# Low-Light Photodetectors for Fluorescence Microscopy
## 3 Single Point Detectors
### High Resolution of Photon Counting
#### 3.4.4 Application of HPDs to Single-Molecule Fluorescence Microscopy  
We applied the cooled-photocathode HPD to single-molecule fluorescence microscopy. We applied the HPD to low-background wide-field single-molecule fluorescence detection with high temporal resolution as a proof-of-principle demonstration. The fluorescence collected by an objective was divided into two, each simultaneously detected by HPD or imaged by EMCCD. The HPD allowed the fluorescence intensity of a mobile single molecule fluorophore to be determined at higher temporal resolution than conventional high-sensitivity CCD cameras. Specifically, the cooled-photocathode HPD detected fluorescence of a single Qdot while performing two-dimensional diffusion with 0.1 ms temporal resolution (Figure 15).  
The HPD also enabled wide-field single-molecule fluorescence lifetime measurement with nanosecond temporal resolution. We succeeded in obtaining time courses of fluorescence lifetime of a single Qdot whose fluorescence images were simultaneously monitored 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 was excited by a pulse laser (wavelength = 520 nm); (**b**) Time courses of the Qdot fluorescence (upper, blue) obtained by an EMCCD and the lifetime (lower, red) simultaneously obtained by an HPD. The lifetime was obtained by fitting each decay curve drawn using data accumulated in three seconds with a single-exponential (red); (**c**) Trajectory of the Qdot obtained by EMCCD. The inset is a fluorescence image of the observed mobile Qdot.  
Figure 15: Wide-field sub-millisecond single-molecule fluorescence detection by a cooled HPD [32]: (**a**) Schematic of the observed mobile Qdot. The Qdot (Qdot655 streptavidin conjugate) was attached to biotinylated phosphoethanolamine (PE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl), via biotin-streptavidin interaction in a 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer formed on a coverslip. The Qdot was excited by a CW laser (wavelength = 532 nm); (**b**) Time course of a mobile Qdot fluorescence intensity was obtained by an HPD with a time resolution of 0.1 ms (red). The repeated fluorescence-on and –off are caused by blinking. The intensity profile matches the intensity simultaneously obtained by EMCCD (blue) with a time resolution of 31.319 ms; (**c**) Trajectory of the Qdot obtained by EMCCD. The inset is a fluorescence image of the observed mobile Qdot.  
A group at Tohoku University incorporated two HPDs into a specially equipped line confocal optical system in which a slit was used instead of a pinhole to improve the time resolution of single-molecule FRET study. They achieved FRET observation of single protein molecules that flowed unidirectionally in a flow cell at a time resolution of 10 us and analyzed the high-speed folding process of protein molecules [36].