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 3 operation, optimization of Leica SP2 LSCM

Beam splitter (BSP)

As mentioned in Part 1, beam splitter is a crucial component in the light path between light source and specimen, between emission from specimen and detector, reflecting some bands and transmitting some others, affecting both excitation and emission.
Because of the two contradictory tasks placed on the single filter, in double or multiple labeling, the reflected wavelength bands for excitation means transmission barrier for emission, a phenomenon known as "emission hole". See Part 1, Filter set in Fluorescent Microscopy.

The choice of  BSP and cut-off value are very important and tricky. Inadequate choice of beam splitter will greatly influence the intensity and specificity of the final results.

For single labeling detecting, the choice is simple: a long pass shorter than emission but longer than excitation, or a band pass filter with cut-off value including the excitation but not emission works fine.

But for simultaneous multiple labeling detecting, a multi-band reflecting filter including all desired excitation bands has to be chosen. A filter in this category is marked for the bands it reflects. For example, a tri-chroic BSP 488/514/546 will reflect these three bands to specimen for excitation. But when emission is concerned, these three bands are also blocked thus lost in the return path. The barrier is usually 10-20 nm wide. Taking this BSP as example, spectra at 480-500, 504-524, 535-555 nm will never reach detector. If this filter is used for FITC, YFP and TRITC simultaneous excitation, FITC (em 497-575nm) and YFP (em 513-590 nm) will lose considerable amount of their emission. If this is intolerable in weak specimen, sequential scan is preferred and a single band BSP can be used each time to eliminate this loss.
As a general rule, if Multi-band BSP has to be used, choose as few bands as possible.

But the limited filters available for choosing is a problem. On a filter wheel, which usually holds 6 filters, if no suitable BSP is available, a neutral beam splitter 30/70 has to be chosen, which does not care about wavelength, simply reflects 30% light from light source to specimen and let 70% emission pass through. The result is compromised but better than nothing.

In confocal microscope, the newly invented non-beam-splitter based AOBS (acousto-optical beam splitter) from Leica solved this problem. See Special features from Leica SP2. (which is not available in this unit).

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This page was last updated 10.11.2009