Low-Light Photodetectors for Fluorescence Microscopy

applied sciences

Review Low-Light Photodetectors for Fluorescence Microscopy

Hiroaki Yokota 1,* , Atsuhito Fukasawa 2, Minako Hirano 1 and Toru Ide 3

1 Biophotonics Laboratory, The Graduate School for the Creation of New Photonics Industries, 1955-1, Kurematsu-cho, Nishi-ku, Hamamatsu-shi, Shizuoka 431-1202, Japan; [email protected] 2 Electron Tube Division, Hamamatsu Photonics K.K., 314-5 Shimokanzo, Iwata-shi, Shizuoka 438-0193, Japan; [email protected] 3 Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University, 3-1-1 Tsushima-naka, Kita-ku, Okayama-shi, Okayama 700-8530, Japan; [email protected] * Correspondence: [email protected]

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 (sC- MOS) 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 Citation: Yokota, H.; Fukasawa, A.; photodetectors (HPDs), in addition to newly emerging photodetectors, such as silicon photomultipli- Hirano, M.; Ide, T. Low-Light ers (SiPMs) (also known as multi-pixel photon counters (MPPCs)) and superconducting nanowire Photodetectors for Fluorescence single photon detectors (SSPDs). In particular, this review shows distinctive features of HPD and Microscopy. Appl. Sci. 2021, 11, 2773. application of HPD to wide-field single-molecule fluorescence detection. https://doi.org/10.3390/ app11062773 Keywords: low-light photodetectors; fluorescence microscopy; time-resolved fluorescence microscopy;

Academic Editor: Jacek Wojtas hybrid photodetector (HPD); single-molecule ﬂuorescence detection

Received: 19 February 2021 Accepted: 17 March 2021 Published: 19 March 2021 1. Introduction The development of the light microscope has enabled investigation of the fine struc- Publisher’s Note: MDPI stays neutral tures of biological specimens under magnification. In recent decades, fluorescence micros- with regard to jurisdictional claims in copy—a form of highly sensitive optical microscopy—has evolved and is a necessary ele- published maps and institutional affil- ment of life science studies [1–3]. Microscopy has elucidated biological processes in vitro iations. 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 [4–7]. Single-molecule Copyright: © 2021 by the authors. fluorescence microscopy led to the emergence of super-resolution microscopy [8–10]. Licensee MDPI, Basel, Switzerland. Low-light photodetectors used in fluorescence microscopy are classified into two cate- This article is an open access article gories: point photodetectors and wide-field photodetectors [5,11]. Point photodetectors, distributed under the terms and notably photomultiplier tubes (PMTs), are most commonly used in laser scanning mi- conditions of the Creative Commons croscopy (LSM). The detectors are also used in point-like excitation and detection to study Attribution (CC BY) license (https:// freely diffusing biomolecules, such as protein molecules and nucleic acids (DNA and RNA) creativecommons.org/licenses/by/ in solution [11]. Wide-field photodetectors, such as electron-multiplying charge-coupled 4.0/).

Appl. Sci. 2021, 11, 2773. https://doi.org/10.3390/app11062773 https://www.mdpi.com/journal/applsci Appl. Sci. 2021, 11, 2773 2 of 20

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. 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, de- scribes 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.

2. Fluorescence Microscopy Fluorescence measurements are characterized by their high sensitivity, up to single- molecule detection. Because biological samples commonly exhibit low contrast, fluorescence microscopy makes good use of fluorescence phenomenon to enhance the contrast. Fluorescence microscopy acquires data of target biological samples through fluorescence emissions characterized not only by intensity and position, but also by lifetime. Fluores- cence microscopy uses the two fluorescence detection methods: steady-state and time- resolved fluorescence detection. 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 FLIM.

2.1. Fluorescence Fluorescence is a photophysical phenomenon of the emission of light through the excitation of a fluorophore from the ground state to an excited electronic state upon the absorption of light, with the light energy equivalent to the energy gap between the two states [10,12]. Figure1 shows a Jablonski diagram that illustrates the electronic energy levels of a fluorophore and the transitions between them are represented by arrows. S0,S1, and S2 represent the singlet ground, and first and second excited electronic states, respectively. The vibrational ground states and higher vibrational states of each electronic state are illustrated with black and gray lines, respectively. The transition from the ground state to the excited state by the light absorption occurs in less than 10−15 s. A fluorophore is usually excited to some higher vibrational level of the excited state. The electron usually rapidly relaxes to the lowest vibrational level of S1. This process is called internal conversion and Appl. Sci. 2021, 11, x FOR PEER REVIEW 3 of 20

Appl. Sci. 2021, 11, 2773 3 of 20 usually excited to some higher vibrational level of the excited state. The electron usually rapidly relaxes to the lowest vibrational level of S1. This process is called internal conver- generallysion and generally occurs within occurs 10 within–12 s. Then, 10–12 s. the Then, excited the stateexcited isrelaxed state is torelaxed the ground to the stateground in astate few in nanoseconds a few nanoseconds (10–9 s), (10 which–9 s), which is accompanied is accompanied by the by radiation the radiation of the of fluorescence the fluores- emission.cence emission. Because Because internal internal conversion conversion is generally is generally complete complete prior prior to emission, to emission, thelast the relaxationlast relaxation step step to the to the ground ground state state accounts accoun forts for most most of of the the overall overall process. process. Thus, Thus, thethe relaxationrelaxation timetime isis calledcalled thethe fluorescencefluorescence lifetime.lifetime. TheThe wavelengthwavelength ofof thethe fluorescencefluorescence isis longerlonger thanthan thethe excitationexcitation wavelengthwavelength becausebecause thethe energyenergy fromfrom thethe absorbedabsorbed photonphoton isis partiallypartially lostlost viavia non-radiativenon-radiative decay.decay. ThisThis shiftshift inin wavelengthwavelength isis calledcalled thethe StokesStokes shift.shift.

Figure 1. Jablonski diagram for the electronic energy levels of a ﬂuorophore and the transitions between them. Figure 1. Jablonski diagram for the electronic energy levels of a fluorophore and the transitions between them.

An electron in the S1 state can also flip its spin thus creating the first triplet state T1, An electron in the S1 state can also flip its spin thus creating the first triplet state T1, which is termed intersystem crossing. Transition from T1 to S0 is forbidden, thus the rate which is termed intersystem crossing. Transition from T1 to S0 is forbidden, thus the rate constants for triplet emission (phosphorescence) are several orders of magnitude smaller constants for triplet emission (phosphorescence) are several orders of magnitude smaller than those for fluorescence, which results in a long-lived dark state called blinking. While than those for fluorescence, which results in a long-lived dark state called blinking. While in the T1 state, the fluorophore may experience photobleaching, which is an irreversible fluorescencein the T1 state, switching-off the fluorophore process. may experience photobleaching, which is an irreversible fluorescenceTable1 shows switching-off characteristics process. of several fluorophores commonly used in fluorescence microscopy,Table 1 includingshows characteristics dyes, a quantum of several dot (Qdot) fluorophores [14–16], andcommonly a fluorescent used in protein fluorescence [17,18]. Photobleachingmicroscopy, including can be adyes, representative a quantum photostability dot (Qdot) [14–16], indicator and of aa fluorophore fluorescent andprotein the higher[17,18]. resistance Photobleaching to photobleaching can be a representati allows longerve photostability or brighter fluorescence indicator of observation. a fluorophore and the higher resistance to photobleaching allows longer or brighter fluorescence obser- Table 1. Characteristicsvation. of fluorophores commonly used in fluorescence microscopy.

Typical Molecular Table 1. CharacteristicsEmission of fluorophores commonly used in fluorescence microscopy. Resistance to Excitation Extinction Quantum Fluorescence Fluorophore Wavelength Photoblea- Wavelength CoefficientMolecularYield Lifetime (ns) (nm) Ching Typical(nm) Excitation Emission Wavelength(M−1cm− 1)Extinction Quantum Fluorescence Resistance to Fluorophore Wavelength (nm) (nm) 150,000 Coefficient Yield Lifetime (ns) Photoblea-Ching Cy3 532 570 −1 −1 0.15 0.2 ++ (550 nm) (M cm ) 150,000 Cy3 532 570 250,000 0.15 0.2 ++ Cy5 633 670 (550 nm) 0.28 0.9 ++ (649 nm) 250,000 Cy5 633 670 2,100,000 0.28 0.9 ++ Qdot655 532, 633 ~655 (649 nm) 0.44 ~20 +++ (532 nm) 2,100,000 GreenQdot655 532, 633 ~655 0.44 ~20 +++ 56,000 (532 nm) fluorescent 488 507 0.60 2.3 + Green fluorescent (484 nm) 56,000 protein (GFP) 488 507 0.60 2.3 + protein (GFP) (484 nm) The markingsThe markings +, ++, +, ++,and and +++ +++ indicate indicate “poor,” “poor,” “moderate,”“moderate,” and and “excellent,” “excellent,” respectively. respectively. Adapted Ad withapted permission with permission from [7]. from [7]. 2.2. Laser Scanning Fluorescence Microscopy (LSM) 2.2. LaserLaser Scanning scanning Fluorescence fluorescence Microscopy microscopy (LSM) (LSM) is one of the most widely used forms of biologicalLaser scanning fluorescence fluorescence microscopy. microscopy LSM creates (LSM) fluorescenceis one of the most images widely by sequentially used forms recordingof biological the fluorescence fluorescence intensitymicroscopy. of each LSM pixel creates by scanningfluorescence a focused images laser by beamsequentially across arecording specimen the using fluorescence confocal optics intensity to obtain of each only pixel in-focus by scanning fluorescence. a focused LSM laser with beam a confocal across illumination setup provides a base for various fluorescence microscopes using steady- Appl. Sci. 2021, 11, x FOR PEER REVIEW 4 of 20

Appl. Sci. 2021, 11, 2773 4 of 20

a specimen using confocal optics to obtain only in-focus fluorescence. LSM with a confocal illumination setup provides a base for various fluorescence microscopes using steady- state andand time-resolvedtime-resolved ﬂuorescencefluorescence microscopy.microscopy. FigureFigure2 2 shows shows schematics schematics of of confocal confocal microscopy andand ﬂuorescencefluorescence microscopymicroscopy withwith confocalconfocal optics.optics.

