Methods - Fluorescence Microscopy
Fluorescence microscopy enables the selective visualization of structures and even single molecules within a cell. Different methods are available for labeling the target structures:
(i) staining with fluorescent dyes, which have a high affinity to the target structure,
(ii) the use of fluorescence-labeled antibodies or molecules as probes, which specifically bind to the structure of interest, and
(iii) the fusion of the protein of interest with the green fluorescent protein (GFP) or any of the numerous available GFP derivatives (CFP, YFP, RFP etc.).
Taking advantage of physical effects, such as the Foerster resonance energy transfer (FRET), interactions can be determined between individual molecules by fluorescence microscopy.
Furthermore, targeted bleaching of defined areas within a specimen (for example, FRAP: Fluorescence Recovery After Photobleaching) allows the observation of the dynamic distribution of fluorescent structures within a cell (for example: vesicle transport, metabolic transport processes between cell compartments).
Using ratiometric fluorescence microscopy, changes in concentrations of individual ions (like Ca2+) or changes in the pH-value within the cell can be examined.
Compared to electron microscopy, the great advantage of fluorescence microscopy is the fact that studies can be performed with living cells without the need for fixation or dehydration of the specimen.
Fast image acquisition combined with high sensitivity makes conventional epifluorescence microscopy particularly suitable to study thin and very sensitive living specimens. However, the disadvantage of this method is that not only focused light but also light from above and below the focal plane will reach the detector. This effect is particularly prominent in thicker samples, leading to a marked decrease in the signal-to-noise ratio and to a significant decrease in image quality. This problem can be overcome using confocal laser scanning microscopy. With this technique, the specimen is illuminated with a focused laser beam scanning a defined area spot by spot and line by line while at the same time a special pinhole near the detector ensures that only light from the focal plane is detected. In this way, very focused images of different optical sections can be generated. Subsequently, the obtained images can be used for reconstruction of the three-dimensional structure of the investigated object.