Two optical configurations are commonly used in single-molecule fluorescence microscopy: point-like excitation and detection to study freely diffusing molecules, and wide field illumination and detection to study surface immobilized or slowly diffusing molecules. Both approaches have common features, but also differ in significant aspects. In particular, they use different detectors, which share some requirements but also have major technical differences. Currently, two types of detectors best fulfil the needs of each approach: single-photon-counting avalanche diodes (SPADs) for point-like detection, and electron-multiplying charge-coupled devices (EMCCDs) for wide field detection. However, there is room for improvements in both cases. The first configuration suffers from low throughput owing to the analysis of data from a single location. The second, on the other hand, is limited to relatively low frame rates and loses the benefit of single-photon-counting approaches. During the past few years, new developments in point-like and wide field detectors have started addressing some of these issues. Here, we describe our recent progresses towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. We also discuss our development of large area photon-counting cameras achieving subnanosecond resolution for fluorescence lifetime imaging applications at the single-molecule level.

, 368 2013 Phil. Trans. R. Soc. B Guerrieri, F. Panzeri, I. Rech, A. Gulinatti, F. Zappa, M. Ghioni and S. Cova Siegmund, Anton S. Tremsin, John V. Vallerga, A. Cheng, M. Levi, D. Aharoni, K. Arisaka, F. Villa, F. X. Michalet, R. A. Colyer, G. Scalia, A. Ingargiola, R. Lin, J. E. Millaud, S. Weiss, Oswald H. W. single-molecule fluorescence microscopy Development of new photon-counting detectors for

A Complementary Metal Oxide Semiconductor (CMOS) camera (1024x1024 pixels) is used to record spontaneous oscillations of hair cell stereocillia in an in-vitro preparation of the bullfrog sacculus with the otolithic membrane removed. The CMOS camera is attached to an Olympus BX51WI Microscope inside of a sound-isolation chamber, with white light transmission illumination using an X-Cite 120 metal halogenide lamp. The combination of the parallel readout of the CMOS chip and the high intensity of illumination allows full frame images of the oscillations to be taken at 1000 frames per second. A weighted, time averaged differential algorithm is used to aid in the visualization of the hair cell movement. To detect the displacement from its center of the stereocillia tip with nanometer position resolution and millisecond time resolution, an average background intensity value was subtracted from each image to remove lamp intensity fluctuations and then a center of intensity algorithm was applied. This combination of our imaging system and data analysis allows for the oscillations of more than one hair cell to be recorded during the same time period, and their frequency components extracted.