CONFOCAL LASER SCANNING MICROSCOPES (CLSM)

Confocal microscopy, also known as confocal laser scanning microscopy (CLSM) or laser scanning confocal microscopy (LSCM), uses the principle of fluorescence excitation to investigate the structural properties of cells and the location of particular structures or proteins within those cells in fixed tissue. CLSM is also used to investigate the movement of biological entities in live cells, be they vesicles or even individual proteins.

In confocal microscopes, the light source is generally one or more lasers. Unlike in traditional wide-field fluorescence microscopy, the excitation light bandwidth is much narrower (2-3 nm rather than 20-30 nm) since it is determined by the source, not the excitation filter. The laser beam is a narrow beam of light and so, in order to illuminate the whole visual field, it has to be rapidly scanned across the area in a series of lines. The fluorescence detected at each point is measured in a detector (PMT) and an image built up. This method has enormous advantages over wide-field imaging in that it is possible to illuminate selected regions of the visual field allowing complex photobleaching protocols to be carried out to investigate the rates of lateral travel of fluorophores and for the excitation of different fluorophores in different regions of the same cell.

The major difference between fluorescence microscopy and CLSM is the pinhole. The pinhole is a device that is placed in front of the PMT to block the passage of out-of-focus light into it. Consequently, the only light to enter the PMT, and thus detected, comes from near the focal plane of the objective lens of the microscope. As this is taken across the area of the sample, it produces an image that is a slice through the object and surrounding material. This is known as optical slicing and allows the observer to see inside the object of interest. This gives a clearer 3D image, with enhanced contrast (i.e. lower signal to noise ratio) and more fine detail observable. In addition to this, CLSM has the ability to control depth of field and collect serial optical sections from thick specimens.