subject: Confocal Microscopy: Ground Breaking Technology [print this page] Confocal Laser Scanning Microscopy or CLSM is one of a series of methods to generate slices from microscopic samples by means of optics. The sample stays intact, and the optical slicing may be repeated many times. The benefit of CLSMis a dramatically increased contrast by removal of out-of-focus haze. Z-sequences of optical slices are sources for subsequent rendering as anaglyphes, depth-coded maps or 3D movies. True Confocal Scaning is also very well compatible with multi-fluorescence imaging, time-lapse imaging, FLIM, FRAP and FCS measurements - plus a whole world of spectral applications.
To perform biomedical applications using confocal microscopes, there is a need for the right kind of light source. This source should have sufficient power, tunable color for simultaneous excitation of a series of samples. Such a source has been invented: the white light laser.
Classical laser sources used for confocal laser scanning microscopy only provides one single narrow emission. Some gas lasers can emit a couple of lines simultaneously. The best known example is the argon gas laser, which may be provide upto 5 lines in the blue-green range. There are solid state lasers that are tunable, but only for the IR range. Beside their high complexity they too can emit only a single wavelength at a time and the tuning process is very slow (many seconds).
White light laser offers visibility for multiple fluorescent stains and optimal excitation. Plus, there is no spectral limitation thanks to synergies with tunable beam splitter and tunable emission detection. The former offers multicolor imaging with maximum spectral flexibility, along with the use and the possibility to use novel dyes. SP or emission detectorsuse secondary dichroic mirrorsfor directing the emission from fluorochromesgiving out different colors to the set of sensors.
The sensors for single point scanning systems such as true confocal laser microscopes are usually photomultiplier tubes. A new development has led to chimeric devices called hybrid detectors or HyDs. These combine the benefits of two technologies for scanning, which are vacuum technology and semiconductor technology, while overcoming their limitations. These detectors use a photocathode to convert the photons into accelerated electrons, as in a photomultiplier tube.
This is backed with resonant scanning systems that allow line frequencies up to 16 kHz, which offers higher scan speed to improve the time resolution. Additionally, with the use of these scanners fluorescence is brighter and the fluorochromes are less photo-stressed, which acts as an extra benefit.
