Quantitative fluorescence microscopy has exploited the principle that the excitation wavelengths of biological molecules is always lower than their emission wavelengths. This has enabled scientists to visualize biological structures and their functions. The addition of immunofluorescence techniques allowed the specificity of antibody-hapten bonds and naturally occurring ligands to be used to generate specific probes. New filter designs made it possible to simultaneously detect multiple fluorophores with even greater specificity. These, however, were only qualitative observations. The latest developments in engineered probes and microscopy methods such as radiometric imaging, fluorescence resonance energy transfer imaging, and fluorescence lifetime imaging have permitted quantitative measurements. This review examines the latest in quantification techniques and their applications.
- Emission wavelengths
- Excitation wavelengths