amp;sdata=TEBfgj6Gg%2Bt02BMyFZCLb8%2Feb5nOyAJmnftouvPd1qY%3D&amp;reserved=0=
> >>>      >      >
> >>>      >      > DNA-PAINT acronym soup review ... Nieves 2018 Genes,
> >>>      >      >
> >>>
> >>
> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.mdpi.com-252F2073-2D4425-252F9-252F12-252F621-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3Diwx3Z1Pgr4a2SObO9F47jEtqlfsYQFk2R4gu0Qv1uJM-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DtJY5yunlGClYXKHrXuCDe5KU8D-qhc1ZS26_KuNdXpE%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288904398&amp;sdata=RUgIPB%2Fi0n6i28lOhKZAGSrlyirYALd0l75r5qhZCMk%3D&amp;reserved=0=
> >>>      >      >
> >>>      >      > Babcock 2018 (4 --> 8 cameras, single molecule
> localization
> >>> microscopy
> >>>      >      > with $1550 CMOS cameras) ...
> >>>      >      >
> >>>
> >>
> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.nature.com-252Farticles-252Fs41598-2D018-2D19981-2Dz-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3DwN-252FHP30wNUE0-252BJWeFJIJFsBk32ZhD4DfKi9gc3ZTz2c-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DTIL4NaeETsqkjycmtWw_CnoEWvrpY1Tkr_SAmz-QkRM%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288904398&amp;sdata=D26AicKRsRoIaCGZdaxaF5OKXfGuo8yS0zvSVgmoBwY%3D&amp;reserved=0=
> >>>      >      >
> >>>      >      > Moffitt et al 2018 ...
> >>>      >      >
> >>>
> >>
> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fscience.sciencemag.org-252Fcontent-252F362-252F6416-252Feaau5324.long-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DFR98V6ldS9Q38wI59kly9U8pCzp92Vzc1J6T8ydCU9w-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3D55vzrzqpm3n5Z7logfovDr0N73h1gF7gldtYUSkZ6c0%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288904398&amp;sdata=CQMKMRjzbJVLgZjtpCzzZSwaNvCbt72baFvqppyebSI%3D&amp;reserved=0=
> >>> and
> >>>      >      > commentary
> >>>
> >>
> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fscience.sciencemag.org-252Fcontent-252F362-252F6416-252F749-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3Do0B379uuh-252FrB6gS9n6lG-252BjAZeztiYHZVmICwX4ghKh8-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DXeBps6CWTMM197evmy1n1uC5qOcvU8mAN95bEPmieiQ%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288904398&amp;sdata=um%2F%2FPNffQLOyrTsQcJymSuR2XwRcVnNMwMgQ6qYy19A%3D&amp;reserved=0=
> >>>      >      >
> >>>      >      > ***
> >>>      >      >
> >>>      >      > Some resolution numbers:
> >>>      >      >
> >>>      >      > 1.4 NA objective lens ... 500 nm wavelength .. dxy =
> 0.61 *
> >>> wqavelength
> >>>      >      > / NA (I routinely drop the 0.61 from 0.61)
> >>>      >      >
> >>>      >      > 0.6 * 500 / 1.4 = 214 nm
> >>>      >      >
> >>>      >      > widefiel deconvolution (re: Goodwin 2014) ~10% better ...
> >> 193
> >>> nm (if
> >>>      >      > pixel size matched or interpolate optimally).
> >>>      >      >
> >>>      >      > point scanning confocal -- Zeiss has a nice PDF, "Zeiss
> 2008
> >>> Principles
> >>>      >      > - Confocal Laser Scanning Microscopy" (see fig 10) on
> >> confocal
> >>>      >      > resolution wrt 1 and smaller pinhole size, source of the
> >>> values below,
> >>>      >      >
> >>>      >      > 1 Airy unit: 0.51 * 500 / 1.4 = 182 nm.
> >>>      >      >
> >>>      >      > 0.5 Airy unit ... 0.44 * 500 / 1.4 = 157 nm ... ~0.25
> >> photons
> >>> throughput
> >>>      >      > (which doesn't matter if target is photostable).
> >>>      >      >
> >>>      >      > 0.2 Airy unit ... you can ask your Zeiss rep about
> AiryScan
> >>> (and
> >>>      >      > FastAiryScan).
> >>>      >      >
> >>>      >      > 0.1 Airy unit ... 0.37 * 500 / 1.4 = 132 nm ... ~0.10
> >> photons
> >>> throughput.
> >>>      >      >
> >>>      >      > Most modern point scanning confocal microscopes have a
> 405
> >> nm
> >>> laser, so
> >>>      >      > if using BV421 (and ignoring potential photobleaching
> for a
> >>> moment),
> >>>      >      >
> >>>      >      > 1 Airy unit: 0.51 * 421 / 1.4 = 153 nm.
> >>>      >      >
> >>>      >      > or in reflection mode, i.e. nanodiamond or nanogold,
> >>>      >      >
> >>>      >      > 1 Airy unit: 0.51 * 405 / 1.4 = 147 nm ... and reflection
> >>> implies no
> >>>      >      > photobleaching, so infinite number of photons (though
> also
> >> no
> >>> blinking,
> >>>      >      > so not usually eligible for precision localization) ...
> >>>      >      >
> >>>      >      > 0.1 Airy unit: 0.37 * 405 / 1.4 = 107 nm
> >>>      >      >
> >>>      >      > and not going completely exotic with NA (i.e. 1.65), if
> >>> perfectly
> >>>      >      > refractive index match with a fairly conventional 1.49 NA
> >>> lens, and
> >>>      >      > inreflectance:
> >>>      >      >
> >>>      >      > 0.1 Airy unit: 0.37 * 405 / 1.49 = 100.57 nm
> >>>      >      >
> >>>      >      > I think I'd rather invest a DNA-PAINT friendly rig than
> deal
> >>> with 157 to
> >>>      >      > 101 nm.
> >>>      >      >
> >>>      >      > DNA-PAINT makes resolution irrelevant, if you use it (and
> >>> don't run out
> >>>      >      > of disk space or time or money), since precision
> >> localization
> >>> is
> >>>      >      > resolution divided by square root of number of photons,
> ex.
> >>> 250 nm XY
> >>>      >      > resolution / sqrt(1,000,000) = 0.25 nm, and could
> increase
> >>> number of
> >>>      >      > photons per target further, but why bother?
> >>>      >      >
> >>>      >      > ***
> >>>      >      >
> >>>      >      > point scanning confocal microscopes are also great
> platforms
> >>> for
> >>>      >      > F-Techniques, such as FastFLIM (aka rapidFLIM, etc, much
> >>> faster than
> >>>      >      > classic TCSPC slow FLIM), FCS, FCCS, see Liu 2008,
> >>>      >      >
> >>>
> >>
> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.ncbi.nlm.nih.gov-252Fpubmed-252F18387308-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DZQTosrSoPFpCa8Y3IdEa9Xz-252B4RHC8JO4gBppHmzkazo-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DmeRUIZdF3FLQZGyok4cxyc4AP4yq2lXX41Vt8q9WMmw%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288904398&amp;sdata=XTEH306i5ez7vKq2cHKCVQlSphNiFIdnqIXmwnOmyNw%3D&amp;reserved=0=
> >>>      >      >
> >>>      >      > ***
> >>>      >      >
> >>>      >      > p.p.s. Disclosures I am ...
> >>>      >      >
> >>>      >      > 1. currently hosting a Leica THUNDER Imager tour event
> (ends
> >>> Monday
> >>>      >      > 3/18/2019 afternoon) ...  see pdf download page,
> >>>      >      >
> >>>      >      > THUNDER Technology Note
> >>>      >      > THUNDER Imagers: How Do They Really Work?
> >>>      >      >
> >>>      >      >
> >>>
> >>
> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.leica-2Dmicrosystems.com-252Fscience-2Dlab-252Fthunder-2Dtechnology-2Dnote-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DauRUORuEKAir87-252Bbw7RHyTR9IxxrLYl1g3bFBiRTXBM-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DLmFGYl_Icu6b9AAIYnHkoiH26bRZ688ODEc9I6k8yco%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288904398&amp;sdata=5ZGLmCZt85tKxH1CJYM%2Bvul5S4qXw9%2FOnyWG80m2QxM%3D&amp;reserved=0=
> >>>      >      >
> >>>      >      > 2. hosting Nikon confocal demos in May 2019.
> >>>      >      >
> >>>      >      > 3. aiming to co-host with ISS a FastFLIM (one day)
> >>> mini-symposium this
> >>>      >      > summer.
> >>>      >      >
> >>>      >      > 4. an unpaid advisor for Gary Brooker for FINCH/CINCH,
> re:
> >>>      >      >
> >>>
> >>
> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.ncbi.nlm.nih.gov-252Fpubmed-252F28261321-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DuVw0kiW1baZV0RJv-252Bk1ObKznhw51pemnCMehjHkUa2g-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DKC1HMkiDzpE2DLsuj1I2nHvopwFVMMc0GVecDV-fWkM%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288904398&amp;sdata=QC0LyEp9hbSKOgpV%2Fi%2B%2FABGDX4az02fRVdMZ4Ovufps%3D&amp;reserved=0=
> >>>      >      >
> >>>      >      >
> >>>      >      > On 3/17/2019 10:43 AM, Mika Ruonala wrote:
> >>>      >      > > *****
> >>>      >      > > To join, leave or search the confocal microscopy
> listserv,
> >>> go to:
> >>>      >      > >
> >>>
> >>
> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Flists.umn.edu-252Fcgi-2Dbin-252Fwa-253FA0-253Dconfocalmicroscopy-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DdHOCjeBda4sSZPf-252FB2-252B5Sv3Q8Qzcs708p4we8vHf-252FIg-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DqZ-tdRwhHG172zv4uHPeAJMNeIIHNxozhQudV-hF2kE%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288904398&amp;sdata=BnCAxorVJgPSFw70Kr%2B401vq4hMlZhvzdkivum9XGNE%3D&amp;reserved=0=
> >>>      >      > > Post images on
> >>>
> >>
> https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fwww.imgur.com-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DcnhzoWOP4RwDR622fn447aQVxW8ZBI3D0utze67RiGg-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DYzF36y8eV4SG3E0LJEDu-9AMm3Nh05dwp_XKhB6BUyU%26e&amp;data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288914407&amp;sdata=ec0HIKuOMVqlLU7knyQ1JdS1B6c1BMCIb%2FYsHIFdYbM%3D&amp;reserved=0=
> >>> and include the link in your
> >>>      >      > posting.
> >>>      >      > > *****
> >>>      >      > >
> >>>      >      > > Hi.
> >>>      >      > > There are software solutions that are able to create
> image
> >>> data from
> >>>      >      > wide-field and even microscope systems with seemingly
> >> similar
> >>> quality to
> >>>      >      > that obtained from confocal systems.
> >>>      >      > >
> >>>      >      > > The comparison of acquisition vs. software is
> essentially
> >> a
> >>> comparison
> >>>      >      > of image acquisition vs. image processing. While a
> software
> >>> solution is way
> >>>      >      > cheaper than a hardware solution if it is able to produce
> >>> image data with
> >>>      >      > equal quality why would anyone choose to invest to a
> >> confocal
> >>> anymore?
> >>>      >      > >
> >>>      >      > > I’m looking forward to a vidid discussion!
> >>>      >      > >
> >>>      >      > >> m
> >>>      >      >
> >>>      >
> >>>      >
> >>>      --
> >>>      Alison J. North, Ph.D.,
> >>>      Research Associate Professor and
> >>>      Senior Director of the Bio-Imaging Resource Center,
> >>>      The Rockefeller University,
> >>>      1230 York Avenue,
> >>>      New York,
> >>>      NY 10065.
> >>>      Tel: office    ++ 212 327 7488
> >>>      Tel: lab        ++ 212 327 7486
> >>>      Fax:            ++ 212 327 7489
> >>>
> >>>
> >>>
> >> --
> >> Benjamin E. Smith, Ph. D.
> >> Imaging Specialist, Vision Science
> >> University of California, Berkeley
> >> 195 Life Sciences Addition
> >> Berkeley, CA  94720-3200
> >> Tel  [phone]
> >> Fax [phone]
> >> e-mail: [hidden email]
> >> http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>
> >>
> >>
>

