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 1 Principles of Confocal microscopy

2. Filter-set and its choice, limitation

A detailed internal structure of the filter-set cube containing above-mentioned filter 5, 8, 9, shown here as A, B, C and its light path is given below:

Arrow 1 represents the excitation light source of mixed wavelength.
Filter A
is the excitation filter,  which is usually a band pass or long pass filter, allows light at certain wavelength range or wavelength longer than cut-off value to go through. For a filter cube designed for simultaneous multiple fluorescence detecting, a long pass filter with cut-off value shorter than the shortest excitation has to be used. That leads more unwanted wavelength to specimen results in more nonspecific background fluorescence.

Arrow 2 represents excitation light which passes through A and reaches the surface of  Beam Splitter B,  BSP is a special filter which reflects certain wavelength away but permit other wavelength to pass. A multiple-bands reflecting filter or neutral percentage splitter can be used for this role. Wavelength falling into the reflecting band or shorter than the cut-off value will be stopped and reflected to the specimen. The fluorescence light emitted from specimen is illustrated as Arrow 3. The emission is longer than excitation wavelength and cut-off value of the BSP, so the returned emission can go through the BSP towards emission filter C.

Unfortunately, these two tasks placed on the single filter are contradictory. Reflecting certain wavelength to specimen for excitation means blocking it from transmission.
Beam splitter is not a magician, it can not distinguish which is excitation and which is emission light. It simply reflects certain band of wavelength towards specimen even if it is the emission coming back from specimen. In single fluorophore labeling, there is no problem. But in scenario of double or multiple labeling, if the shorter emission extends to the wavelength area of the next longer excitation, this part of emission will lost on the way back to detector, all the later fluorophores will suffer some loss. This is known as "emission hole". The more bands a BSP has, the more barrier zones it contains. So, don't use multi-band chroic unless necessary. For details on how to choose BSP, refer part 3, Operation and optimization: Beam Splitter.

Emission filter C. Emission filter is either a band pass filter or long pass filter, the cut-off value is longer than excitation wavelength so the residue of excitation light 2 is further prevented from reaching image plane. Strict band pass results more specific but weaker signal, while long pass leads stronger signal but more background.

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This page was last updated 23.03.2004