http://confocal-microscopy-list.275.s1.nabble.com/General-question-Software-vs-hardware-tp7589316p7589333.html
Many applications overlap but here are three examples of when laser scanning confocal is indispensable:
You cannot tell by widefield whether you are seeing a fluorescent labeled structure or reflection from light emitted by another bright area.
Some of the weird chambers brought in by engineers, transwell chambers, and other oddities.
Perhaps this does not really address the original question, but we have found that where deconvolution requires sitting at another computer with another software package and paying a core a fee for use, it just isn’t going to happen.
Subject: Re: General question: Software vs. hardware
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.
The Mina & Everard Goodman Faculty of Life Sciences Bar-Ilan University, Ramat-Gan 5290002, Israel
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> *****
>
> Hi Mika,
>
> White et al 1987 (
>
https://urldefense.proofpoint.com/v2/url?u=http-3A__jcb.rupress.org_co> ntent_105_1_41.long&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeII
> ZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=QhUrmPqL07FT6Fex_
> r1_9otpqB337-xfUGpGNb7ONvc&s=yVMwIyG9dOwJekwvrhAd-YcpXuwUz0br0S_5PdDo0
> 6A&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__www.ncbi.nlm.nih.
> gov_pubmed_24974028&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeII
> ZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=QhUrmPqL07FT6Fex_r1_9otpqB337-xfUGpGNb7ONvc&s=Ua3LXNZyL2gL-lg5mxuXVCNKXwSMTo41DbEEcPPLBiQ&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__link.springer.com> _book_10.1007-252F978-2D3-2D319-2D97454-2D5&d=DwIFaQ&c=j5oPpO0eBH1iio4
> 8DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0Rkn
> E5bU&m=QhUrmPqL07FT6Fex_r1_9otpqB337-xfUGpGNb7ONvc&s=OLwJ4Tn_dtindX_od
> PzsUb7FqLW0x-HCs7F8Mb3VwyA&e=
>
> Expansion ... X10 protocol ... Truckenbrodt 2019 Nat Protoc,
>
https://urldefense.proofpoint.com/v2/url?u=https-3A__www.nature.com_ar> ticles_s41596-2D018-2D0117-2D3&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc0
> 4rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=QhUrmP
> qL07FT6Fex_r1_9otpqB337-xfUGpGNb7ONvc&s=cUzsjl7SIzMQUNgwgKER9mDPZEMohi
> 5rhFZJwOnal6s&e=
>
> DNA-PAINT acronym soup review ... Nieves 2018 Genes,
>
https://urldefense.proofpoint.com/v2/url?u=https-3A__www.mdpi.com_2073> -2D4425_9_12_621&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuC
> s&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=QhUrmPqL07FT6Fex_r1_
> 9otpqB337-xfUGpGNb7ONvc&s=oaz40_UfNUr03mxrG9Wo1FC400X08izokzftEHhlf9w&
> e=
>
> Babcock 2018 (4 --> 8 cameras, single molecule localization microscopy
> with $1550 CMOS cameras) ...
>
https://urldefense.proofpoint.com/v2/url?u=https-3A__www.nature.com_ar> ticles_s41598-2D018-2D19981-2Dz&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc
> 04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=QhUrm
> PqL07FT6Fex_r1_9otpqB337-xfUGpGNb7ONvc&s=Uu8NR6naGZycBiUtnau3E1wR4y4fN
> ok6ZujZHMvXGWY&e=
>
> Moffitt et al 2018 ...
>
https://urldefense.proofpoint.com/v2/url?u=http-3A__science.sciencemag> .org_content_362_6416_eaau5324.long&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeE
> lZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=Q
> hUrmPqL07FT6Fex_r1_9otpqB337-xfUGpGNb7ONvc&s=J2hElzRpmEhoCdpN3AeONt9_O
> 6naYQrBAiIw8h7zE5c&e= and commentary
>
https://urldefense.proofpoint.com/v2/url?u=http-3A__science.sciencemag> .org_content_362_6416_749&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3E
> xJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=QhUrmPqL07F
> T6Fex_r1_9otpqB337-xfUGpGNb7ONvc&s=9lrtHfkyrZ91YwO1mX9XIgo7iM-qm3zflvT
> o_y1TYWk&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__www.ncbi.nlm.nih.
> gov_pubmed_18387308&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeII
> ZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=QhUrmPqL07FT6Fex_
> r1_9otpqB337-xfUGpGNb7ONvc&s=2wsUl3TkcW6hCeGZ7XS0HuDA1fe27y-YJTFdjfrfS
> 2g&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__www.leica-2Dmicro> systems.com_science-2Dlab_thunder-2Dtechnology-2Dnote&d=DwIFaQ&c=j5oPp
> O0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV
> 7fujQf0RknE5bU&m=QhUrmPqL07FT6Fex_r1_9otpqB337-xfUGpGNb7ONvc&s=xRDjJDP
> NeqFeYwha2XGXbIpLiWeX8IbVAzG-lzoxIRo&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__www.ncbi.nlm.nih.
> gov_pubmed_28261321&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeII
> ZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=QhUrmPqL07FT6Fex_
> r1_9otpqB337-xfUGpGNb7ONvc&s=8YR5g4lC-nPSNY3Qrraw1xlFP0fPAaHtqKsOQbRHr
> NQ&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=http-3A__lists.umn.edu_cg> > i-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=j5oPpO0eBH1iio48Dtse
> > deElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5b
> > U&m=QhUrmPqL07FT6Fex_r1_9otpqB337-xfUGpGNb7ONvc&s=3LqjIRKGooGbWufKQI
> > fCOyBhknMhzM4msy9LD6YtGwo&e= Post images on
> >
https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=> > DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDi
> > PlC50tp7rW2nBkvV7fujQf0RknE5bU&m=QhUrmPqL07FT6Fex_r1_9otpqB337-xfUGp
> > GNb7ONvc&s=LwlU9Ttd8-NvvVIj2xG11Fkz5KupvCoj80DexAghEHY&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
>
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