--
 <https://www.chroma.com/>CHROMA TECHNOLOGY CORP®
*an employee owned
company*
10 Imtec Lane, Bellows Falls, Vermont 05101 USA
800-824-7662 |
FAX: [phone]
www.chroma.com <https://www.chroma.com/> | 
[hidden email] <mailto:[hidden email]>                             

[Arvydas Matiukas]

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*****

Thanks Warren for pointing to this practical solution. How close to 100% circular polarization you achieve using the Berek compensator?
I see Newport makes one or is there a specific model you would recommend.

Thanks,
Arvydas





>>> "Warren R. Zipfel" <[hidden email]> 03/18/19 6:57 PM >>>
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Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFAg&c=ogn2iPkgF7TkVSicOVBfKg&r=DtXPX1Vw9uh8rqlCEC9BTBr2oYBP4dEG1ecsgK6l-4k&m=AAK6Bfabc_s37Yv_aH3Gl0o9UfdZI7gpIk1bC-r4WKc&s=Zx-N1MHif5u2smp-j3KnjFYpnN2z1elBWbRt66dYviI&e= and include the link in your posting.
*****

I use a Berek compensator in the Ti:S beam and either a polarizer after the objective to verify the Berek settings for  X or Y linear polarization (for anisotropy imaging)  or a polarizer and 1/4 waveplate as an analyzer if I need R or L circular polarization.  The more complicated optical path on most setups makes the adjustability of a Berek necessary.

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Arvydas Matiukas
Sent: Monday, March 18, 2019 4:49 PM
To: [hidden email]
Subject: Circularly polarizing laser beam

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Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFAg&c=ogn2iPkgF7TkVSicOVBfKg&r=DtXPX1Vw9uh8rqlCEC9BTBr2oYBP4dEG1ecsgK6l-4k&m=AAK6Bfabc_s37Yv_aH3Gl0o9UfdZI7gpIk1bC-r4WKc&s=Zx-N1MHif5u2smp-j3KnjFYpnN2z1elBWbRt66dYviI&e= and include the link in your posting.
*****

Hello list,

I am struggling to get a precisely circularly polarized beam. No matter how precisely I set fast axis of the quarter wave plate at 45 degrees  to the polarization of laser beam, the ellipticity is always <0.9 (Imin/Imax <0.9).

The laser beam is 588nm (5nm bandwidth) off OPO and pulse broadening fiber (from fs pulses of driving Mai Tai to ps pulses), diameter 4mm. Polarization is 1:70 that is further  improved to 1:600 by Glan polarizer. The zero order 588nm

 quarter wave plate  is aligned normal to the beam and rotated at 45 degrees (other angles only increase ellipticity).


