High Resolution Imaging Using Confocal and 2-photon Molecular Excitation Microscopy

Extract

Fluorescence microscopy has proved to be an invaluable tool for biomedical science since it is possible to visualise small quantities of labelled materials (such as intracellular ions and proteins) in both fixed and living cells. However, the conventional wide field fluorescence microscope suffers from the disadvantage that objects outside the focal plane also fluoresce (in response to the excitation light) and this leads to a marked loss of contrast for objects in the focal plane. This is especially a problem when the fluorescent probe is distributed throughout the thickness of the cell and the cell is thicker than about 1 μm. The confocal microscope overcomes this problem by illuminating the preparation with a point source of excitation light and limiting the collection of light with a pinhole that is confocal with the illumination source. This converts the microscope from an imaging system to a point detector and images are produced by scanning the illuminating and detecting point over the specimen to build an image (in much the same way that a television set produces an image). The basic idea behind the confocal system is shown in Figure 1, and it should be noted that light from points outside the focal plane is defocused at the pinhole and so does not pass through the pinhole efficiently.