FigureFigure 2.2. SchematicsSchematics of of confocal confocal microscopy microscopy and fluorescence fluorescence microscopy with confocal optics: ( a) confocal confocal microscopy; ((bb)) two-photontwo-photon microscopy; microscopy; (c )( fluorescencec) fluorescence correlation correlation microscopy microscopy (FCS); (FCS); (d) fluorescence (d) fluorescence lifetime lifetime imaging imaging microscopy microscopy (FLIM). (FLIM). 2.3. Confocal Microscopy 2.3. Confocal Microscopy Confocal microscopy limits the observed volume to reduce out-of-focus signals. Min- sky introducedConfocal microscopy the concept limits of confocal the observed microscopy volume [19 to] andreduce issued out-of-focus an original signals. patent Min- for asky microscope introduced in the 1961. concept In confocal of confocal microscopy, microsco twopy [19] pinholes and issued that arean original conjugated patent in thefor identicala microscope image in plane 1961. are In placedconfocal at amicrosco focal pointpy, in two the pinholes light path. that Light are from conjugated outside ofin thethe focalidentical plane image is not plane focused are on placed the pinhole(s) at a focal and point only in fluorescencethe light path. very Light near from to the outside sample’s of focalthe focal point plane reaches is not the focused detector on (Figure the pinhole(s)2a). Thus, theand confocal only fluorescence laser scanning very microscopy near to the enablessample’s three focal dimensional point reaches reconstruction the detector of (F specimens.igure 2a). Thus, the confocal laser scanning microscopy enables three dimensional reconstruction of specimens. 2.4. Two-Photon Micriscopy 2.4. Two-PhotonTwo-photon Micriscopy microscopy, which was first reported by the Watt W. Webb group in 1990 [Two-photon20], makes use microscopy, of the phenomenon which was that first two reported photons by are the absorbed Watt W. by Webb a fluorophore group in simultaneously.1990 [20], makes Theuse fluorophoreof the phenomenon can be excited that two by photons light with are one-half absorbed the by energy a fluorophore of each photonsimultaneously. or twice theThe wavelength. fluorophore Thecan two-photonbe excited by excitation light with light one-half is generated the energy by increas-of each ingphoton the photonor twice density the wavelength. using a focused The two-photon high-power excitation femtosecond light is pulse generated laser (Figure by increas-2b). Fluorescenceing the photon emitted density from using the a focusfocused point high-p is detectedower femtosecond by a point pulse detector laser (commonly (Figure 2b). a PMT)Fluorescence and a fluorescence emitted from image the isfocus acquired point by is LSM.detected Two-photon by a point microscopy detector (commonly enables deep a imagingPMT) and because a fluorescence the microscopy image uses is acquired near-infrared by LSM. laser excitationTwo-photon light microscopy that exhibits enables better tissuedeep imaging penetration because and collectsthe microscopy the localized uses near fluorescence-infrared signal. laser excitation light that exhibits better tissue penetration and collects the localized fluorescence signal. 2.5. Fluorescence Correlation Spectroscopy (FCS)

Fluorescence correlation spectroscopy (FCS) is also based on confocal optics with a continuous wave laser(s) and monitors the mobility of molecules, typically translation diffusion into and out of a small volume (Figure2c) [ 12]. FCS analyzes time-dependent fluorescence intensity fluctuations in a tiny observed volume on the order of femtoliter. When a fluorophore diffuses (or fluorophores diffuse) into the illuminated volume, a fluorescence burst is detected due to steady-state fluorescence emission from the fluo- Appl. Sci. 2021, 11, 2773 5 of 20

rophore(s). If the fluorophore diffuses (or fluorophores diffuse) quickly out of the volume, the burst duration is short, whereas if the fluorophore diffuses (or fluorophores diffuse) more slowly the photon burst duration persists for longer. Correlation analysis of the time series enables the diffusion coefficient of the fluorophore to be determined. The fluorescence intensity fluctuation depends on the size and number of the molecules passing through the illuminated volume, which provides information on biomolecular interaction in vitro and in vivo.

2.6. Fluorescence Lifetime Imaging Microscopy (FLIM) Fluorescence lifetime imaging microscopy (FLIM) is an advanced tool that maps the fluorescence lifetime distribution through time-resolved fluorescence detection (Figure2d) [ 21,22]. The fluorescence lifetime can respond to changes in pH, temperature, and ion concentrations such as calcium concentration. Its capability to offer both localization of target fluorophores and the fluorophores’ local microenvironment exhibits its superiority to fluorescence intensity based steady-state imaging because the lifetime of a fluorophore is mostly independent of its concentration. FLIM can be performed using the time-domain method in which the sample is excited with a pulse laser, or the frequency- domain method in which the sample is excited with intensity-modulated light, commonly sine-wave modulation [12].

3. Single Point Detectors 3.1. Performance Indices 3.1.1. Dark Count Dark count refers to the tiny ﬂow of electricity in a photodetector operated under a totally dark condition. This dark current should be minimized. Dark count is caused by several phenomena that vary with photodetectors. The dark count of PMTs results from thermionic emission from the photocathode and dynode, and ionization of residual gases (ion feedback) [23]. The dark count of SPADs and SiPMs results from thermionic emission from the depletion layer. The dark count of HPDs is negligible due to its high electron bombardment gain. The dark count of SSPDs, which is low and dependent on the cooling temperature and bias current [24], results from energy dissipation in the nanowire and the blackbody radiation at room temperature through the optical ﬁber [25].

3.1.2. Instrument Response Function The transit time and its ﬂuctuation are the major determinants of the time response of a photodetector [23]. The transit time refers to the travel time of the photoelectron. The full-width half-maximum (FWHM) of the instrument response function is a standard for the time response. Many detectors exhibit a non-Gaussian instrument response function. The rise and fall times of a detector are evaluated from the waveform. Transit time spread (TTS) is the ﬂuctuation of the transit time of the single photoelectron pulse.

3.1.3. Afterpulse Afterpulses are spurious pulses that may appear subsequent to the input signal [23]. An afterpulse is best characterized by the autocorrelation function of the detected photons. For vacuum tube-based photodetectors, positive ions generated by the ionization of residual gases in a detector create afterpulses that appear several hundreds of nanoseconds to several microseconds later than the input signal. This phenomenon is called ion feedback [23]. Among solid-state photodetectors, SPADs and SiPMs experience high afterpulse noise with high count rate measurements. The afterpulse noise is generated by thermally released trapped carriers [26]. HPDs and SSPDs are free of afterpulses.

3.1.4. Sensitivity The sensitivity of a photodetector is largely determined by the quantum efﬁciency of the photocathode material. Most photocathodes used in vacuum tube-based photodetectors Appl. Sci. 2021, 11, x FOR PEER REVIEW 6 of 20

3.1.3. Afterpulse Afterpulses are spurious pulses that may appear subsequent to the input signal [23]. An afterpulse is best characterized by the autocorrelation function of the detected photons. For vacuum tube-based photodetectors, positive ions generated by the ionization of residual gases in a detector create afterpulses that appear several hundreds of nanoseconds to several microseconds later than the input signal. This phenomenon is called ion feedback [23]. Among solid-state photodetectors, SPADs and SiPMs experience high afterpulse noise with high count rate measurements. The afterpulse noise is generated by thermally released trapped carriers [26]. HPDs and SSPDs are free of afterpulses.

3.1.4. Sensitivity Appl. Sci. 2021, 11, 2773 6 of 20 The sensitivity of a photodetector is largely determined by the quantum efficiency of the photocathode material. Most photocathodes used in vacuum tube-based photodetectors are made of compound semiconductors. GaAsP exhibits the highest quantum effi- ciencyare in made the visible of compound region semiconductors.(about 45%). A GaAsPphotocathode exhibits (extended the highest red quantum GaAsP) efﬁciency that is in morethe sensitive visible regionto a longer (about wavelength 45%). A photocathode than GaAsP (extended is available red for GaAsP) Hamamatsu that is more Photonic sensitive products.to a longer Silicon wavelength and group than III-V GaAsP compound is available semiconductors, for Hamamatsu such Photonicas InGaAs products. and GaAs, Silicon are alsoand used group in III-V solid-state compound photodetectors. semiconductors, Figure such 3 shows as InGaAs the spectral and GaAs, response are alsoof these used in photocathodes.solid-state photodetectors. Figure3 shows the spectral response of these photocathodes.

FigureFigure 3. Spectral 3. Spectral response response of photocathodes. of photocathodes.

