NetNotes


Zeiss and Nikon have about the same range (400–700 or 750 nm). G. Esteban Fernandez g.esteban.fernandez@gmail.com


We use the Leica; it can collect the images for spectral unmix-


ing either parallel (5 channels) or serially (adjustable range and bin width). Depending on the sample and the dyes, I oſten find that the five channels are sufficient. Happy unmixing. Richard Cole richard. cole@health.ny.gov


Tat’s 5 colors. I was wondering what you need the other 29


channels for on a 34 channel system? We have done six colors on the same slide (on a 5 channel system without unmixing, by sequential scanning, pretty much your channels plus Alexa 680). If you man- age more than 10 colors with a reasonable separation I am impressed. With 10 or less, two sweeps in a 5 channel system should be sufficient, no? You would have to go over it twice but you also collect noise only 10 times, not 34 times. I don’t have much experience with computa- tional unmixing, but it does not seem a sure thing to me which way of scanning you would reach the same image quality faster. Maybe I am missing the point. Steffen Dietzel lists@dietzellab.de


> that’s 5 colors. I was wondering what you need the other 29


channels for on a 34 channel system?< To get the right intensities of the different fluorophores, you need to solve a system of linear equations. When the spectra are too close together, this gets more difficult and the error in the result increases. Having more data points can help, especially if you have good enough signal to noise. But splitting the signal into more channels decreases the SNR. It would be interesting to see if there is an optimum number of channels. As I remember, the Zeiss soſtware can do the spectral unmixing on the fly and display the unmixed virtual channels during imaging, not sure about the others. If your features do not overlap, e.g. separate cells and high enough resolution to resolve them, you actually can get away with just 2-3 channels and distinguish many colors, similar to how a RGB com- puter monitor can display them, e.g. CFP, GFP,YFP and RFP with 3 fluorescent channels. By combination of membrane and nuclear staining 10 different cell types can be distinguished using these 3 flu- orescence


channels. www.sciencemag.org/cgi/content/full/science.


aad3439/DC1 Andreas Bruckbauer bruckbaua@aol.com Tere is some literature discussing ways to optimize channel set-


tings for separating a given set of labels by Neher/Neher: https://doi. org/10.1111/j.1365-2818.2004.01262.x and there is an accompanying ImageJ Plugin. I am not sure how well it can be applied to the real- ity of your instrument - this will probably depend on how well you know the actual additive (read-out) noise and QE of your detector, the expected relative brightness of your dye species in the sample and how well you can define the goal of your experiment. It applies to a situa- tion where all dye species can be present within the region represented by each individual pixel and you are interested in relative intensity. In other words, it assumes you wish to follow up with spectral unmixing, which always determines an estimate for the ratio of photons received from the respective dye species. When using APDs, as we do in our microscopes, which have negligible additive noise, it is safe to assume that using all your channels and then optimizing the detection bands (roughly speaking, the goal here is to have the inversion process of the mixing matrix not subtract large multiples of the detection channels from each other which would amplify noise), will give optimum SNR in the result. As Andreas has mentioned, assuming perfect spatial separation


of the species on scales accessible by the resolution of the instrument (i.e. when we can assume that the light in each pixel stems from only one dye species), the task shiſts from unmixing to classification of pixels and two detection channels are theoretically enough when the detection bands are carefully selected. Super-resolution methods like STED can obviously help with this in the spatial domain, while


2019 July • www.microscopy-today.com My experience from years ago when I was a field service rep indi-


cates to me a mist of compressor oil from when the can was charged. Tis is not unusual to see with canned air especially on front surface mirrors and dichroics. You are at the mercy of the bottling company when you use those cans. Sometimes you get one that does that. You might want to try the “Optic Bulb Blower” on the same page as the link you provided and use a micron filter on the intake side so you don’t get dirt into the bulb. If you do go that route, replace the bulb every couple of years because eventually the rubber will break down and contribute its own particles. As long as they are new and internally clean they work well. You should check with the manufacturer of the dichroic to find out what they recommend to clean it. Some dichroics go to pieces when wet so be careful. Dan Focht dan@bioptechs.com


It could be both of the above, so think back on how exactly you


were cleaning your optics: Was it only for very short bursts? Or was it longer… this could have caused a drop in temperature to cause problems, as Martin suggested. Did you always hold the can upright and not rotated (i.e. as it would stand on a shelf)? If not, this greatly increases the chance of things other than air spitting out of the can (such as propellant, as Craig suggested), which would then need to be chemically removed (risky when applied to a dichroic, unfortu- nately). Both of these can be avoided by using a manual air puffer/ blower instead. Good luck in restoring your optics! Nicolai Urban Nicolai.Urban@mpfi.org


As mentioned, it could be condensation dissolving some dirt on the glass and then evaporating. Also, these cans are full of liquid, and


59


multi-color single-molecule based methods, where bursts of photons are classified, rely on temporal separation (vs. spatial in the above case). In both cases the same considerations apply to the classifica- tion process itself: https://scholar.google.com/scholar?hl=en&as_sdt =0%2C5&q=Fluorescence+nanoscopy+goes+multicolor&btnG=). Andreas Schönle a.schoenle@abberior-instruments.com


Confocal Listserver Strange Shape Appears on Dichroic After Blowing with


Compressed Air (Thread started March 28, 2019) I saw that the main dichroic that we are using for a multi-


photon microscope was getting dusty and decided to clean with compressed air. The air duster we use is from Newport (https:// www.newport.com/f/canned-air-duster). Strangely on one of our blows, a white area appeared on our filter forming an elliptical shape which quickly turned into a white/green-ish halo (see image here: https://imgur.com/rlrP0yB) that does not seem to be going away. This is on the opposing side of the AR coating. I was under the impression that using compressed air was a safe way to clean optics. Has anyone else encountered a similar problem? Has the compressed air damaged the coating in some way? Thanks. Steven Hou shou@partners.org


My guess is that as the air from the can expanded, its tempera-


ture dropped, cooling the mirror and allowing water from the air to condense on the surface. If true, I would want to check with the man- ufacturer before assuming that the mirror is unchanged. Good luck. Martin Wessendorf martinw@umn.edu


Oh, Steven, that’s not good. The canned air contains pro-


pellant that can end up contaminating your optics. Use a blub blower instead like this one: https://www.bhphotovideo.com/c/ product/1317992-REG/visibledust_19112366_zee_pro_sensor_ cleaning.html (no commercial interest but I use them in our lab). If the artifact you are seeing is not going away then it is probably contaminant from the can. You will need to remove the filter and clean it. Craig Brideau craig.brideau@gmail.com


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60  |  Page 61  |  Page 62  |  Page 63  |  Page 64  |  Page 65  |  Page 66  |  Page 67  |  Page 68