Any suggestions what I may be missing are welcome.


Thanks,
Arvydas
************************

[Steffen Dietzel]

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Am 18.03.2019 um 19:51 schrieb Feinstein, Timothy N:
> For that to work the algorighms need to automatically (accurately) estimate SNR.  That doesn't seem like an insurmountable challenge, but I haven't seen it yet.

Well, in a perfect world estimating should not be necessary: If the
detector runs in photon counting mode, the SNR could be calculated.

Steffen


--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging

Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany

http://www.bioimaging.bmc.med.uni-muenchen.de

[Craig Brideau]

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I agree with Warren. I've managed to fudge nearly circular with a 1/2 and
1/4 wave combination, by tilting the plates slightly to compensate for
chromatic dependence. The ability of the Berek compensator to tilt lets you
do this in a more controlled manner. You will also find the circular state
is very sensitive to environment. If you have a temperature change or any
new mechanical stresses on the system you will start going back to
elliptical frequently. I used a motorized rotation system with my
half/quarter waveplate set and had to calibrate frequently. Here's my old
paper:
https://www.spiedigitallibrary.org/conference-proceedings-of-spie/8226/82263A/Automated-control-of-optical-polarization-for-nonlinear-microscopy/10.1117/12.908995.full?SSO=1

Craig

On Mon, Mar 18, 2019 at 4:57 PM Warren R. Zipfel <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> I use a Berek compensator in the Ti:S beam and either a polarizer after
> the objective to verify the Berek settings for  X or Y linear polarization
> (for anisotropy imaging)  or a polarizer and 1/4 waveplate as an analyzer
> if I need R or L circular polarization.  The more complicated optical path
> on most setups makes the adjustability of a Berek necessary.
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On
> Behalf Of Arvydas Matiukas
> Sent: Monday, March 18, 2019 4:49 PM
> To: [hidden email]
> Subject: Circularly polarizing laser beam
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hello list,
>
> I am struggling to get a precisely circularly polarized beam. No matter
> how precisely I set fast axis of the quarter wave plate at 45 degrees  to
> the polarization of laser beam, the ellipticity is always <0.9 (Imin/Imax
> <0.9).
>
> The laser beam is 588nm (5nm bandwidth) off OPO and pulse broadening fiber
> (from fs pulses of driving Mai Tai to ps pulses), diameter 4mm.
> Polarization is 1:70 that is further  improved to 1:600 by Glan polarizer.
> The zero order 588nm
>
>  quarter wave plate  is aligned normal to the beam and rotated at 45
> degrees (other angles only increase ellipticity).
>
>
> Any suggestions what I may be missing are welcome.
>
>
> Thanks,
> Arvydas
> ************************
>

[0000001ed7f52e4a-dmarc-request]

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Deconvolution needs Nyquist sampling and this often means lots of z-slices causing bleaching and potentially bleaching live samples. The latest implementation of lattice SIM from Zeiss in the Elyra7 (no commercial interest) has a 'leap mode' which basically skips some z-slices. When I remember correctly, they claim that the missing information is recovered from the out-of-focus part of the signal. I think this would work only for samples where the signal is sufficient sparse so that the in-focus signal is not swamped the out-of-focus signal. When it works, it speeds up image acquisition and reduces bleaching. 
Otherwise the ApoTomes comes to mind, using grid pattern illumination without super-resolution, Elyra7 also has an ApoTome mode.
According to my experience, samples which are very inhomogeneous such as cells in hydrogels, cells on silicon or other weird substrates or plants or dense tissue slices are better imaged with a confocal. Adaptive optics with a guide star approach to set the parameters as in Eric Betzig's lattice light sheet might help to image some of these samples.
best wishes
Andreas


-----Original Message-----
From: Fco. Javier Díez Guerra <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Mon, 18 Mar 2019 20:38
Subject: Re: General question: Software vs. hardware

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*****

Hi,

Very interesting discussion!

Most opinions gather around the idea of rely on hardware and make use of
software as much as you can.

But, not strictly speaking on deconvolution, I would be interested to
listen also to opinions on more blurred mixtures of hardware and
software, such as structured illumination (SIM) in their latest
implementations, for example Lattice SIM.

