# Low-Light Photodetectors for Fluorescence Microscopy
## 1 Introduction  
The development of the light microscope has enabled investigation of the fine structures of biological specimens under magnification. In recent decades, fluorescence microscopy--a form of highly sensitive optical microscopy--has evolved and is a necessary element of life science studies [1; 2; 3]. Microscopy has elucidated biological processes in vitro and in vivo, and also enabled tracking of biomolecules in real time. Development of highly sensitive photodetectors and light sources, in addition to evolution of various illumination methods and fluorophores, has helped microscopy acquire single-molecule fluorescence sensitivity, enabling single-molecule fluorescence imaging and detection [4; 5; 6; 7]. Single-molecule fluorescence microscopy led to the emergence of super-resolution microscopy [8; 9; 10].  
Low-light photodetectors used in fluorescence microscopy are classified into two categories: point photodetectors and wide-field photodetectors [5; 11]. Point photodetectors, notably photomultiplier tubes (PMTs), are most commonly used in laser scanning microscopy (LSM). The detectors are also used in point-like excitation and detection to study freely diffusing biomolecules, such as protein molecules and nucleic acids (DNA and RNA) in solution [11]. Wide-field photodetectors, such as electron-multiplying charge-coupleddevices (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. The two photodetectors are distinct in many aspects. Point photodetectors have high temporal resolution (high sampling frequency) but have no spatial resolution without scanning. Wide-field photodetectors, in contrast, have spatial resolution (typically sub-micrometer precision) but are limited to relatively low frame rates. This review focuses on low-light point photodetectors and presents their fluorescence microscopy applications and recent progress.  
In fluorescence microscopy, the fluorescence emission can be characterized not only by intensity and position but also by lifetime [12]. Fluorescence microscopy uses two fluorescence detection methods: steady-state and time-resolved fluorescence detection. Time-resolved measurements contain more information than is available from steady-state measurements. Fluorescence lifetime measurement by time-resolved detection provides data that is independent of fluorophore concentration and allows us to obtain information on the local ambient environment around the fluorophore, such as pH, ion concentrations, temperature, and fluorescence resonance energy transfer (FRET) efficiency [13]. The laser scanning fluorescence microscope, an indispensable imaging device in the biological sciences, is one of the most widely used fluorescence microscopy. 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. These photodetectors include conventional PMTs, single photon avalanche diodes (SPADs), hybrid photodetectors (HPDs), in addition to newly emerging multi-pixel photon counters (MPPCs) (also known as silicon photomultipliers (SiPMs)) and superconducting nanowire single photon detectors (SSPDs). In particular, 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.