Part 1 Principles
1. Fluorescence microscope
2. Filterset in FL-Mic
3. How concocal differs?
. 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

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
Fluorescence detecting
       General consideration
Multi-channel detecting
       Background  correction
       Cross-talk correction
            Cross excitation
            Cross emission
            Unwanted FRET

Part 3 Operation and

 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?
Are you abusing confocal?

Confocal Microscopy tutorial

Part 3 operation, optimization of Leica SP2 LSCM

PMT gain and offset adjusting

Gain (voltage) on PMT has a 0 to 1000 V adjustable range. Offset of PMT has -100 to 100 range for tuning, lower value has higher cut-off and higher value has lower cut-off.
Combination of gain and offset adjustment on PMT produces a preliminary optimized image with balanced intensity and background noise from which further operation can be done.
The default value for Gain and Offset after LCS launch is zero. They must be changed from default. Raise Gain from zero while lower offset to control background. Otherwise, most probably, you get a black screen.
During adjusting, pay attention to the hardware legend for gain and offset reading. It is better to maintain Gain at the range of above 600 V by lowering offset value a little bit (usually within negative range). Higher PMT voltage give less secondary emission noise (shot noise), but too high PMT voltage gives out more dark current thus more background, while too high offset cut away too much signal and reduce dynamic range of the image.

Since these two parameters counteract each other, it is important to find an appropriate balance point. You might get similar image intensity at a combination of 700 V gain and -20 offset, or 1000 V gain and -100 offset, but obviously, the former has better image quality while the latter suffers from high noise due to high voltage and low dynamic due to high cut-off. Similarly, +100 offset combined with 200 V gain is not good either since you have more shot noise due to low voltage while too much background due to low threshold. So, avoid using this pair at their extreme value.

While turning the corresponding knobs on the control box to adjust gain and offset, keep your ear alert: When you hear a beep sound, it is a warning that you reach one of its end. You can get better result by drawing both gain and offset back from extreme to middle range to find a new balance point. It is not advisable that you push one parameter very hard to the end while draw it back by using another parameter at its end too, and get bad side effects from both.

Keep in mind: Raising gain (voltage) on PMT can amplify weak signal but also amplify noise; raising offset on PMT (threshold) can cut off background noise but signal is equally affected,  the SNR and image quality won't improve. To improve image quality, approaches which increase photon number has to be used, such as average, accumulation, slow scan speed, lower scan format for bigger pixel size and longer pixel time, larger pinhole size, etc..

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This page was last updated 23.03.2004