Best,

Javier



El 18/03/2019 a las 16:11, [hidden email] escribió:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi everyone,
>
> I agree with most that has been said. I am firmly of the belief that you should try to acquire the best possible images and then you can always improve them further by using deconvolution or other methods. The more imaging artifacts and erroneous light you can reject during the imaging process, the less you will have to deal with later. Also, many journals have started asking about providing the raw data for figures as well - for which having superior data right off the bat is a huge benefit.
> 
> One large risk of using software is that you can incur all sorts of artifacts, especially if you do not know what you are doing or if you push the limits of the deconvolution too far. I have had to gently let down some scientists who were excited about seeing this or that in their deconvolved data, with the raw data simply not supporting it. Furthermore, as Timothy pointed out, some deconvolution software automatically applies certain procedures or maybe does not make very clear exactly what has been done - reiterating the statement: you should know what you are doing when using software to improve your images. (Or at least consult with someone who does)
>
> So in conclusion: get the best images you can, and then improve them even further. The results speak for themselves.
>
> Best,
> Nicolai
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On Behalf Of Feinstein, Timothy N
> Sent: Montag, 18. März 2019 09:56
> To: [hidden email]
> Subject: Re: General question: Software vs. hardware
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988468333&sdata=%2B0sr4LxFc4S9c9F6hK8DeKlbHShPwXlrKWzWgWTUp7Y%3D&reserved=0
> Post images on https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988478337&sdata=7oaSK53PubjjY5%2Fsqe8ifzN8JxQsnG6rCZ07bJj2X2A%3D&reserved=0 and include the link in your posting.
> *****
>
> Avi, I really agree with your point.  I feel that people to deconvolve any time spatial information is critical, whether they're using widefield, CLSM, spinning disc, or light sheet.  It's true that deconvolving adds time and data volume and especially cost, but in trade you get an image that is substantially sharper, with reduced noise and background, and more quantitatively accurate*.
>
> Regarding whether to just go with a point scanning confocal, I don't see it as a simple question of better or worse**.  A nuclear-cytoplasmic translocation assay with monolayer cells works just as well on a widefield, and (in my experience!) many types of biosensor assay work better with a properly set up widefield.  The 16-bit depth of widefield images is nice for quantitation, and modern sCMOS cameras have by far the best acquisition speeds.  I don't know whether widefields still have a more linear relationship between sample brightness and detected signal, but the last time I checked that was still true.
>
> (*) Deconvolution is quantitatively useful as long as people make sure to tell the software to preserve the original intensity values.  One of my complaints about Hyvolution was that you could not do that, so I just used the Huygens package that came with it.  I don't know whether Lightning gives you that option...if not then caveat emptor.
>
> (**) My advice mostly applies to turnkey stuff that any lab can implement, not exotic techniques available to folks with specialists or engineers on hand.
>
> Best,
>
>
> T
> Timothy Feinstein, Ph.D. esearch Scientist Department of Developmental Biology University of Pittsburgh
>
>
> On 3/18/19, 5:07 AM, "Confocal Microscopy List on behalf of Avi Jacob" <[hidden email] on behalf of [hidden email]> wrote:
>
>   
>      I'll point out, that you can, of course, deconvolve confocal images too.
>      So, while you can indeed get near confocal quality with well-acquired wf
>      data after deconvolution, you can also get near SR quality when
>      deconvolving a well-acquired stack from a confocal. And then you can also
>      deconvolve a SR stack and get... well you get the idea! It's like an arms
>      race.
>      I have the Hyvolution and had access for a couple of weeks to the Lighting,
>      and now confocal images look blurry to me.
>      Avi
>     
>      --
>      Avi Jacob, Ph.D.
>      Kanbar Light Microscopy Unit
>      The Mina & Everard Goodman Faculty of Life Sciences
>      Bar-Ilan University, Ramat-Gan 529002, Israel
>     
>     
>     
>      On Sun, Mar 17, 2019 at 6:04 PM George McNamara <[hidden email]>
>      wrote:
>     
>      > *****
>      > To join, leave or search the confocal microscopy listserv, go to:
>      > https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988478337&sdata=8UtAwuTn3RvnsxIPIRQhzw%2BYhqpEwpdVbRUgT4i2v0Q%3D&reserved=0
>      > Post images on https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988478337&sdata=7oaSK53PubjjY5%2Fsqe8ifzN8JxQsnG6rCZ07bJj2X2A%3D&reserved=0 and include the link in your posting.
>      > *****
>      >
>      > Hi Mika,
>      >
>      > White et al 1987 ( https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fjcb.rupress.org%2Fcontent%2F105%2F1%2F41.long&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988478337&sdata=nzaoZX0omJibqA4g7XfhlKbeiTPdqXNnUtAstnMp%2F7s%3D&reserved=0 ) made a
>      > compelling case for point scanning confocal microscopes: collect just
>      > the in focus light with instant gratification. The case has not changed,
>      > the hardware (especially data deluge side) has gotten a lot better. I
>      > note that both widefield detectors and PMT/APD/hybrid detectors/others
>      > have gotten a lot better in the 32 years from 1987! As have the optics
>      > and automation.
>      >
>      > Paul Goodwin 2014 ( https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpubmed%2F24974028&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988478337&sdata=IJYfSZDn9%2B8IqtOVPtr4oHIsVQ0DwNU%2FcYDBWHLCtls%3D&reserved=0 ) made
>      > a nice case for quantitative deconvolution microscopy (and on very high
>      > quality specimens, ~10% improvement in resolution compared to simple
>      > widefield), but historically slow.
>      >
>      > Now, with 'instant gratification' spatial deconvolution, thanks to the
>      > GPU revolution (NVidia RTX Titan~16 Teraflops [S.P.], 24 Gb ram, $2500
>      > ... not including the deconvolution software module price), widefield,
>      > spinning disk (and slightly exotic variants like iSIM, DMD based, etc,
>      > see also new THUNDER Imagers [see p.p.s.]), multiphoton (I'm excited
>      > about the price point of recent fiber lasers, which could become much
>      > better price if achieve 'economy of scale'), and of course, point
>      > scanning confocal microscopes.
>      >
>      > Spatial deconvolution (especially if someone $uccessfully implements
>      > joint spatial deconvolution and spectral unmixing, multiple cameras -
>      > for 4 cameras see Babcock 2018, mentions aiming for 8 cameras) helps
>      > with Expansion Microscopy and/or DNA-PAINT, to go super-resolution ...
>      > really single molecule counting (and DNA-PAINT eliminates the classic
>      > issue of PALM/STORM/FPALM of not counting every molecule). Sure,
>      > DNA-PAINT (like STORM etc) have the issue of a whole lotta images
>      > acquired. Data deluge: who cares? Jerome & Price's 10th commandment of
>      > confocal imaging is: "10. Storage Media Is Essentially Free and Infinite".
>      >
>      > More significantly, DNA-PAINT and related methods (single molecule RNA
