NetNotes


not supporting it. Furthermore, as Timothy pointed out, some decon- volution soſtware 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 soſtware 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. Te results speak for themselves. Nicolai Urban Nicolai. Urban@mpfi.org Dear Mika, it would be really helpful if you could be more spe-


cific. What exactly do you want to compare? Tere are so many options out there these days that it is difficult to guess what you mean. Hav- ing said that, as Avi pointed out, it is generally best to first get a good image by physical means and then do the computational improvement like deconvolution. Tat is true for widefield, confocal and also STED super-resolution. So, confocal and decon is better than confocal alone or widefield and decon. Although for some applications widefield and decon might be good enough. Steffen Dietzel lists@dietzellab.de


Many applications overlap but here are three examples of when


laser scanning confocal is indispensable: 1) You cannot tell by wide- field whether you are seeing a fluorescent labeled structure or reflec- tion from light emitted by another bright area; 2) Some thick tissues; and 3) 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 soſtware package and paying a core a fee for use, it just isn’t going to happen. Michael Cammer Michael.Cammer@med.nyu.edu


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. Tis 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. Tey 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 micro- scope with deconvolution (or likely even just a simple high-pass fil- ter) 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. Deconvolution 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). Tis 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 deconvolution. Unless you measure


the Point Spread Function in the sample (such as using TetraSpeck beads) at a higher resolution than you acquire 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 deconvolu- tion allows a computer to guess what information should be removed. Tus, just because the image looks better does not necessarily mean it is correct, otherwise STED, STORM, AFM, and cryo-EM would be obsolete. Benjamin Smith benjamin.smith@berkeley.edu


I agree that one needs to define the problem more carefully,


and it is like the screwdriver vs. wrench question. Tere are also other issues to consider, one of them being photon economy. I’m a


52


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 com- putational technology or the imaging technology, poor choices are made with little understanding. Regarding the Point Spread Function needing to be at a higher


resolution, this makes no sense to me. Te 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 restor- ative deconvolution doesn’t subtract anything. It reassigns light to its purported origin. Tere should be constraints that the total inte- grated optical density be the same before/aſter 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. Tat data is lost and irretrievable. Tis problem is confounded by sample photobleaching. Te 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 reas-


sign fluorescence to its point of origin, but it does this while collecting all available photons in the case of widefield. Tis 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 its point of origin. As others have pointed out, one can also deconvolve confocal and super resolution images. Tis advantage is probably lim- ited 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 jcarmichael@chroma.com


I disagree with Jeff’s statement: Confocal by its very nature


rejects something like 90-98% of available fluorescence photons. Tat data is lost and irretrievable. 1. Trivially, if it is not digitized, it is not data. If it is from a far of focus plane, deconvolution not going to reassign it very well.


2. Reducing to the simplest cases: one vs two sub-resolution fea- tures, 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 depend- ing on the gap size (and wavelength), may be resolvable. Sure, GaAsP or (GaAsP)Hybrid detector has lower QE in the vis- ible (∼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.


3. Field of view and scanning: a. Camera based: at the mercy of whatever objective lens magnification and additional magnification in the instrument, readout (typically) some number of entire


www.microscopy-today.com • 2019 July


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