Solution-based single-molecule fluorescence spectroscopy is a powerful new experimental approach with applications in
all fields of natural sciences. The basic concept of this technique is to excite and collect light from a very small volume
(typically femtoliter) and work in a concentration regime resulting in rare burst-like events corresponding to the transit
of a single-molecule. Those events are accumulated over time to achieve proper statistical accuracy. Therefore the
advantage of extreme sensitivity is somewhat counterbalanced by a very long acquisition time. One way to speed up data
acquisition is parallelization. Here we will discuss a general approach to address this issue, using a multispot excitation
and detection geometry that can accommodate different types of novel highly-parallel detector arrays. We will illustrate
the potential of this approach with fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence
measurements obtained with different novel multipixel single-photon counting detectors.
A Complementary Metal Oxide Semiconductor (CMOS) camera (1024×1024 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.
We report benchmark tests of a new single-photon counting detector based on a GaAsP photocathode and an electron-bombarded
avalanche photodiode developed by Hamamatsu Photonics. We compare its performance with those of
standard Geiger-mode avalanche photodiodes. We show its advantages for FCS due to the absence of after-pulsing and
for fluorescence lifetime measurements due to its excellent time resolution. Its large sensitive area also greatly simplifies
setup alignment. Its spectral sensitivity being similar to that of recently introduced CMOS SPADs, this new detector
could become a valuable tool for single-molecule fluorescence measurements, as well as for many other applications.
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