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 2 application of confocal microscopy

4. Thick specimen

As emphasized in previous section, one forte of confocal microscopy is its power to resolve thick specimen thanks to its depth discrimination property. It can be used for tissue block, small organ, embryo, etc.

The following two images are taken from the same field of the plant of 60 Ám thick by different means. The first one is taken by CCD camera in the wild field microscope configuration. the second one is taken by confocal microscopy with pinhole size at one Airy unit. The difference on the results is striking.

   

It is worth to mention that the difference is not just the sharpness or crisp of the images, but also the  ability of resolving the internal structures. Many fine structures clearly visible in confocal image are obscured or totally invisible in the image taken under wild field microscope.

For even thicker specimen, no satisfied image can be obtained at all.

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This page was last updated 23.03.2004