Cytometry Lab

Confocal Laser Scanning Microscopy (CLSM)

Confocal Laser Scanning Microscopy (CLSM) is a fluorescence based imaging technique that provides greater resolution than standard fluorescence microscopy due to its point illumination and detection properties. The point detection is achieved by the addition of a pinhole in front of the fluorescence detector. Point illumination is achieved using lasers rather the fluorescent lamps as a light source.

Using this technique, it is possible to provide thin x-y sections along the z-axis, which are devoid of out of focus light (optical sectioning). Multiple x-y sections can then be projected to provide an extended focus image.It is also possible to fix the position scanning in the y-direction and allow scanning only in the x-z direction to achieve x-z images.

Furthermore, by scanning only in the x direction over a period of time, it is possible to achieve an x-t scan. This technique of rapid scanning over time is useful in very quick physiological measurements such as [Ca2+] fluxes in twitching muscle cells.

Simplified light path of CLSM. This figure also illustrates how the confocal iris is able to prevent out-of-focus information from arriving at the detector.

 

Advantages of CLSM

1. Clearer images

Due to its point illumination/detection properties, out of focus light is excluded from reaching the detector. Because of this, only in-focus light is detected, hence in focus images are collected.

 

2. Increased sensitivity

In confocal microscopic systems fluorescence from the sample is detected using highly sensitive photomultiplier tubes (PMTs). This is advantageous in that samples of low intensity can be imaged. Furthermore, for samples that are sensitive to photobleaching, lower excitation intensity can be used hence allowing for longer imaging times.

 

3. Reduced photobleaching

Because of the use of point illumination light sources (lasers) to excite the sample, only small areas of the samples are photobleached.

 

4. Three-dimensional reconstruction

Due to its ability to perform optical sectioning, thicker samples can be imaged and reconstructed in an in-focus three-dimensional manner using image analysis software. These images can be displayed as rotating movies as well as stereo-images.

 

5. More accurate quantitation

The low incidence of out of focus light in the digital images generated by CLSM enables more accurate quantitation of images. Quantitating relative fluorescence levels within an image can be achieved using commercial or freeware image analysis software.

 

6. Multiple simultaneous analyses

Multiple fluorescence detectors allow simultaneous analysis of multiple cellular properties and/or markers. The range of useful fluorochromes is further expanded in instruments utilizing multiple lasers. For further information on choosing compatible fluorochrome sets, see "Fluorescence in Cytometry".

 

Limitations of CLSM

1. Slow scan rates

Most confocal microscopes rely on point illumination light sources to excite the samples. For these systems, maximum scan rates of up to 20 frames per second (at lowest resolution; about 7 fps for good resolution images) can be achieved. These systems are not suitable for very rapid physiological events. To overcome this problem one can use a line scan (x-t scan), although the data generated will not be of an x-y (two-dimensional) image.

 

2. Expense

Confocal microscopes are more expensive than conventional fluorescence microscopes due to the advanced electronics, laser excitation sources and tailor-made software required for running the hardware.

 

Important points in basic microscopy that applies to confocal microscopy.

1. Choice of mounting media, coverslips, immersion media and lenses.

This is probably one of the most important aspects of confocal microscopy.

 

2. Sample preparation

An image is as good as the sample. Another very important aspect of all microscopy is the condition of the sample. There is no standard procedure for sample preparation. This relies entirely on the type of sample, as to what kind of fixative is required.

 

3. Choice of microscope Ð upright or inverted

This is dependent heavily on the type of experiments to be performed. Upright microscopes are suitable for fixed samples. Water immersible lenses enable the use of upright microscopes for imaging live samples. Inverted microscopes, although awkward to use for the novice microscopist, are more suited for physiological experiments with living cells and tissues. With less obstruction from the top of the sample, it is possible to set up microinjection, patch-clamping, as well as environmental control chambers.

The WEHI Confocal System has an inverted microscope.