Fluorescence microscopy: Established and emerging methods, experimental strategies, and applications in immunology

KEY WORDS fluorescence; microscopy; imaging ABSTRACT Cutting-edge biophysical technologies including total internal reflection fluores-cence microscopy, single molecule fluorescence, single channel opening events, fluorescence reso-nance energy transfer, high-speed exposures, two-photon imaging, fluorescence lifetime imaging, and other tools are becoming increasingly important in immunology as they link molecular events to cellular physiology, a key goal of modern immunology. The primary concern in all forms of mi-croscopy is the generation of contrast; for fluorescence microscopy contrast can be thought of as the difference in intensity between the cell and background, the signal-to-noise ratio. High informa-tion-content images can be formed by enhancing the signal, suppressing the noise, or both. As improved tools, such as ICCD and EMCCD cameras, become available for fluorescence imaging in molecular and cellular immunology, it is important to optimize other aspects of the imaging system. Numerous practical strategies to enhance fluorescence microscopy experiments are reviewed. The use of instrumentation such as light traps, cameras, objectives, improved fluorescent labels, and image filtration routines applicable to low light level experiments are discussed. New methodolo-gies providing resolution well beyond that given by the Rayleigh criterion are outlined. Ongoing and future developments in fluorescence microscopy instrumentation and technique are reviewed. This review is intended to address situations where the signal is weak, which is important for emerging techniques stressing super-resolution or live cell dynamics, but is less important for con-ventional applications such as indirect immunofluorescence. This review provides a broad integra-tive discussion of fluorescence microscopy with selected applications in immunology. Microsc. Res