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 2 application of confocal microscopy

6. fluorescence detecting in confocal microscopy

    general consideration

Before digital camera is widely available to many labs, confocal microscopy is the only convenience way to get co-localization data from multi fluorescence labeled specimen. Simple co-localization study was the major application of confocal microscopy in the middle and late 1990s.

Since high resolution and high sensitivity digital camera become affordable for many labs, it is not necessary to use confocal for simple co-localization study any more. A digital camera can do equally good job or even better job in case of weak signal. As discussed in part 1, section 6 of this tutorial, the superior suppression of out-focal-plane signal in confocal microscopy is achieved by discard lot of emission signal from specimen. If you have a generally weak signal that can not afford this luxury discard, or if you have a low SNR that can not meet the high SNR demanding from confocal microscopy, confocal will frustrate you. The efforts for "getting a good picture" by confocal from a weak and noisy staining will certainly fail.

If your task is a simple co-localization study, spatial distribution of the labeling or tomography of the cell structure has no impact on your study, if you do not need a z-series for 3-D reconstruction, if you do not have a thick specimen that is difficult to be resolved on conventional microscope, you really don't need confocal microscopy and be bothered by lot of parameter settings and the lengthy image optimization process, bleach your valuable fluorescent signal away before you get a satisfactory image from it. The final results equal to or even worse than what you can easily get from a digital camera do not reward you for the efforts you have paid and the cost of consuming laser.

The folllowing situation in simple fluoresscence detecting need confocal microscopy:

• Multi-channel detection that must be performed simultaneously

In digital camera based fluorescece microscopy, multi-labeling is handled sequentially by change filterset in turn and take separate shot then display them in different channel with or without an overlay.  Time delay in several seconds between channels is inevitable. If the time delay is intolerable in your application, confocal is your only choice. In confocal system with maximum number of PMTs installed, up to six detecting channels can operate simultaneously without any time delay.

• Severe Background suppression

Background fluorescence is sometimes the headache of fluorescence microscopy. It  makes picture looking bad,  buries weak signal in background and reduce general contrast thus reduce obtainable resolution. This problem is more severe in tissue section and thick specimen than in cultured cell specimen. In digital camera based system, the common way to overcome it is simply lessen the excitation intensity or reduce the exposure time.
In confocal system, most of the out-of-focal-plane signal is restricted by pinhole resulting a cleaner background and detected background can be further manipulated by play with parameter on PMTs, i.e. reducing gain and raising offset added on the PMTs, or averaging signal on the detected frames by digital image processing algorithm. It must be pointed out, the assumption here is you have a general strong signal that can survive the discard of most of the original signal due to the confocal effects.

• Cross-talking correction

Cross-talking refers excitation spectra overlaps, emission spectra overlaps, excitation and emission spectra overlaps in multi-labeled specimen. It is a common problem for multi-labeled specimen. When a mixture of fluorophores exists and more than one excitation light apply to the mixture, there is a complex of interactions and it can be divided into three categories:

1. Interaction between different excitation light and fluorophores: cross excitation

2. Interaction between two fluorophores' emission: cross emission

3. Interaction between emission from one fluorophore and another fluorophore: FRET

They will be discussed in following sections.

• Far red emission which can not be detected by your eyes and CCD.

Recently, some new fluorophores appear in the market emitting at longer than 650 nm which is invisible to most of the eyes of human beings and many common CCD chips. These dyes, however, greatly expand the range of choice and possibility of three, or four fluorophores labeling. In this case, confocal can do a decent job for you to detect emission from 650 to 750 nm.