Leica SP8 HyDs linear range to 60 photons per microsecond
20240809F Leica SP8 HyDs linear range to 60 photons per microsecond
Short version: our Leica HyD 2nd generation (SP8) detector: "linear range up to 60 photons/μsec (microsecond)."
GM comment: You can see the Pixel Dwell Time ("PDT") in the Leica LAS X GUI (same section as image dimensions and zoom). If using 10 line accumulation, then "Total Pixel Dwell Time" is 10x the the PDT. I recommend acquiring so positive label pixels are in the 300-1000 photon count range (hence the need for 16-bit mode). If acquiring Z-series at maximum useful resolution -- for 63x/1.35NA lens XYpixel = 50nm, Z-step 150nm (modest laser power, line accumulations and if needed frame accumulations to reach these peak values) -- then activating HyVolution2 will improve image quality further (the result image will by default scale to the full 16-bit dynamic range -- just adjust the Leica lookup table (LUT) to match intensities. Pur Leica confocal sales rep, Kevin Murphy, PhD, likes to turn on gray shaded background in the Leica LAS 3D viewer to better visualize dim features -- both LAS X acquisition and 3D programs can display side by side linked images (the "+" at top right of LAS X GUI, then select window and click on image name).
Long version and web link to issue PDF -- the author has some tedious stuff and of course I always use Photon Counts, not the silly Leica "standard" mode. The author does make the point that the newer Leica STELLARIS HyD X, HyD R (both hybrid detectors) and HyD S (silicon detector) have count faster than our 2nd gen (you are welcome to buy the image core a nicely equipped STERLLARIS -- arrange with ACCM, the dept who purchased the SP8, to use it for trade in credit).
https://academic.oup.com/mt/issue/32/3
PDF Volume 32, Issue 3, May 2024
page 51 (I wrote in the thread - same page - recommend always use HyD photon count mode):
Yes, explaining stuff to users can be tough sometimes, in especially if they are under the false impression that they already
know a lot. On the SP8, our standard advice to any user is to put the HyD gain on 50%. With this setting you are completely in the linear range (up to 60 photons/μsec) when no overexposure occurs = no blue pixels in Glowover LUT. That is easy to remember and there is a clear benefit for the images/image analysis. On the Stellaris I believe the HyDs can take more than 60 photons/μsec, so the numbers would be different. But I don’t recall them right now. The only control the users are then supposed to use to adjust image “brightness” is the laser power. If noisy, then averaging. If a user has strong signal, but is interested in the weak ones, I discuss the use of lower HyD gains but then try to hammer into the user’s head that the strong signals won’t be in the linear range anymore, that is, gray level 250 does not mean 5× as many photons as gray level 50, but more. Depending on the research question, linearity may not be required. And sometimes this is a no-go. For many samples, our users do not need any averaging or accumulation when using a scan speed of 600 or 400 lines/sec (that is with the linear scanner electronics, Klondike upgrade on the SP8). If averaging is applied, typically more than 3 do not lead to a visible improvement. As George already pointed out, there is no difference in time for accumulation and averaging. With a HyD gain of 50, averaging essentially fills the gaps in the histogram. The HyD gain setting has no effect on the detector, therefore it cannot cause damage. The actual voltage is always 8000 V. So, the number of impacting photons solely depends on laser power and sample brightness.
[see online PDF for complete text - and other messages in confocal microscope thread on this topic].
Steffen Dietzel lists@sdietzel.de
