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

Emission collection and Spectral detector adjustment

Leica SP confocal system has an unique emission filter-free spectral detecting system as detailed in section 9 of part 1 of this tutorial.

In the software user interface, the spectral detecting control looks like below:

1. selected laser lines. 2. reference curve for emission spectra. 3. spectra scale.
4. list of reference curve. 5. spectra adjusting slider. 6. linkage to active PMTs.

Under the spectral scale, for each detecting channel, there is a slider for adjusting emission detecting position and range. Drag and move the slide to change its position, mouse-click on one edge of the slider, hold and drag to widen or narrow its width. Or double-click the slider to bring a dialogue, enter numerical value of starting and ending points for the detecting position and band width. This provides a versatile control on emission collection: virtually any position and range within visible spectra: 400 to 750 nm. The curve (2) here is not a setting of parameter but serves as a reference for slider adjusting. There are many curves in the list (4) to for you to choose from.

Practical tips for emission collecting slider adjusting:

• Set the range of collecting according to the reference curve, try to avoid cross-emission as much as possible.

•  position the slider at least 5-15 nm after the excitation laser line to avoid collecting the laser light  itself. The longer the laser wavelength, the farther the slider is away from the laser peak value. Ex. for blue lines 458-488:  5nm after; for green lines 514-568: 10 nm after; for red line 633: 15 nm after.

An interesting experiment result from me by using a mirror-slide on microscope stage, a neutral BSP 30/70 to collect laser directly shows the "diffusing" effect of different laser wavelength.

Laser source	peak position	range collected (end at 10% of peak value)
458 nm	456,5 nm	451-463 nm
476 nm	477,5 nm	471-483 nm
488 nm	488,2 nm	481-497 nm
514 nm	513 nm	506-522 nm
568 nm	568 nm	560-580 nm
633 nm	633,9 nm	623-648 nm

Spectral data of Emission peak in table 1 below can be used as reference numerical values for setting slider position and range.

Table 1. Fluorophore Spectral data and corresponding laser for Excitation (PDF)

Note:
 1. The percentage in table 1 should not be used as the setting for AOTF percentage (as more than one user asked). They speak totally different things. value in table 1 indicate the percent of fluorophore excited under one laser line. AOTF percentage means the percent of light from laser source delivered to the specimen. Maybe, the low value in table 1 implies you have to use high AOTF percentage and vice versa.
2. The range in parenthesis indicates the 20% boundary on both side, at that wavelength, the intensity drop to 20% of its peak value. Value with * means until there, it has not dropped below 20%, further data is not available.

Sequential scan:

In multiple fluorescence labeled specimen, some times, meeting above two criteria reduces signal intensity significantly. If that become intolerable, sequential scan has to be used to collect wider range, where you don't need worry about collecting into next fluorophore' excitation laser or overlapped emission field.

Sequential scan is a must-be choice if the detecting channels (number of PMTs) are fewer than fluorophores in your specimen.

Follow steps below to work with it:

1. Optimize individual detecting channels you are use, one by one, including parameters for:
       Laser line and its intensity by AOTF.
       Beam-splitter
       PMT and LUT for the channel
       Emission collecting range (it can be much wider then simultaneous scan)
       Number of average

2. Save the settings separately under user setting list

3. Check sequential scan check box, which brings a sequential settings list box for holding those saved parameters.

4. Drag all the saved settings from user setting to the list box, or use add button to do the same. Finally save this settings as new item, which can be loaded and re-used in the future.

5. In the sequential scan mode box, choose either between line, frame or stacks.

6. Click either single scan or series scan to start data acquisition.