Part 1 Principles |
Confocal Microscopy tutorialPart 1 Principles of Confocal microscopy8. SNR (Signal to Noise Ratio) in confocal microscopyAs mentioned in last section, PMT (photon multiplier tube) has a large active area with high capacity for photons thus can detecting strong signal with less saturation problem. PMT can multiply received photons thus has high photon sensitivity suitable for detecting weak signal at very low noise level. These features endow PMT with wide dynamic range and good SNR. But in fluorescence microscopy, the total photon number is very
small, less than 1000 photons per pixel time, and even smaller in confocal
microscope, about 10-30 photons/pixel/µs due to the
massive rejecting of signal by pinhole. The dynamic
range can not be calculated from the ratio of full photon capacity / noise
anymore, and solely affected by signal intensity. Similarly, the SNR is affected more by signal intensity (total
photon influx) than
by background noise level as mentioned in part 1
Optical section, When N is 1000 photoelectron per pixel time, if set S: 1.2, d:
100. q: 0.05, SNR is 25. In formula 4, photoelectron number on the numerator has to be squared, it has more weight in the formula and results in a pronounced effect on SNR. This makes confocal microscope very vulnerable to weak signal. The reduced photon number not only weakens signal intensity, but also deteriorates image quality. Raising gain (voltage) on PMT can amplify weak signal but also raise noise, raising offset on PMT (threshold) can cut off background noise but signal is equally affected, the SNR and image quality won't improve. In worst case, the structure details are buried in the noise, you even can't get usable data at all. To improve image quality, approaches which increase photon number has to be used, such as average, accumulation, slow scan speed, lower scan format, larger pinhole size, etc..
This page was last updated 23.03.2004 |