I found that the signals for my dye are weak in tissue sample, what is the maximum limit to which I can increase the detector gain and laser power percent for my dye?
Hi Chem,
unfortunately, percentages do not mean much, since the actual power on the sample strongly depends on the light source you are using, the objective you are employing, and the other settings of the microscope, so you have to experiment a bit with your settings.
As a general guideline, photodetector gain should be kept quite high when you are using your confocal microscope in live mode to find the focal plane and image area you want to image. Just turn it up until you start seeing visible noise in dark areas of the image, this way you know you will not miss any fluorescent object just because the gain is not high enough. Once you find the are of your sample you are interested in, turn down the gain to try to limit noise as much as possible, in order to get a "good" image. Ideally you should lower the gain until dark areas are completely black, and slow down the scan speed and/or increas the excitation power until the bright areas are almost at the maximum possible signal (but be careful not to saturate the image).
As for the excitation power, it really depends on whether your sample is fixed or fresh and living. If it is live, the lower the power, the better, as exposure to high intensities of light can interfere with the biological processes you are trying to observe. If the sample is fixed, however, the limiting factor is photobleaching, meaning the more you acquire images, the more you "burn" the fluorescent molecules, and the less bright the next image will be. I would suggest, when you are just looking at your sample to find the area you want ot image, to keep the power as low as possible, as long as you see something, and only turn it up once you start acquiring the images you want to save.If you are collecting a 3d stack, keep the power lower, as photobleaching happens even in the planes above and below the one you are imaging. Finally, if you can spare the time,slowing down the acquisition speed or averaging more images will in general always lead to better results than simply turning the power up, so try to be patient and only increase the power as a last resort.
Good luck!
I largelt agree with what Paolo said above. The two are basically tradeoffs - higher gain means you can use lower laser power and/or a faster scan speed, but at the expense of more noise in an image. Higher laser power or slower scan will give you stronger fluorescence and therefore allow lower gain and less noise, but at the expense of increase photoxicity and photobleaching.
In terms of percentages, most of the labs I've been in don't turn the laser power above 30% of maximum, but that could be ideosyncratic. But definitely start with lower power before you work up to something higher. (Also, figure out your settings in a region a little away from the area you intend to finally image.) Keep in mind that the UV laser line has a much higher potential for damage, so be particularly careful as to how much power you put behind that.
I assume you use PMT,
There you can increase the gain and laser power till you do not get saturated image,this you can find by LUT settings in confocal,
I recomend to use HyD detector if ur institute has
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