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 2 application of confocal microscopy

6. fluorescence detecting in confocal microscopy

    Cross-talk correction 1: cross-excitation

Cross-talk is a common problem for multiple fluorophore labeling experiment. In a specimen labeled by more than one fluorophores, the interaction between their excitation and emission is complicated. They can be divided into three categories:

1. Cross-excitation: a fluorophore is not just excited by wavelength at its peak value, but also by wavelength at certain range around the peak, which can extends into the area used by other fluorophores. For example

FITC has an excitation peak (100% of relative intensity) at 490 nm, it has range of excitation between 470-540 and is still excited 20% at 546 nm which is the peak value used by TRITC.

General speaking, cross excitation is not harmful in terms of specificity of the emission. Anyway, It does not matter very much by which excitation wavelength the fluorophore is excited, it is still emitted specifically from the fluorophore itself. Since excitation has additive effect, i.e. excitation from different wavelength will be added together, for emission coming from excitation wavelength other than its peak wave, it is just an extra butter on the cake. Although harmless, cross excitation make situation more complicated when dealing with cross emission correction. That means, when setting up PMT's gain and offset level to remove cross emission for a single channel, all lasers used in simultaneous excitation should be taken into consideration.

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This page was last updated 23.03.2004