Part 1 Principles
1. Fluorescence microscope
2. Filterset in FL-Mic
3. How concocal differs?
4
. 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
      microscope

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
6.
Fluorescence detecting
       General consideration
      
Multi-channel detecting
       Background  correction
       Cross-talk correction
            Cross excitation
            Cross emission
            Unwanted FRET


Part 3 Operation and
             Optimization

 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?
FAQ
Are you abusing confocal?

Confocal Microscopy tutorial

Part 3 operation, optimization of Leica SP2 LSCM

Pinhole, resolution and optical section thickness

As emphasized in Part 1 section 5, pinhole is the main player of confocal effects. Through its out-of-focal-plane signal rejection, optical sectioning is possible. When pinhole is indefinite small or in practical, smaller than 0.25 AU;  the thickness of optical section is not influenced by pinhole size any more but solely decided by axial resolution of objective lens in use, reach the thinnest level: ,

the lateral resolution also reaches smallest to , about 1.4 times over conventional optical system.

But when pinhole is smaller than 0.25 AU, because of the additional diffraction and greatly reduced signal intensity and the deteriorated SNR, all these resolution gain are lost in the noisy image. Pinhole size between 0.25 and 1 AU is the usual working range. At this range, for lateral resolution, coefficient 0.37 has to be substituted by value between 0.37 and 0.51 depending on the actual pinhole size in use. The optical section is  thicker than the z-resolution of the objective and is determined by the actual pinhole size in use.
So, in practical, pinhole size is mainly used to control optical section thickness other than to achieve highest lateral or Z-resolution.
The calculation of optical section thickness is not convenience by using this formula because the physical size of pinhole in use has to be known or deduced from the AU in use. Generally speaking, the z-resolution is about 2 times of the lateral resolution. Ref table 2 for resolution of objectives equipped in this microscope.

In Leica LCS software, under Hardware legend, an entry "Voxel-size" can be seen.  The last value represent the sampled section thickness. But this value is simply the quotient of the z-dimension of the image (derived from the starting and ending position of the Z-series) divided by the number of sections you have set. It is not the actual optical section thickness.
In the following example, that is: 12.7m / (40-1)=325 nm.

If the value you get here is too small (less than half of the z-resolution of the objective lens you use), the section number you set is unnecessarily too much.

Occasionally, pinhole size can be used to adjust amount of photon received by PMT to change the signal intensity and increase SNR. In addition to the "optimal" 1 AU, Pinhole  1-3 AU is the range of choice. Bigger pinhole give you stronger signal but with the compromised confocal effects.

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This page was last updated 23.03.2004