TheseThese photocathodes photocathodes are areusually usually used used in a in vacuum a vacuum and and under under the the ambient ambient tempera- temperature ture rangingranging fromfrom roomroom temperaturetemperature downdown to 273 K. As the ambient temperature is low- lowered, ered,the the spectra spectra slightly slightly shift shift to to shortershorter wavelengthwavelength duedue toto thethe bandgapbandgap broadening of the the semiconductors.semiconductors. Thus, Thus, the the spectral spectral respon responsese of these of these photocathodes photocathodes does does not notchange change significantlysignificantly in the in temperature the temperature range. range. 3.1.5. Active Area 3.1.5. Active Area The diameter or surface of the active area of a photodetector determines the observed The diameter or surface of the active area of a photodetector determines the observed area. Confocal optics uses a pinhole in a plane conjugate with the image plane in the area. Confocal optics uses a pinhole in a plane conjugate with the image plane in the sam- sample—the light from the pinhole is easy to focus on a small point detector, such as a ple—the light from the pinhole is easy to focus on a small point detector, such as a SPAD SPAD [13]. Point photodetectors with a larger active area are often useful because the light [13]. Point photodetectors with a larger active area are often useful because the light does does not need to be focused and they can used to observe a large area without scanning. not need to be focused and they can used to observe a large area without scanning. 3.2. Photomultiplier Tube (PMT) Photomultiplier tubes (PMTs) are the most widely used point detectors in fluorescence microscopy. A PMT is a vacuum tube that contains a photocathode, focusing electrodes, an electron multiplier (dynodes), and an anode (Figure4)[ 23]. Incident photons are absorbed by the photocathode, which ejects primary electrons (~3 eV). The electrons are accelerated by a high voltage to hit a series of dynodes. Then, additional electrons (5–10 electrons) are ejected and exponentially amplified. The electron current is then detected by an external electrical circuit. Typical PMTs have 8–10 dynodes with a cathode-to-anode voltage gap of ~1 kV and current gain of 106 to 107. Appl. Sci. 2021, 11, x FOR PEER REVIEW 7 of 20

3.2. Photomultiplier Tube (PMT) Photomultiplier tubes (PMTs) are the most widely used point detectors in fluorescence microscopy. A PMT is a vacuum tube that contains a photocathode, focusing electrodes, an electron multiplier (dynodes), and an anode (Figure 4) [23]. Incident photons are absorbed by the photocathode, which ejects primary electrons (~3 eV). The electrons are accelerated by a high voltage to hit a series of dynodes. Then, additional electrons (5–10 electrons) are ejected and exponentially amplified. The electron current is then detected Appl. Sci. 2021, 11, 2773 by an external electrical circuit. Typical PMTs have 8–10 dynodes with a cathode-to-anode7 of 20 voltage gap of ~1 kV and current gain of 106 to 107.

FigureFigure 4. 4. SchematicSchematic ofof aa photomultiplierphotomultiplier tube tube (PMT). (PMT). The main photocathodes used in PMTs are bi-alkali, with spectral response peaks The main photocathodes used in PMTs are bi-alkali, with spectral response peaks around 400 nm and range of up to 700 nm. The advent of the GaAsP photocathode aroundwith its 400 spectral nm and response range peakof up around to 700 500 nm. nm The invigorated advent of the the LSM GaAsP market photocathode because its with itsspectral spectral response response ranges peak fromaround visible 500 light nm in (500–700vigorated nm) the to nearLSM infrared market lightbecause (900 its nm). spectral responseLaser scanning ranges microscopesfrom visible with light a GaAsP (500–700 photocathode nm) to near were infrared released light to the (900 market nm). in Laser scanningthe late microscopes 2000s. Current with laser a scanningGaAsP photocathode microscopes incorporate were released PMTs to withthe market these multi- in the late 2000s.alkali Current photocathodes. laser scanning microscopes incorporate PMTs with these multi-alkali photocathodes.PMTs have been the leading low-light point photodetector for some time and, thus, a largePMTs number have been of power the suppliesleading low-light and signal poin processingt photodetector circuits for for them some are time available. and, thus, However, troubles can arise from unwanted noise generated by residual gas molecules (ion a large number of power supplies and signal processing circuits for them are available. feedback), and large variation in responsivity and gain caused by difficulty in controlling However,metal evaporation troubles tocan produce arise photocathodesfrom unwanted and noise secondary generated electron by surfaces. residual In gas addition, molecules (ionsingle feedback), photons and can belarge detected variation with in PMTs, responsivity but discrimination and gain of caused single versusby difficulty multiple in con- trollingphotons metal is difficult. evaporation to produce photocathodes and secondary electron surfaces. In addition, single photons can be detected with PMTs, but discrimination of single versus multiple3.3. Single photons Photon is Avalanche difficult. Diode (SPAD) The single photon avalanche diode (SPAD) is a solid-state photodetector composed of 3.3.three Single semiconductor Photon Avalanche layers, Diode called p-layer,(SPAD) i-layer, and n-layer (Figure5)[ 27]. The n-layer has extra electrons, whereas the p-layer has holes. The average gain for an avalanche pho- todiodeThe single (APD) photon is around avalanche 100, which diode is insufficient (SPAD) for is single-photona solid-state photodetector detection. Therefore, composed of SPADsthree semiconductor are usually operated layers, in “Geiger-mode,”called p-layer, wherei-layer, an and applied n-layer bias (Figure voltage is5) greater [27]. The n- layerthan has the extra diode’s electrons, breakdown whereas voltage. the Then,p-layer when has holes. a charge The is average generated gain by anfor incident an avalanche photodiodephoton, the (APD) charge multiplicationis around 100, (or which avalanche) is insufficient occurs until for it single-photon saturates corresponding detection. to aThere- fore,current SPADs typically are usually specified operated by the components. in “Geiger-mode,” These APDs where with a singlean applied pixel are bias referred voltage is greaterto as SPADs,than the and diode’s those breakdown with multiple voltage. pixels are Then, referred when to a as charge silicon is photomultipliers generated by an incident(SiPMs) photon, or multi-pixel the charge photon multiplication counters (MPPCs).(or avalanche) Although occurs Geiger-mode until it saturates driven SPADscorrespond- are suitable for single photon counting, SPADs suffer several drawbacks. The active area ing to a current typically specified by the components. These APDs with a single pixel are cannot be increased because fabrication of a large semiconductor surface increases the referrednumber to of as defects. SPADs, Geiger-mode and those drivenwith multiple SPADs have pixels high are dark referred counts to due as tosilicon the Geiger photomul- tipliersdischarge (SiPMs) and high or multi-pixel afterpulse noise. photon The counters dead time (MPPCs). is relatively Although long (about Geiger-mode 50 ns), during driven SPADswhich are photon suitable counting for single is inoperative photon because counting, the modeSPADs needs suffer to beseveral reset for drawbacks. every single The ac- tivephoton area detection.cannot be To increased reduce dark because counts, SPADsfabrication are typically of a large cooled semiconductor to 210–250 K. surface increases the number of defects. Geiger-mode driven SPADs have high dark counts due to the Geiger discharge and high afterpulse noise. The dead time is relatively long (about 50 Appl. Sci. 2021, 11, x FOR PEER REVIEW 8 of 20

ns), during which photon counting is inoperative because the mode needs to be reset for every single photon detection. To reduce dark counts, SPADs are typically cooled to 210– Appl. Sci. 2021, 11, x FOR PEER REVIEW 8 of 20 250 K.

ns), during which photon counting is inoperative because the mode needs to be reset for Appl. Sci. 2021, 11, 2773 every single photon detection. To reduce dark counts, SPADs are typically cooled8 of 20 to 210– 250 K.

Figure 5. Schematic of a single photon avalanche diode (SPAD).

3.4. Hybrid Photodetector (HPD) Figure 5. Schematic of a single photon avalanche diode (SPAD). Figure The5. Schematic hybrid of photodetector a single photon avalanche (HPD) isdiode a hybrid (SPAD). of an avalanche diode (AD) and a pho- 3.4. Hybrid Photodetector (HPD) tocathode, both of which are in a vacuum tube (Figure 6) [23,26,28–31]. When light is in- 3.4. HybridThe hybrid Photodetector photodetector (HPD) (HPD) is a hybrid of an avalanche diode (AD) and a pho- cident onto the photocathode, photoelectrons are emitted from the photocathode. The tocathode,The hybrid both ofphotodetector which are in (HPD) a vacuum is a tube hybrid (Figure of an6)[ avalanche23,26,28–31 diode]. When (AD) light and is a pho- photoelectronsincident onto the photocathode,are then accelerated photoelectrons by area high emitted negative from the voltage photocathode. to directly The bombard the tocathode, both of which are in a vacuum tube (Figure 6) [23,26,28–31]. When light is in- ADphotoelectrons where electron-hole are then accelerated pairs by are a high generated negative and voltage the to signal directly is bombard amplified. the AD The amplification cidentwhere onto electron-hole the photocathode, pairs are generated photoelectrons and the signal are emitted is ampliﬁed. from The the ampliﬁcation photocathode. is The is termed “electron bombardment gain”. photoelectronstermed “electron are bombardment then accelerated gain”. by a high negative voltage to directly bombard the AD where electron-hole pairs are generated and the signal is amplified. The amplification is termed “electron bombardment gain”.

