KEYWORDS: Fluorescence resonance energy transfer, Molecules, Sensors, Liquid crystal on silicon, Field programmable gate arrays, Data transmission, Imaging systems, Resonance energy transfer, Luminescence, Objectives
Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for extracting distance information
between two fluorophores (a donor and acceptor dye) on a nanometer scale. This method is commonly used to monitor
binding interactions or intra- and intermolecular conformations in biomolecules freely diffusing through a focal volume
or immobilized on a surface. The diffusing geometry has the advantage to not interfere with the molecules and to give
access to fast time scales. However, separating photon bursts from individual molecules requires low sample
concentrations. This results in long acquisition time (several minutes to an hour) to obtain sufficient statistics. It also
prevents studying dynamic phenomena happening on time scales larger than the burst duration and smaller than the
acquisition time. Parallelization of acquisition overcomes this limit by increasing the acquisition rate using the same low
concentrations required for individual molecule burst identification. In this work we present a new two-color smFRET
approach using multispot excitation and detection. The donor excitation pattern is composed of 4 spots arranged in a
linear pattern. The fluorescent emission of donor and acceptor dyes is then collected and refocused on two separate areas
of a custom 8-pixel SPAD array. We report smFRET measurements performed on various DNA samples synthesized
with various distances between the donor and acceptor fluorophores. We demonstrate that our approach provides
identical FRET efficiency values to a conventional single-spot acquisition approach, but with a reduced acquisition time.
Our work thus opens the way to high-throughput smFRET analysis on freely diffusing molecules.
Fluorescence lifetime can be used as a contrast mechanism to distinguish fluorophores for localization or tracking, for studying molecular interactions, binding, assembly, and aggregation, or for observing conformational changes via Förster resonance energy transfer (FRET) between donor and acceptor molecules. Fluorescence lifetime imaging microscopy (FLIM) is thus a powerful technique but its widespread use has been hampered by demanding hardware and software requirements. FLIM data is often analyzed in terms of multicomponent fluorescence lifetime decays, which requires large signals for a good signal-to-noise ratio. This confines the approach to very low frame rates and limits the number of frames which can be acquired before bleaching the sample. Recently, a computationally efficient and intuitive graphical representation, the phasor approach, has been proposed as an alternative method for FLIM data analysis at the ensemble and single-molecule level. In this article, we illustrate the advantages of combining phasor analysis with a widefield time-resolved single photon-counting detector (the H33D detector) for FLIM applications. In particular we show that phasor analysis allows real-time subsecond identification of species by their lifetimes and rapid representation of their spatial distribution, thanks to the parallel acquisition of FLIM information over a wide field of view by the H33D detector. We also discuss possible improvements of the H33D detector's performance made possible by the simplicity of phasor analysis and its relaxed timing accuracy requirements compared to standard time-correlated single-photon counting (TCSPC) methods.
Cross strip and cross delay line readout microchannel plate detectors in 18 mm, 25 mm and 40 mm active area
formats including open face (UV/particle) and sealed tube (optical) configurations have been constructed. These have
been tested with a field programmable gate array based electronics for single event encoding. Using small pore MCPs (6 μm) operated in a pair, we achieve gains of >1 x 106 which is sufficient to provide spatial resolution of ~17 μm FHWM
with the 18 mm and 40 mm cross strip readouts. New cross strip electronics can process high output event rates (> 4
MHz) with high spatial resolution, and self triggered event timing accuracy of ~1.5 ns for sealed tube XS optical
sensors. A peak quantum efficiency of between 13% and 19% at 500 nm has been achieved with SuperGenII
photocathodes with response from 400 nm to 900 nm for both cross strip and cross delay line sealed tubes. Local area
counting rates of up to 40 kHz (100μm spot) have been attained with XS sealed tubes, along with image linearity and
stability to better than 50 μm. 25mm cross delay line tubes achieve ~50 μm resolution and > 2 MHz output event rates.
KEYWORDS: Sensors, Molecules, Imaging spectroscopy, Signal detection, Fluorescence correlation spectroscopy, Photodetectors, Temporal resolution, Single molecule spectroscopy, Point spread functions, Signal to noise ratio
Solution-based single-molecule fluorescence spectroscopy is a powerful new experimental approach with applications in
all fields of natural sciences. Two typical geometries can be used for these experiments: point-like and widefield
excitation and detection. In point-like geometries, the basic concept 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. In widefield geometries, the same issues of background reduction and single-molecule concentration
apply, but the duration of the experiment is fixed by the time scale of the process studied and the survival time of the
fluorescent probe. Temporal resolution on the other hand, is limited by signal-to-noise and/or detector resolution, which
calls for new detector concepts. We will briefly present our recent results in this domain.
Single-molecule spectroscopy is a powerful approach to measuring molecular properties such as size, brightness,
conformation, and binding constants. Due to the low concentrations in the single-molecule regime, measurements with
good statistical accuracy require long acquisition times. Previously we showed a factor of 8 improvement in acquisition
speed using a custom-CMOS 8x1 SPAD array. Here we present preliminary results with a 64X improvement in
throughput obtained using a liquid crystal on silicon spatial light modulator (LCOS-SLM) and a novel standard CMOS
1024 pixel SPAD array, opening the way to truly high-throughput single-molecule spectroscopy.
Solution-based single-molecule spectroscopy and fluorescence correlation spectroscopy (FCS) are powerful techniques
to access a variety of molecular properties such as size, brightness, conformation, and binding constants. However, this
is limited to low concentrations, which results in long acquisition times in order to achieve good statistical accuracy.
Data can be acquired more quickly by using parallelization. We present a new approach using a multispot excitation and
detection geometry made possible by the combination of three powerful new technologies: (i) a liquid crystal spatial
light modulator to produce multiple diffraction-limited excitation spots; (ii) a multipixel detector array matching the
excitation pattern and (iii) a low-cost reconfigurable multichannel counting board. We demonstrate the capabilities of
this technique by reporting FCS measurements of various calibrated samples as well as single-molecule burst
measurements.
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.
Fluorescence lifetime imaging (FLIM) is a powerful approach to studying the immediate environment of molecules. For
example, it is used in biology to study changes in the chemical environment, or to study binding processes, aggregation,
and conformational changes by measuring Förster resonance energy transfer (FRET) between donor and acceptor
fluorophores. FLIM can be acquired by time-domain measurements (time-correlated single-photon counting) or
frequency-domain measurements (with PMT modulation or digital frequency domain acquisition) in a confocal setup, or
with wide-field systems (using time-gated cameras). In the best cases, the resulting data is analyzed in terms of
multicomponent fluorescence lifetime decays with demanding requirements in terms of signal level (and therefore
limited frame rate). Recently, the phasor approach has been proposed as a powerful alternative for fluorescence lifetime
analysis of FLIM, ensemble, and single-molecule experiments. Here we discuss the advantages of combining phasor
analysis with a new type of FLIM acquisition hardware presented previously, consisting of a high temporal and spatial
resolution wide-field single-photon counting device (the H33D detector). Experimental data with live cells and quantum
dots will be presented as an illustration of this new approach.
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