Three-dimensional biological imaging by confocal and two-photon laser scanning microscopy

摘要

The commercial confocal laser scanning microscope (CLSM) has made 3D imaging with submicron resolution broadly available. CLSM combines focused illumination and spatially filtered detection to reject out-of-focus light, yielding images of thin optical sections within thick specimens. Images taken at different focal planes are combined to form a 3D reconstruction. Using this technique, fluorescent biological indicators are now quantified within volumes of approximately 0.1 $mu@m$+3$/. Live cell fluorescence imaging is limited by photodynamic properties of the fluorophores. Finite fluorophore excited state lifetimes limit imaging speed, and multiple spots in the specimen must be illuminated to increase temporal resolution. However, multiple illuminated spots decrease 3D image quality. The trade-off between imaging speed and quality is presented for several microscope designs. Fluorophore photobleaching limits the total signal available for 3D imaging, and photobleaching in out-of- focus planes limits the quality of 3D reconstructions. Femtosecond laser pulses focused to a diffraction limited spot can excite fluorophores by a 2-photon absorption that occurs only in the focal spot. Two-photon excitation limits photobleaching to the plane of focus and allows extended viewing of thick samples. Two-photon absorption can also locally release biologically active `caged' molecules and indicators.