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

10. Special features from Leica SP2 confocal microscope

  • Emission-filter free spectral detecting system

Leica SP2 confocal microscope has two unique hardware innovations. The first one is the emission filter free spectral meter detecting system, introduced in 1999, depicted as below:

Like in picture on the left, the emission is first spread by prism into spectra, this spectra is further selected by a motor-driven slit which can change width or move position laterally as in picture on the right. With this device, the detecting wavelength, detecting band width can be continuously changed within 400 -750 nm to meet emission from any fluorophores without need of add new filter set into the system. a lambda scan within the whole range enable you to determine where is the emission peak of your stain and further optimizing of detecting position and range has more accurate basis.

  • Beamsplitter-free AOBS system

The  beam-splitting-filter free AOBS (acousto optical beam splitter) system is introduce in 2002.

Here the ordinary BSP is replaced by a optical crystal with an ultrasonic field applied. The wavelength and amplitude of the ultrasound can be changed (programmed) to deflect certain band of wavelength and its amount passing through the field (figure on the left). Up to eight excitation bands can be programmed at the same time compared to only three bands in tri-chroic BSP in ordinary filter based system. Besides, the band width of the deflected is very narrow or sharp, around 1 nm only (0.6 - 3 nm proportional to the wavelength concerned), compared to ordinary BSP which has about 10 to 20 nm width of barrier to emission know as "emission hole" (figure on the right).

In short, this function has two major benefits: 1. removal of the limitation on the number of beam splitter available in the system and more accurate control on the reflected bands.  2. Higher efficiency for emission collection.

These two approaches: AOBS and spectra detecting create totally filter-free confocal microscope which provides more versatility and higher efficiency in choosing excitation and collecting emission.

Home |  facilities |  services |  reservation |  confocal tutorial |  other tutorial |  personnel |  link | 
  Statement about this web and tutorial
For problems or questions regarding this web contact e-mail:

This page was last updated 23.03.2004