>      > FISH, scRNAseq -> MERFISH = Moffitt 2018 as example, etc) also enable
>      > multiplex -- with single molecule counting -- to whatever plex is needed
>      > to answer the 'biological question(s)' being posed.
>      >
>      > All that said, the installed base of research grade point scanning
>      > confocal microscopes is large (5000+) and efficient at acquiring high
>      > quality images, to the point that user's sample preparation (and
>      > avoidance of purchasing stuff from 'Santa Crap' and similar companies)
>      > is much more limiting than the microscopes.
>      >
>      > George
>      >
>      > p.s. a couple of references not included in above:
>      >
>      > W. Gray (Jay) Jerome, Robert L. Price 2018... Basic Confocal Microscopy
>      > second edition https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Flink.springer.com%2Fbook%2F10.1007%252F978-3-319-97454-5&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988478337&sdata=sNoUx5yoXFfjFCPvziQDweJ2PJXfRhOtB0Tg0rWbj04%3D&reserved=0
>      >
>      > Expansion ... X10 protocol ... Truckenbrodt 2019 Nat Protoc,
>      > https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.nature.com%2Farticles%2Fs41596-018-0117-3&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988478337&sdata=4WrXBtCidTZfrARUzRcm1UvolmzLg1pG0SZkErLU93E%3D&reserved=0
>      >
>      > DNA-PAINT acronym soup review ... Nieves 2018 Genes,
>      > https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.mdpi.com%2F2073-4425%2F9%2F12%2F621&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988478337&sdata=VW4WoAKHnc2hhYQEgPUqt%2BeFdN%2Bw7fADCLXdgDoU98k%3D&reserved=0
>      >
>      > Babcock 2018 (4 --> 8 cameras, single molecule localization microscopy
>      > with $1550 CMOS cameras) ...
>      > https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.nature.com%2Farticles%2Fs41598-018-19981-z&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988488345&sdata=zxdHdhYM6%2BaCes%2Bq2NoF70EmKSiwOvmHrfW%2BRuSjClA%3D&reserved=0
>      >
>      > Moffitt et al 2018 ...
>      > https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fscience.sciencemag.org%2Fcontent%2F362%2F6416%2Feaau5324.long&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988488345&sdata=z42w%2Fn2NVAWdTD5q7mUrwhOliTRDW4%2BCZQgAEPvnhTM%3D&reserved=0 and
>      > commentary https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fscience.sciencemag.org%2Fcontent%2F362%2F6416%2F749&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988488345&sdata=FgPwqbrQ66L398R0tyiOD%2BTrJovMlb22NCmpnV%2BytgQ%3D&reserved=0
>      >
>      > ***
>      >
>      > Some resolution numbers:
>      >
>      > 1.4 NA objective lens ... 500 nm wavelength .. dxy = 0.61 * wqavelength
>      > / NA (I routinely drop the 0.61 from 0.61)
>      >
>      > 0.6 * 500 / 1.4 = 214 nm
>      >
>      > widefiel deconvolution (re: Goodwin 2014) ~10% better ... 193 nm (if
>      > pixel size matched or interpolate optimally).
>      >
>      > point scanning confocal -- Zeiss has a nice PDF, "Zeiss 2008 Principles
>      > - Confocal Laser Scanning Microscopy" (see fig 10) on confocal
>      > resolution wrt 1 and smaller pinhole size, source of the values below,
>      >
>      > 1 Airy unit: 0.51 * 500 / 1.4 = 182 nm.
>      >
>      > 0.5 Airy unit ... 0.44 * 500 / 1.4 = 157 nm ... ~0.25 photons throughput
>      > (which doesn't matter if target is photostable).
>      >
>      > 0.2 Airy unit ... you can ask your Zeiss rep about AiryScan (and
>      > FastAiryScan).
>      >
>      > 0.1 Airy unit ... 0.37 * 500 / 1.4 = 132 nm ... ~0.10 photons throughput.
>      >
>      > Most modern point scanning confocal microscopes have a 405 nm laser, so
>      > if using BV421 (and ignoring potential photobleaching for a moment),
>      >
>      > 1 Airy unit: 0.51 * 421 / 1.4 = 153 nm.
>      >
>      > or in reflection mode, i.e. nanodiamond or nanogold,
>      >
>      > 1 Airy unit: 0.51 * 405 / 1.4 = 147 nm ... and reflection implies no
>      > photobleaching, so infinite number of photons (though also no blinking,
>      > so not usually eligible for precision localization) ...
>      >
>      > 0.1 Airy unit: 0.37 * 405 / 1.4 = 107 nm
>      >
>      > and not going completely exotic with NA (i.e. 1.65), if perfectly
>      > refractive index match with a fairly conventional 1.49 NA lens, and
>      > inreflectance:
>      >
>      > 0.1 Airy unit: 0.37 * 405 / 1.49 = 100.57 nm
>      >
>      > I think I'd rather invest a DNA-PAINT friendly rig than deal with 157 to
>      > 101 nm.
>      >
>      > DNA-PAINT makes resolution irrelevant, if you use it (and don't run out
>      > of disk space or time or money), since precision localization is
>      > resolution divided by square root of number of photons, ex. 250 nm XY
>      > resolution / sqrt(1,000,000) = 0.25 nm, and could increase number of
>      > photons per target further, but why bother?
>      >
>      > ***
>      >
>      > point scanning confocal microscopes are also great platforms for
>      > F-Techniques, such as FastFLIM (aka rapidFLIM, etc, much faster than
>      > classic TCSPC slow FLIM), FCS, FCCS, see Liu 2008,
>      > https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpubmed%2F18387308&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988488345&sdata=8L6sZRJKBP%2Fp%2FZm5YjGAID6uR8sprK8yKE5jSTIOZes%3D&reserved=0
>      >
>      > ***
>      >
>      > p.p.s. Disclosures I am ...
>      >
>      > 1. currently hosting a Leica THUNDER Imager tour event (ends Monday
>      > 3/18/2019 afternoon) ...  see pdf download page,
>      >
>      > THUNDER Technology Note
>      > THUNDER Imagers: How Do They Really Work?
>      >
>      > https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.leica-microsystems.com%2Fscience-lab%2Fthunder-technology-note&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988488345&sdata=XZDsAx4PBnd7K5BF9dshnIrDk6glSie%2BQZA%2FmvDCBRA%3D&reserved=0
>      >
>      > 2. hosting Nikon confocal demos in May 2019.
>      >
>      > 3. aiming to co-host with ISS a FastFLIM (one day) mini-symposium this
>      > summer.
>      >
>      > 4. an unpaid advisor for Gary Brooker for FINCH/CINCH, re:
>      > https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpubmed%2F28261321&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988488345&sdata=2oiWeA2uoGgy3GrBSjVSiEr%2FOgV3pYlJ43MHOOD6FPg%3D&reserved=0
>      >
>      >
>      > On 3/17/2019 10:43 AM, Mika Ruonala wrote:
>      > > *****
>      > > To join, leave or search the confocal microscopy listserv, go to:
>      > > https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988488345&sdata=5aNe3XPrrvZ2lpSR2%2F1K1ereIMtgE3hpSF1r3HNaaFU%3D&reserved=0
>      > > Post images on https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988488345&sdata=PmBfP4DRz%2FvetYmcPIr%2BZDuWIUNmbZaetHNkQ8Wg%2BU4%3D&reserved=0 and include the link in your
>      > posting.
>      > > *****
>      > >
>      > > Hi.
>      > > There are software solutions that are able to create image data from
>      > wide-field and even microscope systems with seemingly similar quality to
>      > that obtained from confocal systems.
>      > >
>      > > The comparison of acquisition vs. software is essentially a comparison
>      > of image acquisition vs. image processing. While a software solution is way
>      > cheaper than a hardware solution if it is able to produce image data with
>      > equal quality why would anyone choose to invest to a confocal anymore?
>      > >
>      > > I’m looking forward to a vidid discussion!
>      > >
>      > >> m
>      >
>     
>