Figure 6. Schematic of a hybrid photodetector (HPD). Figure 6. Schematic of a hybrid photodetector (HPD). FigureIn 6. theSchematic case of of an a HPDhybrid manufactured photodetector by (HPD). Hamamatsu Photonics, the electron bom- − bardmentIn the gain case is approximately of an HPD 1500 manufactured with the photocathode by Hamamatsu supply voltage Photonics, of 8 kV. The the electron bom- signalsIn the of electron-hole case of an pairsHPD aremanufactured further amplified by Hamamatsu to 80-fold (avalanche Photonics, gain) the by applyingelectron bom- bardmentbardmenta reverse voltagegain gain is ofapproximately is about approximately 400 V to 1500 the avalanche 1500with thewith diode.photocathode the Then,photocathode the supply total gain voltage supply will therefore of voltage −8 kV. The of − 8 kV. The signalssignalsbe as much of of electron-hole electron-hole as about 120,000. pairs pairs are further are further amplified amplified to 80-fold to 80-fold(avalanche (avalanche gain) by applying gain) by applying aa reverse reverse voltage voltage of aboutof about 400 V400 to Vthe to avalanch the avalanche diode.e diode.Then, the Then, total thegain total will thereforegain will therefore 3.4.1. Features of HPD bebe as as much much as asabout about 120,000. 120,000. HPDs have significant advantages over PMTs and other low-light photodetectors in 3.4.1.the detection Features of of fluorescence, HPD as discussed below. However, a few disadvantages of HPDs 3.4.1.also exist. Features The extremely of HPD high cathode supply voltage (−8 kV) is difficult to deal with, whichHPDs can behave problematic significant when advantages incorporated over into PMTs systems. and other low-light photodetectors in the detectionHPDs ofhave fluorescence, significant as discussedadvantages below. over However, PMTs and a few other disadvantages low-light ofphotodetectors HPDs in alsothe exist.detection The extremelyof fluorescence, high cathode as discussed supply bevoltagelow. However,(−8 kV) is di a ffifewcult disadvantages to deal with, of HPDs whichalso exist.can be The problematic extremely when high incorporated cathode intosupply systems. voltage (−8 kV) is difficult to deal with, which can be problematic when incorporated into systems. Appl. Sci. 2021, 11, 2773 9 of 20 Appl. Sci. 2021, 11, x FOR PEER REVIEW 9 of 20

Low LowAfterpulse Afterpulse HPDsHPDs have have a notable a notable feature feature of lower of lower afterpulse afterpulse due to due their to theiruncomplicated uncomplicated internal internal structure.structure. Therefore, Therefore, the themajor major cause cause of ofafterpulses, afterpulses, i.e., i.e., ion ion feedback, feedback, is highly unlikelyunlikely to to occur occurin in HPDs. HPDs. Afterpulses Afterpulses evaluated evaluated for for an an HPD HPD and and a PMTa PMT are are shown shown in in Figure Figure7. This7. This graph graphshows shows the the probability probability at at which which afterpulses afterpulses maymay bebe generatedgenerated by a single single photoelec- photoelectron tron input. In contrast to the the PMT’s PMT’s multiple multiple afterpulses in a time time range range from from 100 ns to 1 1 µs,µs, this this HPDHPD exhibited exhibited only a small number of afterpulses in the time range.

FigureFigure 7. Afterpulse 7. Afterpulse noise noise of PMT of PMTand HPD and HPD [32]. [The32]. data The datawere were obtained obtained through through detection detection of of single singlephotons photons from from a a continuous continuouswave wave (CW)(CW) lightlight sourcesource (wavelength = 470 470 nm). nm). The The cathode cathode supply supplyvoltage voltage and and the the reverse reverse voltage voltage of HPDof HPD were were−8 − kV8 kV and and 398 398 V, V, respectively. respectively.

ComparableComparable low lowafterpulses afterpulses have have been been repo reportedlyrtedly achieved achieved solely solely for for superconduct- superconducting ing nanowirenanowire single single photon photon detectors detectors (SSPDs). (SSPDs). These These SSPDs SSPDs have have micrometer micrometer order order active active areasareas and must and must be operated be operated at a liquid at a liquid helium helium temperature temperature [25], [as25 discussed], as discussed in Section in Section 4.2. 4.2.

HighHigh Resolution Resolution of Photon of Photon Counting Counting HPDsHPDs exhibit exhibit better better pulse pulse height height resolution resolution than PMTs. than PMTs. Gain fluctuation Gain fluctuation of HPDs of HPDsis significantlyis significantly lower owing lower to owing much to higher much el higherectron electronbombardment bombardment gain (about gain 1500 (about at a 1500 photocathodeat a photocathode supply voltage supply of voltage −8 kV) ofthan−8 the kV) first than dynode the first gain dynode of an ordinary gain of an photo- ordinary multiplierphotomultiplier tube (typically tube as (typically low as 5–10). as low The as first 5–10). gain The mostly first determines gain mostly the determines signal-to- the noisesignal-to-noise ratio of the electron ratio ofmultiplication, the electron multiplication, which in turn represents which in turn the detector’s represents capability the detector’s to distinguishcapability between to distinguish one and between multiple one photons. and multiple As a result, photons. HPDs As offer a result, high HPDs resolution offer high of photonresolution counting. of photon As shown counting. in Figure As shown 8, signal in Figurepeaks 8that, signal correspond peaks that to 1, correspond 2, 3, 4, and to 5 1, 2, photoelectrons3, 4, and 5 photoelectronscan be identified can in bethe identified output pulse in the height output distribution. pulse height distribution.

High300 Timing Resolution 2 photoelectrons 1 photo- 3 photoelectrons Timeelectron response characteristics of HPDs are largely determined by the junction capac- itance250 of the internal avalanche diode, provided the diameter of the internal avalanche 4 photoelectrons diode200 is around or larger than 1 mm. The internal avalanche diode with a diameter of 1 mm and a very low capacitance (4 pF), which is incorporated in the current HPD product, 5 photoelectrons realizes150 a fast response. Figure9a shows the time response waveforms of an HPD and a

PMT.# of counts The FWHM for the HPD is 0.6 ns, which is smaller than that for PMT (1.6 ns). 100

0 0 500 1000 1500 2000 2500 3000 3500 4000 Output pulse height (Channel #)

Appl. Sci. 2021, 11, x FOR PEER REVIEW 9 of 20

Low Afterpulse HPDs have a notable feature of lower afterpulse due to their uncomplicated internal structure. Therefore, the major cause of afterpulses, i.e., ion feedback, is highly unlikely to occur in HPDs. Afterpulses evaluated for an HPD and a PMT are shown in Figure 7. This graph shows the probability at which afterpulses may be generated by a single photoelectron input. In contrast to the PMT’s multiple afterpulses in a time range from 100 ns to 1 µs, this HPD exhibited only a small number of afterpulses in the time range.

Figure 7. Afterpulse noise of PMT and HPD [32]. The data were obtained through detection of single photons from a continuous wave (CW) light source (wavelength = 470 nm). The cathode supply voltage and the reverse voltage of HPD were −8 kV and 398 V, respectively.

Comparable low afterpulses have been reportedly achieved solely for superconducting nanowire single photon detectors (SSPDs). These SSPDs have micrometer order active areas and must be operated at a liquid helium temperature [25], as discussed in Section 4.2.

High Resolution of Photon Counting HPDs exhibit better pulse height resolution than PMTs. Gain fluctuation of HPDs is significantly lower owing to much higher electron bombardment gain (about 1500 at a photocathode supply voltage of −8 kV) than the ﬁrst dynode gain of an ordinary photomultiplier tube (typically as low as 5–10). The first gain mostly determines the signal-to- noise ratio of the electron multiplication, which in turn represents the detector’s capability to distinguish between one and multiple photons. As a result, HPDs offer high resolution Appl. Sci. 2021, 11, 2773 of photon counting. As shown in Figure 8, signal peaks that correspond10 of 20 to 1, 2, 3, 4, and 5 photoelectrons can be identified in the output pulse height distribution.

300 2 photoelectrons 1 photo- 3 photoelectrons electron 250

4 photoelectrons Appl. Sci. 2021, 11, x FOR PEER REVIEW 10 of 20 200

5 photoelectrons 150

Figure 8. Output height# of counts distribution of an HPD [32]. The incident pulsed lights (wavelength = 470 nm) were adjusted to make100 the photocathode emit three photoelectrons on average. The cathode supply voltage and the reverse voltage were −8 kV and 380 V, respectively. 50 High Timing Resolution 0 Time response characteristics0 500 1000 of 1500HPDs 2000 are 2500 largely 3000 determined 3500 4000 by the junction capacitance of the internal avalancheOutput diode, pulse provided height the (Channel diameter #) of the internal avalanche diode is around or larger than 1 mm. The internal avalanche diode with a diameter of 1 mm and a very low capacitanceFigure 8. (4Output pF), which height is distribution incorporated of an in HPD the [32 current]. The incident HPD product, pulsed lights realizes (wavelength = 470 nm) were adjusted to make the photocathode emit three photoelectrons on average. The cathode a fast response. Figure 9a shows the time response waveforms of an HPD and a PMT. The supply voltage and the reverse voltage were −8 kV and 380 V, respectively. FWHM for the HPD is 0.6 ns, which is smaller than that for PMT (1.6 ns).

Figure 9.FigureTime response9. Time response characteristics characteristics of an HPD of and an a HPD PMT [and32]: a (a PMT) time [32]: response (a) time waveforms response of anwaveforms HPD and of a PMT; (b) transitan time HPD spread and a (TTS) PMT; of ( anb) HPDtransit and time a PMT. spread The data(TTS) were of an obtained HPD usingand a aPMT. pulse The laser data (wavelength were obtained = 405 nm) with a pulse widthusing ofa pulse 77 ps. laser The cathode (wavelength supply = voltage 405 nm) and with the reversea pulse voltage width ofof HPD 77 ps. were The− cathode8 kV and supply 390 V, respectively. voltage and the reverse voltage of HPD were −8 kV and 390 V, respectively. The TTS determines the instrument response function of HPD. The following three factors mainly affect the TTS for HPD: (i) the transit time within the photocathode; (ii) the The TTS determines the instrument response function of HPD. The following three variation in the time taken for the photoelectrons to move in the vacuum from the pho- factors mainly affecttocathode the TTS to the for avalanche HPD: (i) the diode; transit and (iii)time the within electron the transit photocathode; time within (ii) the the avalanche variation in the timediode. taken The TTSfor the for anphotoelectrons alkali photocathode to move is about in the 50 vacuum ps [31]. The from measured the photo- raw TTS for a cathode to the avalancheGaAsP photocathode diode; and in an(iii) HPD the iselectron about 113 transit ps, which time is largerwithin than the that avalanche for alkali because diode. The TTS forthe an GaAsP alkali layer photocathode is thicker than is the about alkali 50 layer. ps [31]. This The raw TTSmeasured value included raw TTS the for laser pulse a GaAsP photocathodewidth (77 in ps) an andHPD temporal is about resolution 113 ps, which of the measurementis larger than system that for (30 alkali ps), so be- the net TTS cause the GaAsPshould layer beis muchthicker smaller. than the Figure alkali9b shows layer. the This time raw response TTS value waveforms included of an the HPD and a laser pulse widthPMT. (77 Theps) and raw TTStemporal for the resolution GaAsP photocathode of the meas inurement a PMT is system 300 ps, which(30 ps), is so larger than that of an HPD. These characteristics are very important for time-resolved ﬂuorescence the net TTS should be much smaller. Figure 9b shows the time response waveforms of an detection because it contributes to accurate ﬂuorescence lifetime measurement. HPD and a PMT. The raw TTS for the GaAsP photocathode in a PMT is 300 ps, which is larger than that of an HPD. These characteristics are very important for time-resolved fluorescence detection because it contributes to accurate fluorescence lifetime measurement.

