resin and the membrane (even though it is also infiltrated with resin). While the resin compresses somewhat during sec- tioning, it stretches again on the water bath. Te filter doesn’t follow this stretching and thus wrinkles are formed. Te inter- esting thing is that Transwell filters have been successfully used for high pressure freezing- freeze substitution and resin embedding (Morphew et al., Journal of Microscopy, vol. 212, pt. 1, October 2003) without this wrinkling being a problem. It can be that for these filters dehydration in acetone will allow for a better resin infiltration of the filter or that a harder resin mixture (less compression during sectioning) may prevent the wrinkles from forming.Rob Mesman

In response to Amalia Pisolli’s questions about working with

cells on Transwell filters, I published a short paper in Microscopy Today a few years ago about embedding cell monolayers, both in flasks and on Transwells. doi:10.1017/S1551929513000485 MT May 2013. Tere is a typo in the resin formula: NMA should be 6.0 g. Te resin is different in the filters than in the cells, but this method has worked well for me for many years. It’s the only reason I still keep any propylene oxide in the lab. Acetonitrile, which we’ve switched to for most purposes, doesn’t make the filters curl. Lee Cohen-Gould

Confocal Microscopy Concerns Confocal Microscopy Listserver

Confocal imaging scan speed I’d like to put a finer point on a question about scanning

speed. Like some others I encourage users to just set the fastest speed and leave it there. Leica SP8s constrain the field of view with faster speed, so I suggest SP8 users choose 700 Hz as a good balance between speed and field of view. My reasoning comes strictly from my own testing. I found that you pay a lot more in bleaching when you try to get a brighter signal by slowing the scan speed than if you just turn up the laser power. Since the slowest speeds drastically increase bleaching, I use them for bleaching/ photoactivation of a region of interest (ROI) and that’s about it. As I understand it, the strong relationship between scan speed and bleaching has to do with longer laser dwell time increasing the chance of exciting fluorophores to the triplet state. Hence, this is why resonant scanning plus line averaging is gentler on a live sample than a galvo scan of comparable signal-to-noise. Is that right? Tanks! Timothy Feinstein

I have always thought that the longer the pixel dwell time

the more accurate the representation the image is of the sam- ple. However, there must be an optimal scan speed that relates to Nyquist sampling (Nyquist indeed first theorized about temporal sampling). Perhaps modern electronics perform ana- log-to-digital conversion (ADC) and digital-to-analog (DAC) so quickly that sampling speed just isn’t a factor in modern confocal imaging as far as the electronics are concerned. Tat being said there must be a “sweet spot” for temporal sampling although it isn’t really discussed much. I default to a scan speed of 1.02 µsec/pixel. I would be happy to hear how and why I am wrong. Brian Armstrong

I teach our users what scan speed means and why you some-

times use a fast speed and sometimes slow. I don’t have a hard and fast rule, but do have recommendations depending on the sys- tem for settings that work well for most fixed samples. For live cell/tissue imaging, there are oſten compromises to be made, i.e.,


scan speed usually higher/faster and no averaging and low laser power particularly if we need high temporal resolution. We will also sometimes open up the pinhole as I mentioned in an earlier post. I think it’s important for the users to understand what scan speed means so that they can make their own decisions. Te level of noise in the image is the main thing that determines what scan speed and averaging we use. If the voltage/master gain is low, then we can go fast and don’t have to use averaging. However, if the voltage/master gain is high and we have fixed samples with good quality, then we have choices to make, i.e., do we increase laser power, slow scan speed down, use averaging or most oſten a combination of all three? If users are acquiring z stacks, then scan speed is very important as the time for acquisition is then multi- plied. At this time investigators might decide to skimp on sam- pling in z (acquire fewer slices) so understanding what they can do to enable a faster scan speed for good quality images is helpful as long as they are also mindful of photobleaching. I stress to the users that they should always check the quality of their image by looking at 100% of the pixels. If they don’t do this and have the image window set to display the entire image in a smaller space, they will not see the noise and will be disappointed later. For label- ling of punctate structures, it’s even more important not to have excess noise present. Jacqueline Ross

Te spinning-disk people say that the critical point about

bleaching is maximum laser power, and I tend to believe it. I think what you describe could be true for continuous wave- length lasers, but maybe not for pulsed lasers where the fluors have more time to drop back to ground state before the next excitation hits. In any case the laser power is important. But I admit I have not thoroughly tested this, and should it really make a difference whether the pixel dwell time is 0.5 or 1 or 2 μs? In both cases there is ample time to go to T-states. Resonant scanning with times < 50 ns may be different. Has anyone pub- lished a paper on this? Fast scan speed and averaging or higher laser power versus slow scan? I encourage our users to test for bleaching by recording the same image (stack) twice and check for intensity decrease in the second stack. If there is none, time can be saved by higher laser power (up to the point of saturation). Fortunately not many of our users use FITC or APC, and mod- ern fluors are pretty stable. Steffen Dietzel

Tat is the nut of my question. Has anyone ever published

a paper on this? Fast scan speed and averaging or higher laser power versus slow scan? I have tested this with gas and solid state lasers and as far as I can tell, strictly from my own experience, you get more sample loss by slowing the scan than if you raise laser power to get a comparable increase in signal. However I’d gladly submit to any properly done testing out there. In prin- ciple if a 100 MHz laser has a 10 ns pulse interval, that’s short enough to doubly excite most common fluors. It seems reason- able then to think that a solid state laser could also endanger the sample more by scanning slowly than by scanning fast with higher pulse amplitude. Timothy Feinstein

Point scanning systems are (hopefully) shot noise limited,

so what (hopefully) matters is the number of photons per pixel. If you scan fast at high power, or slow at lower power, or fast at low power but average, and in each case get (in expectation) N photons per pixel, you have a signal to noise ratio (SNR) of N^0.5 in all instances. If you want a better SNR more photons are required and it won’t matter how you do that. Scanning speed becomes a factor in shot noise limited confocal systems • 2020 January

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