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Nicolai.Urban@mpfi.org on
URL: http://confocal-microscopy-list.275.s1.nabble.com/General-question-Software-vs-hardware-tp7589316p7589331.html
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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
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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:
> *****
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> *****
>
> 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
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> 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
>