NetNotes


Edited by Bob Price University of South Carolina School of Medicine Bob.Price@uscmed.sc.edu


Selected postings are from discussion threads included in the


Microscopy (http://www.microscopy.com) and Confocal Microscopy (https://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy) listserv- ers from February 15, 2019 to April 30, 2019. Postings may have been edited to conserve space or for clarity. Complete listings and sub- scription information can be found at the above websites.


Software and Image Analysis


Confocal Microscopy Listserver General Question: Hardware vs. Software (Thread Started


March 17, 2019) Tere are soſtware 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. Te comparison of acquisition vs. soſtware is essentially a comparison of image acquisi- tion vs. image processing. While a soſtware 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 in a confocal any- more? I’m looking forward to a vivid discussion! Mika Ruonala mika@icit.bio Are you talking about deconvolving widefield fluorescence


stacks? Tis is a very old debate. I prefer confocal - fewer artifacts. Mike Model mmodel@kent.edu


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 widefield data aſter deconvolution, you can also get near Super Resolution (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 Hyvo- lution and had access for a couple of weeks to the Lighting, and now confocal images look blurry to me. Avi Jacob avijacob@gmail.com


To throw my ha’penn’eth’s worth in, I am on the side of con-


focal for a lot of the same reasons: no artefacts in the first place, plus deconvolution can be applied to confocal images for a minor improvement (and I believe is recommended in Pawley’s excel- lent book). All of the things that would make a widefield system comparable (multiple cameras, fast GPUs, pixel shift free filter- cubes, etc.) just bring the price of the widefield close to a confo- cal anyway. Admittedly the running costs are cheaper though. So to summarize- confocal + deconvolution is my preference. Glyn Nelson glyn.nelson@ncl.ac.uk


As Mike said, this is indeed a very old debate, but there are


well-characterized, objective costs and benefits to each approach (scientific, not just monetary). Deconvolution is not just “computa- tional confocal at the expense of artifacts”… it’s a false comparison. By rejecting out-of-focus fluorescence, confocal microscopes reduce the *shot noise* contributed to the image by background. Deconvolu- tion, by contrast, attempts to “reassign” that out of focus informa- tion (provided you have a very accurate representation of the actual Point Spread Function in your sample), but there will come a point


50 doi:10.1017/S1551929519000592


with thicker samples at which the shot noise contributed by out-of- focus fluorescence overwhelms the SNR in the image, and deconvo- lution will fail (figure 4 in the first paper below). However, for thin samples with minimal out-of-focus fluorescence, the increased col- lection efficiency and minimized illumination/detector noise of widefield+decon has benefits for detection of weak signals (figure 2 in the paper below). Tis tradeoff was well-characterized by Swedlow and Murray


(https://www.ncbi.nlm.nih.gov/pubmed/11830634) and followed up with a treatment on the photon-efficiency of different optical section- ing


techniques (https://www.ncbi.nlm.nih.gov/pubmed/18045334).


As usual, there is no one technique that is universally “better” or pref- erable. It will depend on the samples you are imaging and the rela- tive levels of in-focus and out-of-focus information. Talley Lambert tlambert03@gmail.com


Avi, I really agree with your point. I feel that people deconvolve


any time spatial information is critical, whether they’re using wide- field, 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 back- ground, 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. Te 16-bit depth of widefield images is nice for quantitation, and modern sCMOS cam- eras have by far the best acquisition speeds. I don’t know whether widefield systems 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 soſtware to preserve the original intensity val- ues. 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. Timothy N. Feinstein tnf8@pitt.edu


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. Te 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 soſtware 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


www.microscopy-today.com • 2019 July


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