Large Active Area HPDs have a large effective area of more than several millimeters in diameter, enabling high photon collection efficiency. The active area of HPDs is comparable with that of PMTs, which is a marked contrast to SPADs with an effective diameter of only 10 micrometers. Table 2 lists the characteristics of PMTs, SPADs, and HPDs.

Appl. Sci. 2021, 11, 2773 11 of 20

Large Active Area HPDs have a large effective area of more than several millimeters in diameter, enabling high photon collection efﬁciency. The active area of HPDs is comparable with that of PMTs, which is a marked contrast to SPADs with an effective diameter of only 10 micrometers. Table2 lists the characteristics of PMTs, SPADs, and HPDs.

Table 2. Characteristics of point photodetectors [23,27,32]. Appl. Sci. 2021, 11, x FOR PEER REVIEW 11 of 20

Single Photon Photomultiplier Hybrid Avalanche Diode Tube (PMT) Photodetector (HPD) (SPAD) Table 2. Characteristics of point photodetectors [23,27,32]. Afterpulse High High Low PhotomultiplierTransit time Tube spread (PMT) Single Photon Avalanche Diode (SPAD) Hybrid Photodetector (HPD) ~300 ps ~300 ps ~100 ps Afterpulse High(TTS) High Low Transit time spread (TTS) Diameter ~300 ps of active ~300 ps ~100 ps ~5 mm ~several 100 µm ~5 mm Diameter of active area ~5 mmarea ~several 100 µm ~5 mm Operating Operating temperature ~270 K ~270 K 210–250 K 210–250 K Room Room temperature temperature temperature 3.4.2. HPD and Fluorescence Microscopy 3.4.2. HPD and Fluorescence Microscopy The features of HPDs mentioned in Section 3.4.1 show the preeminence of HPDs in fluorescenceThe features microscopy of HPDs applications, mentioned insuch Section as LSM, 3.4.1 FLIM, show and the FCS. preeminence In practice, of HPDs HPDs are in ﬂuorescenceincorporated microscopyinto commercial applications, microscopes such and as LSM, products FLIM, of andBecker FCS. &In Hickl practice, GmbH HPDs and arePicoQuant. incorporated into commercial microscopes and products of Becker & Hickl GmbH and PicoQuant. FCS FCS In FCS, the presence of afterpulses deforms the correlation spectra. To avoid this is- In FCS, the presence of afterpulses deforms the correlation spectra. To avoid this sue, the fluorescence signal is commonly divided into two and detected by two detectors, issue, the ﬂuorescence signal is commonly divided into two and detected by two detectors, and the cross-correlation between the two signals is calculated. This procedure is compli- and the cross-correlation between the two signals is calculated. This procedure is compli- catedcated andand decreasesdecreases thethe signal-to-noisesignal-to-noise ratioratio ofof eacheach acquiredacquired datapoint.datapoint. InIn contrast,contrast, thethe afterpulse-freeafterpulse-free feature of thethe HPDHPD makesmakes FCSFCS measurementmeasurement simpler,simpler, andand aa singlesingle HPDHPD providesprovides betterbetter data data than than PMT PMT [13 [13,26].,26]. As As shown shown in Figurein Figure 10, the10, autocorrelationthe autocorrelation spectrum spec- acquiredtrum acquired by an HPDby an is HPD of good is of quality, good quality, whereas whereas the spectrum the spectrum acquired acquired by PMT containsby PMT overlappedcontains overlapped afterpulse afterpulse noise below noise the below 2 µs region. the 2 µs region.

FigureFigure 10.10. Autocorrelation datadata obtainedobtained byby anan HPDHPD andand aa PMTPMT [32[32].]. TheThe datadata werewere acquired acquired using us- 100ing nM100 AlexanM Alexa Fluor Fluor 532 dye532 solution.dye solution.

FLIMFLIM FLIMFLIM utilizesutilizes time-correlatedtime-correlated singlesingle photonphoton countingcounting (TCSPC)(TCSPC) andand acquiresacquires higherhigher temporaltemporal resolutionresolution data data (

time measurement data obtained by an HPD and a PMT. Afterpulses are also problematic for FLIM. The afterpulse raises the baseline of the PMT data. Consequently, the dynamic Appl. Sci. 2021, 11, x FOR PEER REVIEW 12 of 20 range of PMT is lower by an order of magnitude than that of HPD and seriously interrupts determination of the decay time constant. Appl. Sci. 2021, 11, x FOR PEER REVIEW 12 of 20

Figure 11.FigureFluorescence 11. Fluorescence lifetimeFigure measurement lifetime 11. Fluorescence measurement data obtained lifetime data measurement by anobtained HPD (a )databy and an obtained a HPD PMT (( baby).) and Thean HPD dataa PMT (a were) and (b acquired). a The PMT (b using). The ﬂuoresceindata dye were solution. acquired Adapteddata using were with fluorescein acquired permission using dye from sofluoresceinlution. [13]. Adapted dye solution. with Adapted permission with frompermission [13]. from [13]. Compared with SPADs, HPDs can obtain brighter FLIM images due to the larger Compared with ComparedSPADs, HPDs with SPADs,can obtain HPDs brighter can obtain FLIM brighter images FLIM due images to the due larger to the larger activeactive areaarea ofof thethe HPD.HPD. FigureFigure 1212 shows FLIM images acquired acquired by by an an HPD HPD and and a aSPAD. SPAD. active area of theAlthough HPD. Figure the quantum 12 shows yields FLIM of theimages photodetectors acquired wereby an similar, HPD and the imagea SPAD. acquired by Although the quantumAlthough yields the quantum of the photodetec yields of thetors photodetec were similar,tors were the similar, image the acquired image acquired by by thethe HPD HPD collected collected twicetwice asas manymany photonsphotons asas acquiredacquired by the PMT. the HPD collected twice as many photons as acquired by the PMT.

FigureFigure 12. 12.FLIM FLIM images images acquired acquired byby HPDHPD ((aa)) andand SPADSPAD ((bb).). AdaptedAdapted withwith permissionpermission from [13]. [13].

Figure 12. FLIM images acquired 3.4.3.by3.4.3. HPD OtherOther (a) and TypesTypes SPAD ofof HPDsHPDs (b). Adapted with permission from [13]. ThisThis subsubsectionsubsubsection briefly brieﬂy refers refers to to other other types types of HPDs of HPDs that that were were developed developed by Ha- by 3.4.3. Other TypesHamamatsumamatsu of HPDs Photonics Photonics (Table (Table 3) and3) and applications applications of the of HPD the to HPD single-molecule to single-molecule fluorescence ﬂuo- This subsubsectionrescencemicroscopy. briefly microscopy. These refers other Theseto types other other of types HPDs types of are HPDs of cooled HPDs that HPD are we cooled andre developed MPPC HPD (SiPM)-incorporated and by MPPC Ha- (SiPM)- HPDs. mamatsu Photonicsincorporated (Table 3) HPDs. and applications of the HPD to single-molecule fluorescence Table 3 lists the characteristics of these HPDs. microscopy. These other types of HPDs are cooled HPD and MPPC (SiPM)-incorporated Table 3. Other types of HPDs [32–35]. HPDs. Table 3. Other types of HPDs [32–35]. Table 3 lists the characteristicsType of HPD of these HPDs. Feature Possible Application Type of HPD Feature Possible Application Photocathode-cooledPhotocathode-cooled Low Low thermal noisenoise FCS FCS Table 3. Other types MPPCof HPDs (SiPM)-incorporated [32–35]. Low voltage operation Easy to install into systems MPPC (SiPM)-incorporated Low voltage operation Easy to install into systems Type of HPD Feature Possible Application Photocathode-cooledTableThe cooled3 lists theHPD, characteristicsLow in which thermal the ofphotocathodenoise these HPDs. is cooled by FCS the Peltier element, reduces thermalThe cooledelectronic HPD, noise in whichfrom the the photocathode photocathode to is one-tenth cooled by that the of Peltier the non-cooled element, reduces HPDs MPPC (SiPM)-incorporated Low voltage operation Easy to install into systems thermal(Figure electronic13). This HPD noise is from supposed the photocathode to be useful tofor one-tenth the detection that ofof theextremely non-cooled low HPDslight, including single molecule detection. The cooled HPD, in which the photocathode is cooled by the Peltier element, reduces thermal electronic noise from the photocathode to one-tenth that of the non-cooled HPDs (Figure 13). This HPD is supposed to be useful for the detection of extremely low light, including single molecule detection. Appl. Sci. 2021, 11, 2773 13 of 20 Appl. Sci. 2021, 11, x FOR PEER REVIEW 13 of 20

Appl. Sci. 2021, 11, x FOR PEER REVIEW(Figure 13). This HPD is supposed to be useful for the detection of extremely low13 light,of 20 including single molecule detection.

