Fluorescence CM (FCM) is a version of CM in which the inspected specimen is doped with a high-quantum-yield fluorescent dye that strongly absorbs at the wavelength of the exciting laser beam. Excited dye molecules fluoresce at somewhat longer wavelength. The difference between the fluorescence and absorption wavelengths is called the Stokes shift. If the Stokes shift is sufficiently large, the exciting and fluorescence signals can be efficiently separated by filters so that only the fluorescence light would reach the detector. If the specimen is heterogeneous, the concentration of the fluorescent probe is coordinate-dependent, which results in a high-contrast image. The FCM technique makes it possible to visualize features in living cells and tissues; it is successfully applied in flow cytometry and even for single molecule detection.
The illuminated voxel is a diffraction limited spot within the specimen produced by a focused laser beam. Fluorescence light passes through the pinhole aperture located in the focal plane that is conjugate to the illuminated point of specimen. The signal that reaches the detector from the regions above and below the voxel are of much weaker intensity since the corresponding beams diverge and cover an area much larger than the area of the pinhole. Two basic features, namely, (a) illumination of a single voxel at a time, and (b) blocking out-of-the-voxel fluorescence signal, improve the resolution of FCM as compared to an ordinary microscope.
In essence, the traditional FCM technique visualizes the concentration (density) distribution of fluorescence probe in the sample.