[phil laissue-3]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Dear all,
Alison mentions an important point - phototoxicity is a crucial
consideration. And as many have pointed out in this thread, it's hard to
have general recommendations, as everything is sample-specific. In
principle, I agree that it's best to address a problem early in the
pipeline (sample optimisation, then hardware (image acquisition modality),
then image processing). However, 'confocal = less artefacts' doesn't hold
when it comes to live imaging. The high power density of a spot-scanner
means that you might be studying light-induced pathophysiology - a
different kind of artefact that can be hard to assess. Widefield with its
low power density and fast, sensitive acquisition is a far better first
option in many cases (if compared to a spot-scanning confocal with standard
PMTs, not GaAsP, HyD or APD, or CLEM (Manders) etc.).
My personal favourite is light-sheet microscopy, which combines the best of
both worlds (optical sectioning of confocal with the fast, sensitive
acquisition of widefield), often with a very reasonable price tag.
Cheers, Philippe


_____________________________________
Philippe Laissue, PhD
Lecturer in Bioimaging
School of Biological Sciences, Room 4.38
University of Essex, Colchester CO4 3SQ, UK
(0044) 01206 872246 / [phone] 6056
[hidden email]
website <https://laissue.github.io/>


On Mon, 18 Mar 2019 at 15:07, Alison North <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi all,
>
> So this is a very timely discussion because I have been discussing with
> my staff whether there are data sets that should NOT be deconvolved, and
> if so, how does one decide that?  I too attended Jim Pawley's wonderful
> course (he was certainly a huge character as well as an incredibly
> fantastic microscopist, and he will certainly be remembered by all!), so
> I have generally worked under the assumption that one should deconvolve
> all confocal data.  But I am also very aware of the potential for
> artifacts if a data set isn't "good enough" for deconvolution.
> Obviously the ideal situation is to acquire an optimal data set -
> well-prepared sample, bright staining, Nyquist sampling etc. etc., and a
> high S:N ratio - and by sticking to these rules, our deconvolved
> DeltaVision images or confocal images of fixed samples have always
> looked great.  But nowadays we are faced with different scenarios,
> particularly when you are attempting to do very rapid imaging of live,
> weakly expressing cells, while attempting to minimize phototoxicity.  In
> that case you can end up with pretty lousy S:N ratios, because
> maintaining cell viability or imaging fast enough is more critical.
> For example, I have a lovely new iSIM in my lab, for which the initial
> resolution increase is achieved by the hardware, but the second step in
> resolution increase is via deconvolution.   The whole point of this
> instrument is for rapid, super-resolution imaging - so we can't simply
> increase exposure times to improve S:N, turning up the laser power will
> obviously kill the cells, and we can't increase the pixel size or we'll
> lose the resolution.  And I assume a lot of the new types of
> super-resolution instrument out there must leave you facing the same
> issue, since live cell imaging invariably forces you to compromise
> somewhere within the imaging triangle (or hexagon, or whatever we've got
> up to now!).
>
> Therefore my question is, are there papers out there which have compared
> deconvolution algorithms and looked at the potential for artifacts on
> really low S:N images, which we could use to advise our researchers on
> what is the minimum you can get away with before you really shouldn't be
> deconvolving the data set at all? Also, are there papers showing the
> effect of undersampling in the z-axis on the resulting deconvolved
> images (as is often the case on our spinning disk system)?  I haven't
> managed to find any yet (though I confess I've been too busy with other
> stuff to spend too many hours searching!), so if anybody could point me
> to some good references I'd be most grateful.  I have spoken with
> several renowned microscopists about whether deconvolution is always a
> good idea under such circumstances, and the gut reaction appears to be
> no, but I could do with some hard and fast validation for teaching
> purposes.
>
> Many thanks in advance!
>
> Alison
>
>
>
> On 3/18/2019 9:56 AM, Feinstein, Timothy N wrote:
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=vfjahhKueherhfphxmr-Smw_FhB4QUlg-FsBATM1kl0&e=
> > Post images on
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=edOjY0NZEIhVq-zWzgNHHDaTrutHrPj1Rl6rcwE_B4M&e=
> and include the link in your posting.
> > *****
> >
> > Avi, I really agree with your point.  I feel that people to deconvolve
> any time spatial information is critical, whether they're using widefield,
> CLSM, spinning disc, or light sheet.  It's true that deconvolving adds time
> and data volume and especially cost, but in trade you get an image that is
> substantially sharper, with reduced noise and background, and more
> quantitatively accurate*.
> >
> > Regarding whether to just go with a point scanning confocal, I don't see
> it as a simple question of better or worse**.  A nuclear-cytoplasmic
> translocation assay with monolayer cells works just as well on a widefield,
> and (in my experience!) many types of biosensor assay work better with a
> properly set up widefield.  The 16-bit depth of widefield images is nice
> for quantitation, and modern sCMOS cameras have by far the best acquisition
> speeds.  I don't know whether widefields still have a more linear
> relationship between sample brightness and detected signal, but the last
> time I checked that was still true.
> >
> > (*) Deconvolution is quantitatively useful as long as people make sure
> to tell the software to preserve the original intensity values.  One of my
> complaints about Hyvolution was that you could not do that, so I just used
> the Huygens package that came with it.  I don't know whether Lightning
> gives you that option...if not then caveat emptor.
> >
> > (**) My advice mostly applies to turnkey stuff that any lab can
> implement, not exotic techniques available to folks with specialists or
> engineers on hand.
> >
> > Best,
> >
> >
> > T
> > Timothy Feinstein, Ph.D. esearch Scientist
> > Department of Developmental Biology
> > University of Pittsburgh
> >
> >
> > On 3/18/19, 5:07 AM, "Confocal Microscopy List on behalf of Avi Jacob" <
> [hidden email] on behalf of [hidden email]> wrote:
> >
> >
> >      I'll point out, that you can, of course, deconvolve confocal images
> too.
> >      So, while you can indeed get near confocal quality with
> well-acquired wf
> >      data after deconvolution, you can also get near SR quality when
> >      deconvolving a well-acquired stack from a confocal. And then you
> can also
> >      deconvolve a SR stack and get... well you get the idea! It's like
> an arms
> >      race.
> >      I have the Hyvolution and had access for a couple of weeks to the
> Lighting,
> >      and now confocal images look blurry to me.
> >      Avi
> >
> >      --
> >      Avi Jacob, Ph.D.
> >      Kanbar Light Microscopy Unit
> >      The Mina & Everard Goodman Faculty of Life Sciences
> >      Bar-Ilan University, Ramat-Gan 529002, Israel
> >
> >
> >
> >      On Sun, Mar 17, 2019 at 6:04 PM George McNamara <
> [hidden email]>
> >      wrote:
> >
> >      > *****
> >      > To join, leave or search the confocal microscopy listserv, go to:
> >      >
> https://urldefense.proofpoint.com/v2/url?u=https-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Flists.umn.edu-252Fcgi-2Dbin-252Fwa-253FA0-253Dconfocalmicroscopy-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3DefIAtI3pbBSoyhJucB0DkDu4RXkFkgBZfGrO4PiXrfc-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=9Uk5ZZzc8LP7V7Oj2aFSRbVluY0mpE-7XPLYdsyviMk&e=
> >      > Post images on
> https://urldefense.proofpoint.com/v2/url?u=https-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fwww.imgur.com-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3DMNM8i3iDaQuMA9LF766-252FyvCyp94jmie4IaC5qIEWclA-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=Gy99yAWcbr6KHUA0kYKb4K-wOHL4iNGJsDecMfZ8X-M&e=
> and include the link in your posting.
> >      > *****
> >      >
> >      > Hi Mika,
> >      >
> >      > White et al 1987 (
> https://urldefense.proofpoint.com/v2/url?u=https-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fjcb.rupress.org-252Fcontent-252F105-252F1-252F41.long-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3Da6oZCfeD7Gt4nYMS89PcklnveCdDLLkksafp3tCyld0-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=67LyqPF2rBiBDSWJVZeG7TpJz1o4MRDf8ZyLysbeKx4&e=
> ) made a
> >      > compelling case for point scanning confocal microscopes: collect
> just
> >      > the in focus light with instant gratification. The case has not
> changed,
> >      > the hardware (especially data deluge side) has gotten a lot
> better. I
> >      > note that both widefield detectors and PMT/APD/hybrid
> detectors/others
> >      > have gotten a lot better in the 32 years from 1987! As have the
> optics
> >      > and automation.
> >      >
> >      > Paul Goodwin 2014 (
> https://urldefense.proofpoint.com/v2/url?u=https-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.ncbi.nlm.nih.gov-252Fpubmed-252F24974028-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3DGDve8BuxhLDAdBPKywRQoMTYV-252BqSk9aMcz2WrZCHPU8-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=PZ10eaR5B-jwZmf5y920Z41iJYUJcji_VsufABIeX84&e=
> ) made
> >      > a nice case for quantitative deconvolution microscopy (and on
> very high
> >      > quality specimens, ~10% improvement in resolution compared to
> simple
> >      > widefield), but historically slow.
> >      >
> >      > Now, with 'instant gratification' spatial deconvolution, thanks
> to the
> >      > GPU revolution (NVidia RTX Titan~16 Teraflops [S.P.], 24 Gb ram,
> $2500
> >      > ... not including the deconvolution software module price),
> widefield,
> >      > spinning disk (and slightly exotic variants like iSIM, DMD based,
> etc,
> >      > see also new THUNDER Imagers [see p.p.s.]), multiphoton (I'm
> excited
> >      > about the price point of recent fiber lasers, which could become
> much
> >      > better price if achieve 'economy of scale'), and of course, point
> >      > scanning confocal microscopes.
> >      >
> >      > Spatial deconvolution (especially if someone $uccessfully
> implements
