Part 1 Principles
1. Fluorescence microscope
2. Filterset in FL-Mic
3. How concocal differs?
4
. What is confocal?
5. Resolution in confocal
6. Optical sectioning
7. Confocal image formation
    and time resolution
8. SNR in confocal
9. Variations of confocal
      microscope

10. Special features from
     Leica sp2 confocal

Part 2 Application
1. Introduction
2. Tomographic view
    (Microscopical CT)

3. Three-D reconstruction
4. Thick specimen
5. Physiological study
6.
Fluorescence detecting
       General consideration
      
Multi-channel detecting
       Background  correction
       Cross-talk correction
            Cross excitation
            Cross emission
            Unwanted FRET


Part 3 Operation and
             Optimization

 1. Getting started
 2. Settings in detail
 
     Laser line selection
      Laser intensity and 
         AOTF control

      Beam splitter
      PMT gain and offset 
      Scan speed
      Scan format, Zoom
        and Resolution

     Frame average, and
         Frame accumulation
     Pinhole and Z-resolution
     Emission collecting rang
        and Sequential scan


When Do you need confocal?
FAQ
Are you abusing confocal?

Confocal Microscopy tutorial

Part 1 Principles of Confocal microscopy

8. SNR (Signal to Noise Ratio) in confocal microscopy

As 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/s 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.
It is determined by formula:
30 photon influx corresponds to only 5 bits grey level.

Similarly, the SNR is affected more by signal intensity (total photon influx) than by background noise level as mentioned in part 1 Optical section,
 Formula 4 describes different factors in SNR.

Where
N:  photoelectron number per pixel time.
S:  secondary noise from random variation of multiplication.
d:  dark current of PMT.
q:  laser noise.

When N is 1000 photoelectron per pixel time, if set S: 1.2, d: 100. q: 0.05, SNR is 25.
At 400 Hz scan speed, 512 format, pixel time is about 5 s and N is about 150 assuming photon influx is 30 photons/pixel/s, from formula above,  with S, D, Q unchanged, SNR is 7.89 only.

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..

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This page was last updated 23.03.2004