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

4. What is confocal?

As mentioned on previous section, a point light soure illumination within the specimen will improve image quality by eliminating stray lighe intererence. Laser is a good candidate for providing a point-like light soure. Unlike a mixture of wavelength, laser provides light at discreted band of wavelength and has very high intensity which are also advantages for fluorescence exitation. For this reason, laser is widely usd in confocal microscope.

In practical, a point-like light source is achieved by using a laser light passing through a illumination pinhole. This point-like light source is directed to the specimen by a beam splitter (or AOBS in Leica's BSP-free system) to form a point-like illumination in the specimen. The point-illumination move or scan on the specimen by the help of a scanner. The reflected emission light from specimen's focal plane passes through the detecting pinhole and form point-like image on detector PMT (photon multiply tube).

PMT converts detected photon into electron.
It is possible to amplify weak signal by manipulating the voltage (gain) on the tube. It is also possible to cut off background signal by set certain threshold (Offset) on the tube.

PMT has large active area to receive photons thus high saturate point, and PMT has low dark current thus low background,  together, which provide high dynamic range that is defined as the ratio of maximum allowed intensity / dark current. Besides, PMT has very high refresh rate since there is no charge accumulate on it. It detects event at nano- seconds level.

The electrons convert from photons form analogue current which is then digitized by analogue-digital converter (ADC) and send to the digital image processor.

Taking together, in confocal system:

  • A point light source for illumination

  • A point light focus within the specimen

  • A pinhole at the image detecting plane

These three points are optically conjugated together and aligned accurately to each other in the light path of image formation,  this is confocal.  Confocal effects result in supression of out-of-focal-plane light, supression of stray light in the final image

Confocal images have following features:

  • void of interference from lateral stray light: higher contrast.

  • void of supperimpose of out-of-focal-plane signal: less blur, sharper image.

  • images derived from optically sectioned slices (depth discrimination)

  • Improved resolution (theoretically) due to better wave-optical performance.

Is confocal effect a free cake?
No, confocal effect is obtained at a cost of greatly reduced detecting volume (total signal amount), increase vulnerability to noise, reduced dynamic range, etc.  For detailed discussion, refer section 6: Optical sectioning.

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This page was last updated 23.03.2004