> >      > joint spatial deconvolution and spectral unmixing, multiple
> cameras -
> >      > for 4 cameras see Babcock 2018, mentions aiming for 8 cameras)
> helps
> >      > with Expansion Microscopy and/or DNA-PAINT, to go
> super-resolution ...
> >      > really single molecule counting (and DNA-PAINT eliminates the
> classic
> >      > issue of PALM/STORM/FPALM of not counting every molecule). Sure,
> >      > DNA-PAINT (like STORM etc) have the issue of a whole lotta images
> >      > acquired. Data deluge: who cares? Jerome & Price's 10th
> commandment of
> >      > confocal imaging is: "10. Storage Media Is Essentially Free and
> Infinite".
> >      >
> >      > More significantly, DNA-PAINT and related methods (single
> molecule RNA
> >      > FISH, scRNAseq -> MERFISH = Moffitt 2018 as example, etc) also
> enable
> >      > multiplex -- with single molecule counting -- to whatever plex is
> needed
> >      > to answer the 'biological question(s)' being posed.
> >      >
> >      > All that said, the installed base of research grade point scanning
> >      > confocal microscopes is large (5000+) and efficient at acquiring
> high
> >      > quality images, to the point that user's sample preparation (and
> >      > avoidance of purchasing stuff from 'Santa Crap' and similar
> companies)
> >      > is much more limiting than the microscopes.
> >      >
> >      > George
> >      >
> >      > p.s. a couple of references not included in above:
> >      >
> >      > W. Gray (Jay) Jerome, Robert L. Price 2018... Basic Confocal
> Microscopy
> >      > second edition
> https://urldefense.proofpoint.com/v2/url?u=https-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Flink.springer.com-252Fbook-252F10.1007-25252F978-2D3-2D319-2D97454-2D5-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3D-252FMQlH4t81Pk9tNTHR8fsn1tQc0JAc2nc-252BnO2gdk60Us-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=1JXaiNNf-bzSdjywfRJ8gowB9Yq0uGNulQMlGeSMxkE&e=
> >      >
> >      > Expansion ... X10 protocol ... Truckenbrodt 2019 Nat Protoc,
> >      >
> https://urldefense.proofpoint.com/v2/url?u=https-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.nature.com-252Farticles-252Fs41596-2D018-2D0117-2D3-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3DCF-252Fp9frimbrxZiMDwLWZjzuyoyQbCuKp4EcvaL3Wmmw-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=YeXbqWFX0mMxO3nVRt59pt6RJYlwIPBMZRePQbiF1yU&e=
> >      >
> >      > DNA-PAINT acronym soup review ... Nieves 2018 Genes,
> >      >
> https://urldefense.proofpoint.com/v2/url?u=https-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.mdpi.com-252F2073-2D4425-252F9-252F12-252F621-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3Diwx3Z1Pgr4a2SObO9F47jEtqlfsYQFk2R4gu0Qv1uJM-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=tJY5yunlGClYXKHrXuCDe5KU8D-qhc1ZS26_KuNdXpE&e=
> >      >
> >      > Babcock 2018 (4 --> 8 cameras, single molecule localization
> microscopy
> >      > with $1550 CMOS cameras) ...
> >      >
> https://urldefense.proofpoint.com/v2/url?u=https-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.nature.com-252Farticles-252Fs41598-2D018-2D19981-2Dz-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3DwN-252FHP30wNUE0-252BJWeFJIJFsBk32ZhD4DfKi9gc3ZTz2c-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=TIL4NaeETsqkjycmtWw_CnoEWvrpY1Tkr_SAmz-QkRM&e=
> >      >
> >      > Moffitt et al 2018 ...
> >      >
> https://urldefense.proofpoint.com/v2/url?u=https-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fscience.sciencemag.org-252Fcontent-252F362-252F6416-252Feaau5324.long-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DFR98V6ldS9Q38wI59kly9U8pCzp92Vzc1J6T8ydCU9w-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=55vzrzqpm3n5Z7logfovDr0N73h1gF7gldtYUSkZ6c0&e=
> and
> >      > commentary
> https://urldefense.proofpoint.com/v2/url?u=https-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fscience.sciencemag.org-252Fcontent-252F362-252F6416-252F749-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3Do0B379uuh-252FrB6gS9n6lG-252BjAZeztiYHZVmICwX4ghKh8-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=XeBps6CWTMM197evmy1n1uC5qOcvU8mAN95bEPmieiQ&e=
> >      >
> >      > ***
> >      >
> >      > Some resolution numbers:
> >      >
> >      > 1.4 NA objective lens ... 500 nm wavelength .. dxy = 0.61 *
> wqavelength
> >      > / NA (I routinely drop the 0.61 from 0.61)
> >      >
> >      > 0.6 * 500 / 1.4 = 214 nm
> >      >
> >      > widefiel deconvolution (re: Goodwin 2014) ~10% better ... 193 nm
> (if
> >      > pixel size matched or interpolate optimally).
> >      >
> >      > point scanning confocal -- Zeiss has a nice PDF, "Zeiss 2008
> Principles
> >      > - Confocal Laser Scanning Microscopy" (see fig 10) on confocal
> >      > resolution wrt 1 and smaller pinhole size, source of the values
> below,
> >      >
> >      > 1 Airy unit: 0.51 * 500 / 1.4 = 182 nm.
> >      >
> >      > 0.5 Airy unit ... 0.44 * 500 / 1.4 = 157 nm ... ~0.25 photons
> throughput
> >      > (which doesn't matter if target is photostable).
> >      >
> >      > 0.2 Airy unit ... you can ask your Zeiss rep about AiryScan (and
> >      > FastAiryScan).
> >      >
> >      > 0.1 Airy unit ... 0.37 * 500 / 1.4 = 132 nm ... ~0.10 photons
> throughput.
> >      >
> >      > Most modern point scanning confocal microscopes have a 405 nm
> laser, so
> >      > if using BV421 (and ignoring potential photobleaching for a
> moment),
> >      >
> >      > 1 Airy unit: 0.51 * 421 / 1.4 = 153 nm.
> >      >
> >      > or in reflection mode, i.e. nanodiamond or nanogold,
> >      >
> >      > 1 Airy unit: 0.51 * 405 / 1.4 = 147 nm ... and reflection implies
> no
> >      > photobleaching, so infinite number of photons (though also no
> blinking,
> >      > so not usually eligible for precision localization) ...
> >      >
> >      > 0.1 Airy unit: 0.37 * 405 / 1.4 = 107 nm
> >      >
> >      > and not going completely exotic with NA (i.e. 1.65), if perfectly
> >      > refractive index match with a fairly conventional 1.49 NA lens,
> and
> >      > inreflectance:
> >      >
> >      > 0.1 Airy unit: 0.37 * 405 / 1.49 = 100.57 nm
> >      >
> >      > I think I'd rather invest a DNA-PAINT friendly rig than deal with
> 157 to
> >      > 101 nm.
> >      >
> >      > DNA-PAINT makes resolution irrelevant, if you use it (and don't
> run out
> >      > of disk space or time or money), since precision localization is
> >      > resolution divided by square root of number of photons, ex. 250
> nm XY
> >      > resolution / sqrt(1,000,000) = 0.25 nm, and could increase number
> of
> >      > photons per target further, but why bother?
> >      >
> >      > ***
> >      >
> >      > point scanning confocal microscopes are also great platforms for
> >      > F-Techniques, such as FastFLIM (aka rapidFLIM, etc, much faster
> than
> >      > classic TCSPC slow FLIM), FCS, FCCS, see Liu 2008,
> >      >
> https://urldefense.proofpoint.com/v2/url?u=https-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.ncbi.nlm.nih.gov-252Fpubmed-252F18387308-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DZQTosrSoPFpCa8Y3IdEa9Xz-252B4RHC8JO4gBppHmzkazo-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=meRUIZdF3FLQZGyok4cxyc4AP4yq2lXX41Vt8q9WMmw&e=
> >      >
> >      > ***
> >      >
> >      > p.p.s. Disclosures I am ...
> >      >
> >      > 1. currently hosting a Leica THUNDER Imager tour event (ends
> Monday
> >      > 3/18/2019 afternoon) ...  see pdf download page,
> >      >
> >      > THUNDER Technology Note
> >      > THUNDER Imagers: How Do They Really Work?
> >      >
> >      >
> https://urldefense.proofpoint.com/v2/url?u=https-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.leica-2Dmicrosystems.com-252Fscience-2Dlab-252Fthunder-2Dtechnology-2Dnote-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DauRUORuEKAir87-252Bbw7RHyTR9IxxrLYl1g3bFBiRTXBM-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=LmFGYl_Icu6b9AAIYnHkoiH26bRZ688ODEc9I6k8yco&e=
> >      >
> >      > 2. hosting Nikon confocal demos in May 2019.
> >      >
> >      > 3. aiming to co-host with ISS a FastFLIM (one day) mini-symposium
> this
> >      > summer.
> >      >
> >      > 4. an unpaid advisor for Gary Brooker for FINCH/CINCH, re:
> >      >
> https://urldefense.proofpoint.com/v2/url?u=https-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.ncbi.nlm.nih.gov-252Fpubmed-252F28261321-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DuVw0kiW1baZV0RJv-252Bk1ObKznhw51pemnCMehjHkUa2g-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=KC1HMkiDzpE2DLsuj1I2nHvopwFVMMc0GVecDV-fWkM&e=
> >      >
> >      >
> >      > On 3/17/2019 10:43 AM, Mika Ruonala wrote:
> >      > > *****
> >      > > To join, leave or search the confocal microscopy listserv, go
> to:
> >      > >
> https://urldefense.proofpoint.com/v2/url?u=https-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Flists.umn.edu-252Fcgi-2Dbin-252Fwa-253FA0-253Dconfocalmicroscopy-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DdHOCjeBda4sSZPf-252FB2-252B5Sv3Q8Qzcs708p4we8vHf-252FIg-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=qZ-tdRwhHG172zv4uHPeAJMNeIIHNxozhQudV-hF2kE&e=
> >      > > Post images on
> https://urldefense.proofpoint.com/v2/url?u=https-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fwww.imgur.com-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DcnhzoWOP4RwDR622fn447aQVxW8ZBI3D0utze67RiGg-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=YzF36y8eV4SG3E0LJEDu-9AMm3Nh05dwp_XKhB6BUyU&e=
> and include the link in your
> >      > posting.
> >      > > *****
> >      > >
> >      > > Hi.
> >      > > There are software solutions that are able to create image data
> from
> >      > wide-field and even microscope systems with seemingly similar
> quality to
> >      > that obtained from confocal systems.
> >      > >
> >      > > The comparison of acquisition vs. software is essentially a
> comparison
> >      > of image acquisition vs. image processing. While a software
> solution is way
> >      > cheaper than a hardware solution if it is able to produce image
> data with
> >      > equal quality why would anyone choose to invest to a confocal
> anymore?
> >      > >
> >      > > I’m looking forward to a vidid discussion!
> >      > >
> >      > >> m
> >      >
> >
> >
> --
> Alison J. North, Ph.D.,
> Research Associate Professor and
> Senior Director of the Bio-Imaging Resource Center,
> The Rockefeller University,
> 1230 York Avenue,
> New York,
> NY 10065.
> Tel: office    ++ 212 327 7488
> Tel: lab        ++ 212 327 7486
> Fax:            ++ 212 327 7489
>

