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

Scan speed

Scan speed is specified as 200, 400, 800 and 1000 Hz in Sp2 confocal microscope. It refers to the frequency of  two galvanometers which drive scanning mirror shading laser light across specimen.

In practical, the value tells how many lines per second are scanned on the specimen. This value determines the time resolution of a confocal system and higher Hz means higher time resolution. For example, at 400 Hz, scan a 512 line image needs 1.28 seconds, scan a 4096 line image needs 10.24 seconds. !000 Hz is 2.5 times faster than 400 Hz.

But again, high speed is not a free cake. It reduces the pixel sampling time considerably. Lets take a simple math: at 200 Hz, one line has 5 ms to use, for a 512 pixel sampling along a line, (refer Scan format), one pixel has 9.7 s to spend. At 1000 Hz, it is 1 ms per line, and 1.9 s per pixel. This means the photoelectron collected and image intensity is 5 times less, the SNR and image quality deteriorate dramatically according to formula 4. Image intensity can be compensated by raise gain on PMT, but SNR and image quality can not be improved in this way as discussed in SNR section. Some other way have to be used to increase photon input, such as: stronger laser illumination, this leads more bleaching; bigger pinhole size, this leads to loss of z-resolution, or to increase pixel time by more average and accumulation. But these approaches use more time and cancel the time advantage of high scan frequency.

Usually, 400 Hz is good starting point for 512 sampling rate. If you choose a higher scan format, even with average, the SNR is still not satisfactory, you might need to reduce scan speed to improve SNR. Compare to average, slow speed leads more bleaching. So average is the first choice for noise reduction.
When time is more important than image quality, you have to use higher scan speed. This is not the case for fixed specimen which you have plenty of time to use.

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This page was last updated 23.03.2004