Science, Technology and Engineering

Facilities - Microscope Imaging & Analysis Facility 

Department of Biochemistry

Confocal Microscopy and Fluorescence Photobleaching

The essential feature of the confocal laser scanning light microscope is that it allows optical sectioning of living, functioning tissue, (i.e., it has the ability to make a selective image of just one plane inside the specimen). The principle of confocal microscopy involves focal illumination of a single point in the specimen, which is imaged on a detector pinhole, thus suppressing out-of-focus contributions. Scanning the preparation with a laser beam in the x, y or z directions produces an image which is only related to the in-focus specimen plane.

The image created from the modulated detected intensity has a higher lateral resolution than the corresponding image obtained by conventional light microscopy. The images can be readily stored in a computer system, can be compared with other images obtained simultaneously at different wavelengths or by conventional microscopy, and can be analysed with sophisticated software. With 4th generation confocal scanning laser microscopes coupled to powerful computers, it is now possible to view a specimen from all possible spatial positions and combine structural data with quantitative physiological data to obtain a complex representation of the structure-function relationships in a living, functioning biological specimen.

A major advance in our understanding of the molecular dynamics of cellular systems has derived from recent elegant studies in which the gene encoding the green fluorescent protein (GFP) has been appended to a gene encoding a protein of interest and transfected into a living cell. The ability to monitor the fluorescence of the GFP-tagged protein without fixation or addition of exogenous probes has allowed us to observe the complex organization and reorganisation of cellular components in real-time throughout the life cycle of the cell.

The technique of fluorescence recovery after photobleaching (FRAP) has recently undergone something of a renaissance in studies of the molecular motions of GFP-tagged proteins in cellular system. Confocal microscope-based photobleaching studies allow an additional refinement in that they offer superb resolution and allow the selective capture of light from within a particular plane within the specimen, thus introducing the possibility of studying the dynamics of proteins within intracellular compartments. Further information on some of the biophysical aspects of photobleaching studies of GFP-labelled proteins and details of some of the recent applications of this technique to the study of the molecular motions of cellular components can be obtained from Dr Nick Klonins.