Fluorescence refers to the ability of an atom or molecule to absorb light and emit at different wavelengths. Because slight variations in the surrounding environment can cause large changes in fluorescence properties, reporter molecules are used as sensitive probes. At the microscopic level, fluorophores are linked to cellular biochemistry. Illumination and detection at the proper wavelengths can therefore reveal variations in pH, ion concentration, nucleic acids, and proteins. Resultant images appear "bright" on a dark background.
Light levels resulting from fluorescent molecules are usually very low, but overexposure to illumination can cause sample photobleaching. Photometrics high-performance CCD cameras allow researchers to minimize photobleaching by providing extremely high sensitivity.
Whereas video microscopy can be useful in subjective analysis, higher precision and sensitivity are needed to produce quantitative results. Camera response linearity over a wide range of intensities is required when numerical algorithms (e.g., deconvolution, ratiometric analysis, morphometry) are applied to a single image or an image stack. Furthermore, higher-spatial-resolution cameras provide detailed information within a large field of view. Finally, the wide dynamic range of Photometrics CCD cameras allows dim objects (e.g., neuronal processes) and relatively bright objects (e.g., cell bodies) to be viewed and measured within the same image.
Image courtesy of Photometrics.
Above, bovine pulmonary artery endothelial cells (Molecular Probes Fluocells #2) are shown as a three-color composite image. Images were individually acquired in the spectral bands corresponding to the fluorophores BODIPY, Texas Red, and DAPI showing the distribution of tubuli, F-actin, and nuclei, respectively. Sequential acquisitions of this type allow separate image optimization and can therefore compensate for differences in probe concentration and camera sensitivity.