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Daniel:Notebook/HiResChrPaint/2013-11-26
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=Microscope Practice= [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] Practicing more with the confocal today. I played around with several image gathering techniques using the practice slides from Leica. Today I tested out the 63x objective, which will likely be the one I'll need for FISH. Of note, I played with the gain, offset, and pinhole to try and get the best histogram distribution possible. I think a microscopy class would really help on this point (mental note), but from what I remember in earlier classes the goal is to prevent pixels from being saturated. There is a histogram function on the software that allows you to view the image histogram (for each PMT). Near the bottom, it reports min/max values for pixels. The ideal is to keep the max as close to 255 as possible without actually hitting it. Once you do hit 255, you have saturated pixels and you can no longer distinguish between those saturated pixels. Below are two images, one with high saturation and one where I adjusted the PMT gains to have no saturation, but rather max values around 245. '''Saturated Image''' [[Image:63x_112613_unsaturatedgreen.jpg|600px]] '''Unsaturated Image''' [[Image:63x_112613_unsaturatedimage.jpg|600px]] ==Bright Field Practice== In the afternoon, I worked with the brightfield option of the camera (use PMT trans). I also learned more about saving options, including adding scale bars and saving the overlay. These are baby steps, but I want to be confident in my microscopy techniques before I can properly evaluate the FISH methods. {| class="wikitable" |- |[[Image:BFTest_112613_overlay_noscalebar.jpg|500px]]|[[Image:BFTest_11261_overlay_imposedscalebar.jpg|500px]]| |- |[[Image:BFTest_112613_overlay_customscalebar.jpg|500px]]| |- |Images taken at 20x objective using several different PMTs as well as bright field imaging. '''Top left''': no scale bar; '''top right''': scale bar added by Leica; '''bottom left''': scale bar added by me |}
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