Abstract
Over the years, fluorescence microscopy has evolved and has become a necessary element of life science studies. Microscopy has elucidated biological processes in live cells and organisms, and also enabled tracking of biomolecules in real time. Development of highly sensitive photodetectors and light sources, in addition to the evolution of various illumination methods and fluorophores, has helped microscopy acquire single-molecule fluorescence sensitivity, enabling single-molecule fluorescence imaging and detection. Low-light photodetectors used in microscopy are classified into two categories: point photodetectors and wide-field photodetectors. Although point photodetectors, notably photomultiplier tubes (PMTs), have been commonly used in laser scanning microscopy (LSM) with a confocal illumination setup, wide-field photodetectors, such as electron-multiplying charge-coupled devices (EMCCDs) and scientific complementary metal-oxide-semiconductor (sCMOS) cameras have been used in fluorescence imaging. This review focuses on the former low-light point photodetectors and presents their fluorescence microscopy applications and recent progress. These photodetectors include conventional PMTs, single photon avalanche diodes (SPADs), hybrid photodetectors (HPDs), in addition to newly emerging photodetectors, such as silicon photomultipliers (SiPMs) (also known as multi-pixel photon counters (MPPCs)) and superconducting nanowire single photon detectors (SSPDs). In particular, this review shows distinctive features of HPD and application of HPD to wide-field single-molecule fluorescence detection.
Highlights
The development of the light microscope has enabled investigation of the fine structures of biological specimens under magnification
Low-light photodetectors used in fluorescence microscopy are classified into two categories: point photodetectors and wide-field photodetectors [5,11]
This review focuses on low-light point photodetectors and presents their fluorescence microscopy applications and recent progress
Summary
The development of the light microscope has enabled investigation of the fine structures of biological specimens under magnification. Wide-field photodetectors, such as electron-multiplying charge-coupled devices (EMCCDs) and scientific complementary metal-oxide-semiconductor (sCMOS) cameras, are used in wide-field illumination and detection to study surface-immobilized or slowly-diffusing biomolecules and organelles These biomolecules and organelles include protein molecules, nucleic acids, and lipids, and nucleus and mitochondria, respectively. LSM with a confocal illumination setup provides a base for various fluorescence microscopes using steady-state and time-resolved fluorescence detection, such as two-photon microscopy and fluorescence lifetime imaging microscopy (FLIM). This review introduces these point detectors, describes their operating principles, and compares their specifications. This review shows distinctive features of HPD, and notes the applications of HPD to wide-field single-molecule fluorescence detection and the development of multi-pixel photodetectors
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