Figure 13. Dependence of dark count rate of the developed cooled HPD on the applied Peltier Figure 13. Dependence of dark count rate of the developed cooled HPD on the applied Peltier Figurecurrent 13. [32]. Dependence Application ofof 1.2 dark A Peltier count current rate ofcooled the developedthe HPD to about cooled 283 HPDK at an on ambient the applied tem- Peltier peraturecurrent [ 32of ].298 Application K. of 1.2 A Peltier current cooled the HPD to about 283 K at an ambient currenttemperature [32]. Application of 298 K. of 1.2 A Peltier current cooled the HPD to about 283 K at an ambient tem- peratureThe of MPPC-incorporated 298 K. HPD was developed to solve the difficulties of operating the HPDThe MPPC-incorporatedunder high voltages HPD such was as developed8 kV. The MPPC-incorporated to solve the difficulties HPD of operating can operate the withHPDThe a under lower MPPC-incorporated highvoltage voltages that operates such as HPDPMTs 8 kV. due Thewas to MPPC-incorporated developedthe MPPC’s high to solve gain, HPD an the cand is difficulties operateable to detect with of operating singlea lower photons. voltage The that first operates prototype PMTs of due an MPPC to the (SiPM)-incorporated MPPC’s high gain, and HPD is with able toa GaAsP detect thesingle HPD photons. under The high first voltages prototype such of an MPPCas 8 kV. (SiPM)-incorporated The MPPC-incorporated HPD with a GaAsP HPD can operate photocathode with a diameter of 3 mm and a 25.4-mm (1-inch) bi-alkali photocathode withphotocathode a lower voltage with a diameterthat operates of 3 mm PMTs and adue 25.4-mm to the (1-inch) MPPC’s bi-alkali high gain, photocathode and is able to detect type of MPPC (SiPM)-incorporated HPD were developed by Hamamatsu Photonics, and type of MPPC (SiPM)-incorporated HPD were developed by Hamamatsu Photonics, and singleBarbato photons. et al. evaluated The first its characteristics prototype of [33, an34]. MPPC As shown (SiPM)-incorporated in Figure 14, the HPD operatesHPD with a GaAsP Barbato et al. evaluated its characteristics [33,34]. As shown in Figure 14, the HPD operates photocathodewith lower voltages with (photocathode a diameter ofvoltage 3 mm ~−3 andkV and a 25.4-mmMPPC bias (1-inch) voltage ~ bi-alkali+70 V) than photocathode with lower voltages (photocathode voltage ~−3 kV and MPPC bias voltage ~ +70 V) those of the current HPD product that incorporates an AD (photocathode voltage ~−8 kV typethan of those MPPC of the (SiPM)-incorporated current HPD product thatHPD incorporates were developed an AD (photocathode by Hamamatsu voltage Photonics, and and avalanche photodiode bias voltage ~+450 V). The low voltage operation capability Barbato~−8 kV et and al. evaluated avalanche photodiode its characteristics bias voltage [33, ~+45034]. As V). shown The low in voltageFigure operation14, the HPD operates facilitates installation of the HPD into various apparatus, including fluorescence micro- withcapability lower facilitates voltages installation (photocathode of the HPD voltage into various ~−3 kV apparatus, and MPPC including bias fluorescence voltage ~ +70 V) than scopes. Recently, Hamamatsu Photonics developed an HPD with a 50.8-mm (2-inch) di- microscopes. Recently, Hamamatsu Photonics developed an HPD with a 50.8-mm (2-inch) thoseameter of [35].the current HPD product that incorporates an AD (photocathode voltage ~−8 kV anddiameter avalanche [35]. photodiode bias voltage ~+450 V). The low voltage operation capability facilitates installation of the HPD into various apparatus, including fluorescence microscopes. Recently, Hamamatsu Photonics developed an HPD with a 50.8-mm (2-inch) diameter [35].

Figure 14. Schematic of multi-pixel photon counters (MPPC)-incorporated HPD. Figure 14. Schematic of multi-pixel photon counters (MPPC)-incorporated HPD.

Figure 14. Schematic of multi-pixel photon counters (MPPC)-incorporated HPD.

Appl. Sci. 2021, 11, 2773x FOR PEER REVIEW 1414 of of 20

Appl. Sci. 2021, 11, x FOR PEER REVIEW 14 of 20

3.4.4. Application Application of of HPDs HPDs to to Single-Molecule Single-Molecule Fluorescence Microscopy 3.4.4.We Application appliedapplied thethe of cooled-photocathode HPDscooled-photocathode to Single-Molecule HPD HPD toFluorescence to single-molecule single-molecule Microscopy fluorescence fluorescence microscopy. microscopy.We appliedWe We applied applied the HPD the the tocooled-photocathode HPD low-background to low-background wide-field HPD wide-fieldto single-molecule single-molecule single-molecule fluorescence fluorescence fluorescence detection micros- detectionwithcopy. high We with temporalapplied high the temporal resolution HPD to resolution aslow-background a proof-of-principle as a proof-of-principle wide-field demonstration. single-molecule demonstration. The fluorescencefluorescence The fluo- rescencecollecteddetection collected bywith an high objective by temporal an objective was resolution divided was divided into as a two, proof-of-principle into each two, simultaneously each simult demoaneouslynstration. detected detected The by HPDfluo- by HPDorrescence imaged or imaged collected by EMCCD. by by EMCCD. an Theobjective The HPD HPD was allowed dividedallowed the intothe fluorescence fluorescencetwo, each intensity simult intensityaneously of aof mobile a mobiledetected single sin- by glemoleculeHPD molecule or imaged fluorophore fluorophore by EMCCD. to be to determined beThe determined HPD allowed at higher at higherthe temporal fluorescence temporal resolution resolutionintensity than of than conventionala mobile conven- sin- tionalhigh-sensitivitygle molecule high-sensitivity fluorophore CCD cameras. CCD to cameras. be Specifically, determined Specifically, the at cooled-photocathode higher the cooled-photocathode temporal resolution HPD detected HPD than detected conven- fluores- fluorescencecencetional ofhigh-sensitivity a single of a Qdot single whileCCD Qdot cameras. performing while performing Specifically, two-dimensional two-dimensional the cooled-photocathode diffusion diffusion with 0.1 HPD mswith temporal detected 0.1 ms temporalresolutionfluorescence resolution (Figure of a single15 (Figure). Qdot 15). while performing two-dimensional diffusion with 0.1 ms temporal resolution (Figure 15).

Figure 15. Wide-fieldWide-field sub-millisecond single-molecule fluo fluorescencerescence detection by a cooled HPD [[32]:32]: ( a) Schematic of the observedFigure 15. mobilemobile Wide-field Qdot.Qdot. sub-millisecond TheThe Qdot Qdot (Qdot655 (Qdot655 single-molecule streptavidin streptavidin conjugate)fluo conjrescenceugate) was wasdetection attached attached by to biotinylateda to cooled biotinylated HPD phosphoethanolamine [32]: phosphoethanolamine (a) Schematic of (PE), the ( 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(capobservedPE), 1,2-dioleoyl-sn-glycero-3- mobile Qdot. The Qdotphosphoethanolamine-N-(cap (Qdot655 streptavidin biotinyl), conj ugate)biotinyl), via was biotin-streptavidin viaattached biotin-streptavidin to biot interactioninylated interaction phosphoethanolamine in a 1,2-dioleoyl-sn- in a 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer formed on a coverslip. The Qdot was excited by a CW laser glycero-3-phosphocholine(PE), 1,2-dioleoyl-sn-glycero-3- (DOPC)phosphoethanolamine-N-(cap lipid bilayer formed on a coverslip. biotinyl), The via Qdot biotin-streptavidin was excited by ainteraction CW laser (wavelength in a 1,2-di- (wavelength = 532 nm); (b) Time course of a mobile Qdot fluorescence intensity was obtained by an HPD with a time =oleoyl-sn-glycero-3-phosphocholine 532 nm); (b) Time course of a mobile (DOPC) Qdot lipid fluorescence bilayer formed intensity on was a coverslip. obtained byThe an Qdot HPD was with excited a time by resolution a CW laser of resolution(wavelength of 0.1= 532 ms nm);(red). ( bThe) Time repeated course fluorescence-on of a mobile Qdot and –offfluorescence are caused intensity by blinking. was obtainedThe intensity by an profile HPD matcheswith a time the 0.1 ms (red). The repeated fluorescence-on and –off are caused by blinking. The intensity profile matches the intensity intensityresolution simultaneously of 0.1 ms (red). obtained The repeated by EMCCD fluorescence-on (blue) with and a –offtime are resolution caused by of blinking.31.319 ms; The (c )intensity Trajectory profile of the matches Qdot ob- the simultaneously obtained by EMCCD (blue) with a time resolution of 31.319 ms; (c) Trajectory of the Qdot obtained by tainedintensity by simultaneouslyEMCCD. The inset obtained is a fluoresce by EMCCDnce image (blue) of with the observed a time resolution mobile Qdot. of 31.319 ms; (c) Trajectory of the Qdot ob- EMCCD.tained by The EMCCD. inset is The a fluorescence inset is a fluoresce image ofnce the image observed of the mobile observed Qdot. mobile Qdot. The HPD also enabled wide-field single-molecule fluorescence lifetime measurement The HPD also enabled wide-field single-molecule fluorescence lifetime measurement with nanosecondThe HPD also temporal enabled resolution. wide-field Wesingle-mol succeededecule in fluorescence obtaining time lifetime courses measurement of fluores- with nanosecond temporal resolution. We succeeded in obtaining time courses of fluores- cencewith nanosecondlifetime of a singletemporal Qd otresolution. whose fluorescence We succeeded images in obtaining were simultaneously time courses monitored of fluorescence lifetime of a single Qdot whose fluorescence images were simultaneously monitored bycence an EMCCDlifetime of (Figure a single 16). Qd ot whose fluorescence images were simultaneously monitored by an EMCCD (Figure 16). by an EMCCD (Figure 16).

