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 Microsopy tutorial

Part 3 operation, optimization of Leica SP2 LSCM

Are you abusing confocal microscope?

There are typically two kinds of abusing: Wrongly choosing critical parameters for confocal data acquisition and wrongly choosing of application.

As discussed earlier, wrongly setup of laser intensity, PMT gain and offset and emission signal collecting range of the PMT can greatly influence the specificity, intensity of the final result. In the worst case, they even lead to false positive, false co-localization, or false negative results. See cross-talk correction.

To make right setting for these parameters, whenever possible, prepare single labeled positive and negative control for each fluorophore you are using, stain control slides together with experiment slides to ensure they have same staining condition and similar intensity. Then use control slide to set proper PMT gain, offset and collecting range.

Another type of abusing confocal is the wrongly choosing of application. As mentioned in Part 2. Introduction:  and fluorescence detecting, using confocal microscopy for a simple co-localization study where the spatial localization or tomography information of the antigen has little value in the results, and using confocal microscope to get "good pictures" from weak, noisy signal are two common wrongly use of confocal microscopy. Confocal microscopy is expensive in terms of either the cost incurred, the time you unnecessarily spend, or the signal bleached on your specimen.

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This page was last updated 23.03.2004