http://confocal-microscopy-list.275.s1.nabble.com/General-question-Software-vs-hardware-tp7589316p7589324.html
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
> *****
> 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 Mika,
>
> White et al 1987 (
http://jcb.rupress.org/content/105/1/41.long ) 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://www.ncbi.nlm.nih.gov/pubmed/24974028 ) 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://link.springer.com/book/10.1007%2F978-3-319-97454-5>
> Expansion ... X10 protocol ... Truckenbrodt 2019 Nat Protoc,
>
https://www.nature.com/articles/s41596-018-0117-3>
> DNA-PAINT acronym soup review ... Nieves 2018 Genes,
>
https://www.mdpi.com/2073-4425/9/12/621>
> Babcock 2018 (4 --> 8 cameras, single molecule localization microscopy
> with $1550 CMOS cameras) ...
>
https://www.nature.com/articles/s41598-018-19981-z>
> Moffitt et al 2018 ...
>
http://science.sciencemag.org/content/362/6416/eaau5324.long and
> commentary
http://science.sciencemag.org/content/362/6416/749>
> ***
>
> 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://www.ncbi.nlm.nih.gov/pubmed/18387308>
> ***
>
> 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://www.leica-microsystems.com/science-lab/thunder-technology-note>
> 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://www.ncbi.nlm.nih.gov/pubmed/28261321>
>
> On 3/17/2019 10:43 AM, Mika Ruonala 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.
> > 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
>