A fluorescence microscope is an optical microscope equipped to excite specific fluorophores and detect their emitted light, allowing visualization of labelled structures with high contrast.
Principles and instrumentation
A fluorescence microscope illuminates a specimen with intense light at selected wavelengths to excite fluorochromes and collects the emitted light that occurs when the fluorophores return to the ground state. The instrument typically uses a high-pressure mercury or xenon lamp, or modern LEDs, to produce broad-spectrum light. Excitation filters select narrow wavelength bands corresponding to the absorption spectra of the fluorophore. A dichroic mirror reflects the excitation light toward the objective lens and transmits the longer-wavelength emission light back to the eyepieces or camera. Emission filters isolate the fluorescence signal by blocking remaining excitation light. Because the light source and detection path are aligned through the same objective (epifluorescence geometry), only the labelled structures appear bright against a dark background. Advanced designs include confocal scanning systems that use pinholes to reject out-of-focus light, and total internal reflection configurations that restrict excitation to a thin layer near the coverslip. Super-resolution methods improve spatial resolution below the diffraction limit by controlling fluorophore activation and detection. Calibration, alignment of optical components and proper selection of fluorochromes are essential for quantitative measurements.
Applications and notable types
Fluorescence microscopy is used widely in cell biology and microbiology. Direct immunofluorescence assays detect viral antigens in patient samples, whereas indirect immunofluorescence identifies autoantibodies by using secondary antibodies with fluorescent tags. Fluorescence in situ hybridization uses fluorescently labelled DNA or RNA probes to visualize genetic sequences in chromosomes or identify bacteria in environmental samples. Researchers monitor the localization and dynamics of proteins fused to green fluorescent protein or other fluorescent tags in living cells. Epifluorescence microscopes are used to count bacteria in water samples using nucleic acid stains such as DAPI and SYBR Green. Confocal microscopes generate optical sections of thick specimens, producing three-dimensional reconstructions of tissues or biofilms. Multiphoton and total internal reflection microscopes enable imaging of live cells deep within tissues or near membranes. Despite their versatility, fluorophore photobleaching and phototoxicity require minimizing exposure times and using antifade reagents.
Fluorescence microscopes provide sensitive visualization of specific molecules by converting light interactions into images with high contrast. They are essential tools in modern biology, medicine and environmental monitoring. Proper filter sets, alignment and sample preparation are fundamental for reliable fluorescence imaging.
Related Terms: Flow cytometry, FACS, Confocal microscope, Immunofluorescence, Fluorophore