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 3 operation, optimization of Leica SP2 LSCM

Laser intensity adjusting and AOTF control

There are two ways to control laser intensity: One is by laser's power supply and another is AOTF control.

Power control knob: By power supply control knob on the switchboard, you can set the laser power from minimum to maximum. Higher power gives higher excitation thus higher emission and vice versa. But higher power consume more laser life time and produce more laser noise. So, a good starting point is at 10 o'clock position (about 30% of its maximum), then try adjust other parameter such as AOTF and PMT to get enough intensity. Reserve this as the last sorts if all others fail to deliver the intensity you need.

AOTF refers Acousto-Optical Tunable Filter which is an adjustable quarts filter works at frequency of as high as sound, that is "Acousto-".  AOTF enable quick on and off the laser, change laser lines and their intensity percentage by setting it to 0% through 100%. The change is very quick and can be operated on-line even during the scanning process. AOTF works like a filter in the light path, making certain percentage available downstream. High or low percentage in AOTF control does not affect laser power from its source and does not change the laser noise level.
AOTF setting can be changed or programmed by the software. This is advantageous in automated process such as sequential scan, time lapse, multiple ROIs scan.

(In non-AOTF system, you have to stop the scan, change laser line in use, insert glass neutral density filter to attenuate the intensity, making automation impossible).