W1 with 2 pinholes?

Do people have experience imaging with the Yokogawa W1 with the 2 pinhole sizes? 25um and 50um? In principle 25um should be better for some objectives/NA/wavelengths, but in practice, what differences have you seen? Let’s just leave out the cost for now - if the options were the same price (or not a burden to purchase) would you buy the 2 pinhole version? This is a live fly embryo imaging lab replacing a CSU-10. Thanks! (can DM at lisa.cameron@duke.edu)

Hi! Hope you’re well. I have experience using a dual disk system, 25um and 50um pinholes (not a W1 though, its an X1). It is interesting to be able to switch back and forth. In general, the 50um disk is recommended for high mag, high NA objectives (e.g. 100X 1.40 oil). If you have enough signal, and can image with the 25um disk on a high mag, high NA objective, it definitely does make a difference, less background, image is cleaner with more contrast, increased signal to noise, obviously this will depend on many factors, I’ve done this with lamin A GFP, DNA damage, Ecadherin, and various cytoskeletal probes, particularly with a 40X 1.30 oil; something like a DNA damage probe or something very punctate, I feel made a bit of a difference, but perhaps not as obvious as other probes. Of course, to get the same signal, more laser intensity or longer exposures are required compared to the 50um pinhole disk. The 25um pinhole results in a thinner volume represented in the slice, so for thin samples that are bright, it is great…again, if you can get enough signal. Explaining the difference in practical terms…On my lamin A GFP cell line, with the 50um disk, you see the nuclear lamina, and also some signal ‘within’ the nucleus, that is the lamina above/below the focal plan, but with the 25um disk, the lamin A appears as perfect rings, a line scan really shows the difference. I hope this makes sense. How often a user will take the time and care to compare the resulting data between the two disks is always something to consider, but I think its a great option to have. If the group images the fly embryos a lot in those ‘middle’ magnifications, and there is plenty of signal, its a nice feature and could help clean up the data (depending on the probes they are imaging) a bit. So, without a price different or burden, I’d get the dual.

Thanks for the feedback. Definitely helped.