Dear researchers,
I'm working with FITC and Rhodamine B dyes and I use Sequential scanning. I will adjust the image quality through Smart Gain and Smart Offset and maintain QLUT in reddish region, avoiding blue (over exposure). What is the thumb rule for getting an image with correct brightness and contrast? I'm using Leica CLSM.
Thanks
Parthasarathi
1. Use a laser intensity that will give you pixel values between 50% and 99% of the dynamic range. This will achieve appropriate contrast and your data will be good enough for quantitative analysis.
2. Set the pinhole width to 1 Airy unit to minimize the presence of out-of-focus light in your images. If not enough light is getting through and you have to use a laser intensity which causes bleaching, you can increase the pinhole width.
Martin is totally correct. The way I explain it is that you want to fully use, but not exceed, the dynamic range of the detector. I generally use the histogram function with log scale and increase laser power and gain until achieving close to the max greyscale value (255 for 8 bit), but just a little below. With your method using the color coded LUT, backing off until the blue saturation indicator just disappears will achieve the same thing.
Many thanks Christopher and Martin. I use laser intensity of 20 % and feel higher intensity may induce photobleaching. I will work with the pinhole. May I know how to calculate/fix the pixel values between 50% and 99%?
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