*****
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Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Arvydas,

Where exactly are you measuring the circular polarization, right after the polarizer or after passing other optical elements? Also, where have you been measuring your linear polarization? Circular polarization can be messed up very easily, which is why I always implemented it at the last possible moment before passing the light into the objective lens. Dichroic mirrors especially can mess up your polarization, as can dielectric mirrors, in particular if you hit them off of a 45° angle. Furthermore, wave-plates are optimized only for very specific wavelengths - even if they state a large spectral range, then they will be slightly different depending on the precise wavelength of light. Hence I recommend you not go for a perfect 45° orientation of your 1/4-wave plate, but instead optimize it (and the 1/2 wave plate before) to achieve the best possible circular polarization by trial and error. Think of it as performing a beam walk, just with the polarization. If you need extra precision, angling the wave-plates in respect to the laser beam will allow you to optimize for your specific wavelength.

Obtaining (and maintaining) high-quality circular polarization can be quite tricky, and requires slight readjustment every now and again.
Good luck!

Nicolai

P.S. If you need some specific advise about the above, just shoot me an email.
-----------------------------------------------------------------
Nicolai T. Urban
Max Planck Florida Institute for Neuroscience
1 Max Planck Way
Jupiter, FL 33458


-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Craig Brideau
Sent: Dienstag, 19. März 2019 13:20
To: [hidden email]
Subject: Re: Circularly polarizing laser beam

*****
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*****

I agree with Warren. I've managed to fudge nearly circular with a 1/2 and
1/4 wave combination, by tilting the plates slightly to compensate for chromatic dependence. The ability of the Berek compensator to tilt lets you do this in a more controlled manner. You will also find the circular state is very sensitive to environment. If you have a temperature change or any new mechanical stresses on the system you will start going back to elliptical frequently. I used a motorized rotation system with my half/quarter waveplate set and had to calibrate frequently. Here's my old
paper:
https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.spiedigitallibrary.org%2Fconference-proceedings-of-spie%2F8226%2F82263A%2FAutomated-control-of-optical-polarization-for-nonlinear-microscopy%2F10.1117%2F12.908995.full%3FSSO%3D1&amp;data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C413d206440a441615d5508d6ac909130%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636886134294384536&amp;sdata=p3kQApmUSidTotfFx%2FlgLod5C2bm9wFftsll5yo8UBM%3D&amp;reserved=0

Craig

On Mon, Mar 18, 2019 at 4:57 PM Warren R. Zipfel <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
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> .umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&amp;data=02%7C01%7CN
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> 6u6p06DNARFry4catfl70bO5u7vxbC5FIcQg%3D&amp;reserved=0
> Post images on https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&amp;data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C413d206440a441615d5508d6ac909130%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636886134294384536&amp;sdata=yaZWwZewft2tlISsj6VvmFEDD0%2F%2BmJjjTr0cmJmDNpQ%3D&amp;reserved=0 and include the link in your posting.
> *****
>
> I use a Berek compensator in the Ti:S beam and either a polarizer
> after the objective to verify the Berek settings for  X or Y linear
> polarization (for anisotropy imaging)  or a polarizer and 1/4
> waveplate as an analyzer if I need R or L circular polarization.  The
> more complicated optical path on most setups makes the adjustability of a Berek necessary.
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On
> Behalf Of Arvydas Matiukas
> Sent: Monday, March 18, 2019 4:49 PM
> To: [hidden email]
> Subject: Circularly polarizing laser beam
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
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> .umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&amp;data=02%7C01%7CN
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> *****
>
> Hello list,
>
> I am struggling to get a precisely circularly polarized beam. No
> matter how precisely I set fast axis of the quarter wave plate at 45
> degrees  to the polarization of laser beam, the ellipticity is always
> <0.9 (Imin/Imax <0.9).
>
> The laser beam is 588nm (5nm bandwidth) off OPO and pulse broadening
> fiber (from fs pulses of driving Mai Tai to ps pulses), diameter 4mm.
> Polarization is 1:70 that is further  improved to 1:600 by Glan polarizer.
> The zero order 588nm
>
>  quarter wave plate  is aligned normal to the beam and rotated at 45
> degrees (other angles only increase ellipticity).
>
>
> Any suggestions what I may be missing are welcome.
>
>
> Thanks,
> Arvydas
> ************************
>