Figure 16. Wide-field single-molecule fluorescence lifetime measurement by a cooled HPD: (a) Schematic of the observed mobile Qdot. Sample preparation for the observation was identical to that described in the caption of Figure 15. The Qdot FigureFigure 16.16. Wide-fieldWide-field single-moleculesingle-molecule fluorescencefluorescence lifetimelifetime measurementmeasurement byby aa cooledcooled HPD:HPD: ((aa)) SchematicSchematic ofof thethe observedobserved was excited by a pulse laser (wavelength = 520 nm); (b) Time courses of the Qdot fluorescence (upper, blue) obtained by mobilemobile Qdot.Qdot. SampleSample preparationpreparation forfor thethe observationobservation waswas identicalidentical toto thatthat describeddescribed inin thethe captioncaption ofof FigureFigure 15 15.. TheThe QdotQdot anwas EMCCD excited andby a the pulse lifetime laser (lower,(wavelength red) simultaneously = 520 nm); (b) Time obtain coursesed by anof theHPD. Qdot The fluorescence lifetime wa s(upper, obtained blue) by obtainedfitting each by was excited by a pulse laser (wavelength = 520 nm); (b) Time courses of the Qdot fluorescence (upper, blue) obtained by an decayan EMCCD curve anddrawn the using lifetime data (lower, accumulated red) simultaneously in three seconds obtain withed bya single-exponential an HPD. The lifetime (red); wa (sc )obtained Trajectory by of fitting the Qdot each EMCCD and the lifetime (lower, red) simultaneously obtained by an HPD. The lifetime was obtained by fitting each decay obtaineddecay curve by EMCCD. drawn using The insetdata isaccumulated a fluorescence in threimagee seconds of the observed with a single-exponential mobile Qdot. (red); (c) Trajectory of the Qdot curveobtained drawn by EMCCD. using data The accumulated inset is a fluorescence in three seconds image with of athe single-exponential observed mobile (red); Qdot. (c ) Trajectory of the Qdot obtained by EMCCD. The inset is a fluorescenceA group image ofat theTohoku observed University mobile Qdot. incorporated two HPDs into a specially equipped line confocalA group optical at systemTohoku in University which a slit incorporated was used instead two HPDs of a intopinhole a specially to improve equipped the time line confocal optical system in which a slit was used instead of a pinhole to improve the time Appl. Sci. 2021, 11, 2773 15 of 20

Appl. Sci. 2021, 11, x FOR PEER REVIEW 15 of 20

A group at Tohoku University incorporated two HPDs into a specially equipped resolutionline confocal of single-molecule optical system inFRET which study. a slit Th wasey achieved used instead FRET of observation a pinhole to of improve single pro- the teintime molecules resolution that of single-moleculeflowed unidirectionally FRET study. in a flow They cell achieved at a time FRET resolution observation of 10 of µs single and µ analyzedprotein molecules the high-speed that flowed folding unidirectionally process of protein in amolecules flow cell at[36]. a time resolution of 10 s and analyzed the high-speed folding process of protein molecules [36]. 4. Emerging Point Detectors 4. Emerging Point Detectors This section introduces newly emerging photodetectors that can be used for fluores- This section introduces newly emerging photodetectors that can be used for fluorescence microscopy. These photodetectors are silicon photomultipliers (SiPMs) also known cence microscopy. These photodetectors are silicon photomultipliers (SiPMs) also known as multi-pixel photon counters (MPPCs) and superconducting nanowire single photon as multi-pixel photon counters (MPPCs) and superconducting nanowire single photon detectorsdetectors (SSPDs). (SSPDs).

4.1.4.1. Silicon Silicon Photomultiplier Photomultiplier (SiPM) (SiPM) SiliconSilicon photomultipliers photomultipliers (SiPMs), which offeroffer singlesingle photon photon detection detection capability, capability, are are an anemerging emerging photodetector photodetector in in a varietya variety of industriesof industries and and biological biological ﬂuorescence fluorescence microscopy. microscopy.Caccia Caccia summarized summarized applications applications of SiPMs of SiPM tos biophotonics to biophotonics including including ﬂuorescence fluorescence mi- microscopycroscopy [37 [37].]. SiPM SiPM consist consist of of a SPAD a SPAD array. array. The The sum sum of pulses of pulses from from all SPADs all SPADs is the is SiPM the SiPMoutput. output. Note Note that thethat number the number of SiPM of SiPM output outp is singleut is single irrespective irrespective of the numberof the number of SPADs of SPADsin the SiPM.in the ManySiPM. photonsMany photons can be simultaneouslycan be simultaneously detected detected by the SPADs. by the FigureSPADs. 17 Figure shows 17a schematicshows a schematic of the operating of the operating principle principle of the SiPMs. of the SiPMs.

FigureFigure 17. 17. SchematicSchematic of of the the operating operating principle principle of of silicon silicon photomultipliers photomultipliers (SiPMs): (SiPMs): SiPMs SiPMs consist consist a aSPAD SPAD array array and and every every SPAD SPAD acts acts as as an an element element that that generates generates an an all-or-nothing all-or-nothing current current pulse. pulse. When Whenone or one more or more photons photons are absorbed, are abso arbed, current a current pulse ispulse generated. is generate A currentd. A current pulse ispulse also is produced also pro- by ducedthe dark by count,the dark whereas count, awhereas current pulsea current is not pulse generated is not generated when photon when absorption photon absorption fails. The output fails. is Thethe output sum of is the the SPADs. sum of Adapted the SPADs. with Adapted permission with from permission [38] © The from Optical [38] © Society. The Optical Society.

SiPMsSiPMs have have several several advantages advantages over over PMTs, PMTs, including including low low fabrication fabrication cost, cost, low low oper- op- atingerating voltage, voltage, and andextremely extremely high highdamage damage thresholds thresholds (high durability). (high durability). In addition, In addition, silicon diodessilicon have diodes high have quantum high quantumefficiency efficiencyin the near-infrared in the near-infrared region used region for deep used tissue for deepim- aging.tissue Due imaging. to these Due factors, to these SiPMs factors, can SiPMsbe better can suited be better for high-speed suited for high-speedimaging. Although imaging. SiPMsAlthough can detect SiPMs intense can detect light, intense their light,dark count their darkrate countis larger rate than is larger that of than PMTs, that which of PMTs, is thewhich major is the tradeoff. major tradeoff.The advantage The advantage of high ofdama highge damage thresholds thresholds is reportedly is reportedly distinct distinct for confocalfor confocal fluorescence fluorescence microscopy microscopy and two-phot and two-photonon microscopy microscopy in clinical in clinical sites where sites where sur- gicalsurgical marking marking inks inksemit emitintense intense fluorescence fluorescence [39]. SiPMs [39]. SiPMs have not have yet not been yet widely been widely used forused biological for biological imaging. imaging. Giacomelli Giacomelli et al. evaluated et al. evaluated the performance the performance of commercial of commercial SiPMs bySiPMs comparing by comparing the SiPMs the with SiPMs a GaAsP with a GaAsPPMT for PMT LSM. for They LSM. reported They reported that the that SiPM the sensi- SiPM tivitysensitivity exceeds exceeds the PMT the PMT sensitivity sensitivity for moderate- for moderate- to -highspeed to -highspeed LSM, LSM, whereas whereas the the PMT PMT exhibitedexhibited better better sensitivity sensitivity due due to to its its lower lower dark dark counts counts for for low low speed speed LSM LSM [39]. [39 Modi]. Modi et al.et also al. also compared compared SiPMs SiPMs products products with with a Ga a GaAsPAsP PMT PMT for for two-photon two-photon imaging imaging of of neural neural activityactivity [38]. [38]. They They showed showed that that SiPM SiPM exhibited exhibited a asignal-to-noise signal-to-noise ratio ratio that that was was comparable comparable toto or or better better than than PMTs PMTs in in usual usual calcium calcium imag imaging,ing, though though dark dark coun countsts of of the the SiPMs SiPMs were were higherhigher than than that that of of the the PMT. PMT. They They concluded concluded that that the the low low pulse pulse height height variability variability of of the the SiPMsSiPMs surpassed surpassed the the weak weak point point and and resulted resulted in in high high performance. performance.

Appl. Sci. 2021, 11, x FOR PEER REVIEW 16 of 20 Appl. Sci. 2021, 11, 2773 16 of 20

4.2. SSPD 4.2. SSPD In recent decades, the superconducting nanowire single photon detector (SSPD) has becomeIn an recent increasingly decades, popular the superconducting device, with nanowireapplications single ranging photon from detector sensing (SSPD) to quan- has become an increasingly popular device, with applications ranging from sensing to quantum tum communications for single photon detection with high efficiency, precise timing, and communications for single photon detection with high efﬁciency, precise timing, and low low noise [24,40]. The crucial high-speed feature of the SSPD is represented by the TTS noise [24,40]. The crucial high-speed feature of the SSPD is represented by the TTS (<50 ps) (<50 ps) the fluctuation between the true arrival time of a photon and the electrically reg- the ﬂuctuation between the true arrival time of a photon and the electrically registered istered arrival time recorded by the system. Korzh et al. showed that the use of low-la- arrival time recorded by the system. Korzh et al. showed that the use of low-latency tency materials lowered the TTS of the SSPD, and demonstrated that the temporal resolu- materials lowered the TTS of the SSPD, and demonstrated that the temporal resolution tion can be 2.6 ± 0.2 ps for visible wavelengths and 4.3 ± 0.2 ps at 1550 nm using a special- can be 2.6 ± 0.2 ps for visible wavelengths and 4.3 ± 0.2 ps at 1550 nm using a specialized ized niobium nitride SSPD [41]. niobium nitride SSPD [41]. Figure 18 shows a schematic of the SSPD. The SSPD consists of superconducting nan- Figure 18 shows a schematic of the SSPD. The SSPD consists of superconducting owire with a thickness of a few nanometers that senses photons. Single photon absorption nanowire with a thickness of a few nanometers that senses photons. Single photon absorp- bytion the by SSPD the SSPDsuppresses suppresses superconductivity, superconductivity, which which in turn in generates turn generates a voltage a voltage spike spikethat canthat be can used be usedto detect to detect the photon. the photon.

