Confocal laser scanning microscopy (CLSM)

Like the scanning electron microscope, a laser scan microscope scans microscopic objects point by point by a finely focussed laser beam. The light which is reflected or absorbed by the specimen is detected by photomultipliers and is used to modulate the light intensity of a display monitor which is synchronized to the laser scanning motion. The image of the specimen in the required contrasting method is then formed on a display monitor. The advantages of this optical scanning method are that less stray light is produced and that in addition to various optical contrasting methods brightness and contrast of the image can be varied electronically.

Using fluorescent filters or beam splitters in the photomultiplier path, fluorescence images can be obtained. Due to the high intensity in the laser spot, excitation of specimens with low fluorescence yield is possible and can be detected by the photomultiplier. The total energy applied to the specimen during a scan is much lower than conventional fluorescence microscopy. Furthermore, optical sections through the specimen can be reassembled to 3D-reconstructions.

Due to the high intensity of the laser beam and the fine resolution of the fluorescence image further possibilities arise; for excample, very small areas can be exicited or destructed, as realized in FRET (fluorescence resonance energy transfer), FRAP (fluorescence recovery after photobleaching) and FLIM (fluorescence lifetime imaging).

The confocal system allows the observation over longer periods of time in bright field and fluorescence mode, particularly important for sensitive specimen.

For confocal microscopy we have two CLSMs from Leica:

CLSM 1 is a TCS SP2 laser scanner combined with an inverted DM-IRE2 microscope. The system has three lasers, Ar/ArKr, Green-HeNe and Red-HeNe, and can be set up for microinjection/-manipulation and tissue culture containers.

CLSM 2 is a  TCS SP5 laser scanner combined with an upright microscope stand and equipped with a UV diode, an Argon laser and a white light laser. In addition to the normal scanner, an ultrafast resonant scanner allows for observation of high fluorescence dynamics.

In addition to conventional fluorescence and bright field, the microscopes have a full set of DIC and phase objectives; a strong mercury lamp allows for advanced contrast techniques.

> Chapter "Confocal laser scanning microscopy (CLSM)" in "Lichtmikroskopie online" (in German)

> Leica CLSM 1 and CLSM 2