NetNotes


Interesting question! I will point out that even if you find


a dye that is not fluorescent, the fact that it works as a bright- field dye means that it is absorbing light. For example, a dye that appears blue is absorbing green and red light and trans- mitting blue light, which will still interfere with fluorescent measurements using green or red excitation. You may be bet- ter off looking at a transmitted-light contrast technique, such as oblique illumination, if phase or DIC are not available. On our stereomicroscopes, for example, we can adjust the angle of


the incident light using a mirror below the sample, and


with oblique illumination we get nice contrast for otherwise hard-to-see tissues or cells. I am not exactly sure this can be done on a standard brightfield microscope. Or perhaps clos- ing the aperture diaphragm (so-called “dirty brightfield”)? James Jonkman james.jonkman@uhnresearch.ca


DAB is not fluorescent, but it quenches fluorescence. So,


it will mask any fluorescent dye you use to label the same areas. Sylvie le Guyader sylvie.le.guyader@ki.se


Many thanks, James and Sylvie. Good to know that DAB


quenches fluorescence. Sudan Black staining seems to reduce back- ground autofluorescence in some studies (sounds good). Bright- field images using that compound seem well contrasted! Provided there are no red flags for Sudan Black that I am missing, it looks like this might be useful. I will also check if we can use some of the sug- gested illumination modes. I love how it is called “dirty brightfield.” Jelle Postma j.postma@science.ru.nl


Maybe try Trypan Blue? It should penetrate fixed tissue and


make it darker. It may somewhat quench fluorescence because it has a broad absorption spectrum, but its concentration can be adjusted. Mike Model mmodel@kent.edu


As others have observed, this is an interesting question. Te


best bet may be phase-contrast or DIC as others have suggested, or simply to stop the brightfield condenser down while using a high- NA objective. Cheap, easy, and effective in many tissues. As others have noted, any dye will absorb light, potentially either in the exci- tation range or emission range of one or more of the fluorophores you want to see. Sudan Black will absorb fluorescence, which is why it is used for quenching lipofuscin autofluorescence. You might be able to get enough of a contrast-increase to make it worth it. So why do you not want to use a fluorescent dye? Tere are several DNA-binding dyes that span a large part of the spectrum. Do you really need all of the spectrum to be available for other labeling? Martin Wessendorf wesse001@umn.edu


Dear Martin and Mike, thanks for thinking along and for


the practical tips! The requirement for the dye being non-fluo- rescent is indeed not set in stone. We could sacrifice a fluores- cent channel for a tissue-level stain. To create a more standard- ized procedure for users here, and to keep maximum flexibility for them to use combinations of fluorescent stains, I am check- ing around to see if the idea of a nonfluorescent stain is even realistic. Equally for Trypan Blue and Sudan Black, it looks like they both absorb light, but if I go with this route to solve the problem, the hope is that enough of the target’s fluorescence will still be able to see it. In short, the more fluorescent windows available, the more events of interest we can trace in the same brain slice. But the brightfield screening of slices before looking at fluorescence is performed on a simple system near the ani- mals. Jelle Postma j.postma@science.ru.nl


2022 July • www.microscopy-today.com


A colleague who regularly pretreats brain slices with Sudan


Black to suppress autofluorescence indicates it makes them a little black. Mike Model mmodel@kent.edu


A colleague of mine provided two interesting resources that


are relevant to the question about brightfield stains: StainsFile, Te Internet Resource For Histotechnologists by Bryan Llewellyn (https://stainsfile.info/xindex.html) and a book “Romeis - Mik- roskopische Technik” that seems to be available only in German (https://link.springer.com/book/10.1007/978-3-642-55190-1). Andriy Chmyrov andriy.chmyrov@gmail.com


Eosin is fluorescent, but hematoxylin is not. So, hematoxylin-


only staining might work. However, it does quench fluorescence as do other brightfield dyes. I think that more molecules of a brightfield dye are required to make it visible when compared to a fluorescent dye. Tus, quenching is probably unavoidable. Tere are several fluorescent channels available, that is, 1-DAPI, 2-green-GFP/488, 3-orange-Cy3, 4-near red, Cy3.5 or AF594, 5-far red - Cy5 or AS 635P, 6-far far red, AF 680 or CF680R, without considering spectral or lifetime unmixing. So, DAPI plus 5 different antibody stains are possible. Some investigators want several fluorochromes, but when considering the number of structures in a sample that can be stained the numbers oſten crumble. Tere are a limited number of hosts for primary antibodies. Steffen Dietzel lists@sdietzel.de


Colocalization Maximum Intensity Projections (MIPS) Confocal Listserver Hi everyone, I’m looking for good analogies, tutorials, lessons,


graphics, ideas, etc., to convince our users that they should be using our Imaris license to determine colocalization in confocal Z-stacks instead of maximum intensity projections (MIPS) in FIJI. Since they see ‘differences’ in treated versus untreated in their MIP colocalization analyses, they are happy. I’m pulling my hair out that their analyses are not done properly, and we have soſtware that can handle 3D data sets. Are there any good resources, publications, or data sets to share to show why investigators should NOT be using MIPs to quantify co- localization in confocal Z-stacks? My hand-waving representations and hand-drawn artistic abilities don’t seem to be convincing enough. Tanks so much. Kathryn Spencer kspencer@scripps.edu


An orthogonal section (XZ, YZ) of their 3D stack should show


that many ‘colocalizations’ in MIPS are actually overlays of foci that could be microns apart. Tis manages to convince most of our users. Fred Indig indigfr@grc.nia.nih.gov


Te method used to most efficiently answer colocalization


depends on the question being asked. For example, if only curi- ous about lateral colocalization in two dimensions, then a MIP is valid. For 3D non-parametric colocalization (the more common type of colocalization many are interested in), one of the easiest ways is to binarize the two channels of interest, make an AND stack of the two stacks, and then measure the ratio of the AND stack mean intensity to the mean intensity of the channel of interest. 1 = perfect colocalization, 0 = no colocalization. Benjamin Smith benjamin.smith@berkeley.edu


Please look at the top section of http://microscopynotes. com/imagej/colocalizationspotssimulation/index.html for an il-


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