Figure 18. Schematic of the operating principle of the superconducting nanowire single photon detec- Figure 18. Schematic of the operating principle of the superconducting nanowire single photon tor (SSPD): The SSPD consists of a superconducting nanowire with a thickness of a few nanometers detector (SSPD): The SSPD consists of a superconducting nanowire with a thickness of a few na- a nometersthat senses that photons. senses Aphotons. bias electrical A bias currentelectrical ﬂows current through flows the through nanowire the during nanowire the operation:during the ( ) the operation:entire area (a is) the in the entire superconducting area is in the superconduct state prior toing photon state absorption; prior to photon (b) single absorption; photon (b absorption) single photontakes place absorption in the superconductingtakes place in the nanowire; superconducting (c) superconductivity nanowire; (c) superconductivity is locally suppressed is locally by energy suppressedexcitation via by theenergy photon excitation absorption; via the (d )photon the bias absorption; current makes (d) the the bias suppressed current areamakes resistive the sup- and the pressedresistive area area resistive expands and across the theresistive nanowire, area expa whichnds in across turn generates the nanowire, a voltage which spike; in turn (e) asgenerates current is adiverted, voltage spike; the resistive (e) as current area relaxes is diverted, to the superconductingthe resistive area state.relaxes Adapted to the superconducting with permission fromstate. [42]. AdaptedCopyright with 2020, permission American from Chemical [42]. Co Society.pyright 2020, American Chemical Society.

TheThe SSPD SSPD is is free free of afterpulses because itit returnsreturnsto to the the superconducting superconducting state state without with- outgenerating generating afterpulses afterpulses after after photon photon detection. detection. The The afterpulsing-free afterpulsing-free characteristic characteristic of theof theSSPD SSPD is a is clear a clear advantage advantage for time-resolvedfor time-resolved fluorescence fluorescence microscopy. microscopy. The SSPDThe SSPD has beenhas beenused used for fluorescencefor fluorescence microscopy microscopy and and applied applied to to FCS FCS [43 [43,44].,44]. AlthoughAlthough the SSPD has has thesethese excellent excellent features, features, its its operation operation is is inconvenient inconvenient due due to to its its small small active active area area and and low low operatingoperating temperature. temperature. The The very very small small active active area area (~10 (~10 µm)µm) makes makes the the optical optical alignment alignment difficultdifficult and and the extraordinaryextraordinary lowlow operating operating temperature temperature (≤ (4≤4 K) K) requires requires the the liquid liquid helium he- liumcooling cooling system. system. DetailedDetailed comparisons comparisons have have been been made made between between SSPDs, SSPDs, SPADs, SPADs, and and other other photonphoton- countingcounting technologies technologies in in [27,40,44]. [27,40,44]. The characteristics of SiPMs and SSPDs are summarized in Table4. Appl. Sci. 2021, 11, x FOR PEER REVIEW 17 of 20

The characteristics of SiPMs and SSPDs are summarized in Table 4.

Table 4. Characteristics of emerging photodetectors [24,27].

SiPM SSPD Afterpulse High Low Transit time spread (TTS) ~300 ps ~50 ps Active area ~3 mm ~10 µm Operating temperature 250 K–room temperature ≤4 K Appl. Sci. 2021, 11, 2773 17 of 20

5. Summary and Outlook Table 4.ThisCharacteristics paper of provides emerging photodetectors an overview [24,27]. of low-light point photodetectors used in fluorescence microscopy and introducesSiPM several point detectors SSPD and their operating principles, focusingAfterpulse on HPDs that exhibit high High timing resolution Low and low afterpulse. In addition, we Transit time spread (TTS) ~300 ps ~50 ps demonstrateActive areaapplication of HPD ~3 to mm wide-field single-molecule ~10 µm fluorescence detection. ≤ OperatingFluorescence temperature imaging 250 with K–room a point temperature photodetector 4needs K scanning to acquire an image and5. Summary thus temporal and Outlook resolution of the imaging is often limited. To overcome this limitation, excitationThis paper methods provides an other overview than of low-light point pointexcitation, photodetectors such used as inmultifocal fluores- excitation line excitation,cence microscopyhave been and proposed introduces several [45,46]. point Regarding detectors and photodetectors, their operating principles, imagers with a large number focusing on HPDs that exhibit high timing resolution and low afterpulse. In addition, we ofdemonstrate pixels have application been of reported HPD to wide-field by many single-molecule groups fluorescence [47–50]. detection.For example, Zickus et al. reported scan-lessFluorescence wide-field imaging with FLIM a point using photodetector a camera needs consisting scanning to acquire of a an500 image × 1024 SPAD array at a rate and thus temporal resolution of the imaging is often limited. To overcome this limita- oftion, 1 excitationHz [51]. methods Michalet other thanet al. point firstly excitation, reported such as the multifocal evaluation excitation of line a multi-pixel (8 × 8) HPD developedexcitation, have by been Hamamatsu proposed [45,46 Photonics]. Regarding photodetectors,[11]. Fukasa imagerswa, an with author a large of this paper, and his col- number of pixels have been reported by many groups [47–50]. For example, Zickus et al. leagues,reported scan-less reported wide-field another FLIM multichannel using a camera consisting HPD that of a 500 was× 1024 composed SPAD ar- of 32 channels (two lines ofray 16 at apixels) rate of 1 Hzon [a51 ].chip, Michalet in which et al. firstly the reported size of the each evaluation pixel of was a multi-pixel 0.8 × 0.8 mm (Figure 19) [52]. It was(8 × 8) confirmed HPD developed that by Hamamatsuthe timing Photonics resolution [11]. Fukasawa, and afterpulse an author of thischaracteristics paper, of the multichan- and his colleagues, reported another multichannel HPD that was composed of 32 chan- nelnels (twoHPD lines are of identical 16 pixels) on to a chip,the conventional in which the size ofsing eachle pixel channel was 0.8 HPD.× 0.8 mm Wollman et al. reported an 1024-element(Figure 19)[52]. It was SSPD confirmed array that (a the 32 timing × 32 resolution row-column and afterpulse multiplexing characteristics ofarchitecture) [53]. Fast ac- the multichannel HPD are identical to the conventional single channel HPD. Wollman et al. quisitionreported an 1024-element methods SSPDfor FLIM array (a 32and× 32 multi-pixel row-column multiplexing photodetectors architecture) have [53]. been reviewed by Liu et al.Fast [22]. acquisition These methods point for detector FLIM and multi-pixel arrays can photodetectors be widely have applied been reviewed to simultaneous by multiparame- Liu et al. [22]. These point detector arrays can be widely applied to simultaneous multipa- terrameter observation, observation, including including simultaneous simultaneous multi-wavelength multi-wavelength fluorescence observation. fluorescence observation.

Figure 19. A photograph of the developed multichannel HPD. Adapted with permission from [52]. FigureCopyright 19. 2016, A Elsevier. photograph of the developed multichannel HPD. Adapted with permission from [52]. Copyright 2016, Elsevier.

Low-light photodetectors find more applications in various fields not limited to biological fluorescence microscopy [23]. These applications include flow cytometry and polymerase chain reaction (PCR) in life science, positron emission tomography (PET) for medical diagnosis, elementary particle (neutroino etc.) detection and collision experiments in high energy physics, and semiconductor wafer inspection in industry. In the near Appl. Sci. 2021, 11, 2773 18 of 20

Low-light photodetectors find more applications in various fields not limited to biological fluorescence microscopy [23]. These applications include flow cytometry and polymerase chain reaction (PCR) in life science, positron emission tomography (PET) for medical diagnosis, elementary particle (neutroino etc.) detection and collision experiments in high energy physics, and semiconductor wafer inspection in industry. In the near future, combination of fluorescence microscopy and other modalities may make low- light photodetectors evolve further and may provide more detailed information on target biological specimens.

Author Contributions: Conceptualization, H.Y.; writing—original draft preparation, H.Y.; writing— review and editing, H.Y., A.F., M.H., and T.I.; investigation, H.Y. and A.F.; formal analysis, H.Y. and A.F.; supervision, H.Y.; visualization, H.Y. and A.F. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Science Research Promotion Fund of the Promotion and Mutual Aid Corporation for Private Schools of Japan (to H.Y., M.H., and T.I.) and the research grant of Tokai Foundation for Technology (to H.Y. and A.F.). Data Availability Statement: The data that support the findings of this study are available from the corresponding author upon reasonable request. Acknowledgments: We would like to thank Yoshihiro Takiguchi for his advice on pulse lasers. Conflicts of Interest: The author declares that there are no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Abbreviations

AD avalanche diode APD avalanche photodiode CW continuous wave DOPC 1,2-dioleoyl-sn-glycero-3-phosphocholine EMCCD electron-multiplying charge-coupled device FCS fluorescence correlation spectroscopy FLIM fluorescence lifetime imaging microscopy FRET fluorescence resonance energy transfer FWHM full-width half-maximum GFP green fluorescent protein HPD hybrid photodetector LSM laser scanning microscopy MPPC multi-pixel photon counter PCR polymerase chain reaction PE phosphoethanolamine PET positron emission tomography PMT photomultiplier tube Qdot quantum dot sCMOS scientific complementary metal-oxide-semiconductor SiPM silicon photomultiplier SPAD single photon avalanche diode SSPD superconducting nanowire single photon detector TCSPC time-correlated single photon counting TTS transit time spread

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