Given the complexity of biological systems, it is necessary to go beyond ensemble measurements and attain information at the single molecule level to accurately probe molecular properties. Single molecule imaging can examine real-time conformational dynamics [1], which is often the underlying cause of heterogeneity in molecular distributions in terms of dipole orientations, spectra, or intramolecular distances, in both stable and unstable systems.
A combination of polarisation-resolved detection and 2-colour alternating laser excitation (ALEX) allows quantification of the anisotropy and stoichiometry of the fluorophores present [2]. As a result, it is possible to accurately quantify energy transfer (e.g. FRET). This technique provides a rapid approach for probing the fluorophore’s environment in terms of viscosity, interactions between molecules, and ligand-substrate binding.
Here we present an optimized TIRF microscope in conjunction with ALEX and steady state fluorescence anisotropy detection [3] for single molecule imaging. Validation and determination of the limits of the technique will be by measurement of isolated fluorescent proteins. Preliminary data of single molecules with a fluorescence anisotropy read-out will be presented and future prospects discussed.
1. Santoso Y, et al. (2010) Conformational transitions in DNA polymerase I revealed by single-molecule FRET. PNAS 107: 715–720.
2. Kapanidis A, et al. (2004) Fluorescence-aided molecule sorting: Analysis of structure and interactions by alternating-laser excitation of single molecules PNAS.
101 8936-8941.
3. Devauges V, et al. (2014) Steady-state acceptor fluorescence anisotropy imaging under evanescent excitation for visualisation of FRET at the plasma membrane.
PLoS One 9: e110695.
Imaging viscosity and its spatiotemporal patterns can provide valuable insight into the underlying physical conditions of biochemical reactions and biological processes in cells and tissues. One way to measure viscosity and diffusion is the use of fluorescence recovery after photobleaching (FRAP). We combine FRAP with FLIM and time-resolved fluorescence anisotropy imaging (tr-FAIM), by acquiring time- and polarization-resolved fluorescence images in every frame of a FRAP series. This allows us to simultaneously monitor translational and rotational diffusion. This approach can be applied to measuring diffusion in homogeneous and heterogeneous environments, and in principle also allows the study of homo-FRET. Another way to measure viscosity and diffusion is through specific flexible dyes, e.g. fluorescent molecular rotors, whose fluorescence quantum yield and fluorescence lifetime depend on the viscosity of the environment, in combination with fluorescence lifetime imaging (FLIM). We show that a bodipybased fluorescent molecular rotor targeting mitochondria reports on their viscosity, which changes under physiological stimuli. Both methods can optically measure viscosity and diffusion on the micrometer scale.
We report the simultaneous combination of three powerful techniques in uorescence microscopy: Fluorescence Lifetime Imaging (FLIM), Fluorescence Anisotropy Imaging (FAIM) and Fluorescence Recovery After Photobleaching (FRAP), also called F3 microscopy. An exhaustive calibration of the setup was carried out with several rhodamine 6G (R6G) solutions in water-glycerol and from the combination of the FAIM and FRAP data, the hydrodynamic radius of the dye was directly calculated. The F3 data was analyzed with a home-built MATLAB script, and the setup is currently explored further with Green Fluorescent Protein (GFP). Some molecular dynamic (MD) simulations are currently being run in order to help with the interpretation of the experimental anisotropy data.
KEYWORDS: Fluorescence resonance energy transfer, Sensors, Luminescence, Single photon, Time resolved spectroscopy, CMOS sensors, Spectroscopy, Molecules, Molecular energy transfer, Time correlated photon counting
We demonstrate a new 512x16 single photon avalanche diode (SPAD) based line sensor with per-pixel TCSPC histogramming for time-resolved, time-zoomable, FRET spectroscopy. The line sensor can operate in single photon counting (SPC) mode as well as time-correlated single photon counting (TCSPC) and per-pixel histogramming modes. TCSPC has been the preferred method for fluorescence lifetime measurements due to its collection of full decays as a histogram of arrival times. However, TCSPC is slow due to only capturing one photon per exposure and large timestamp data transfer requirements for offline histogramming. On-chip histogramming improves the data rate by allowing multiple SPAD pulses (up to one pulse per laser period) to be processed in each exposure cycle, along with secondly reducing the I/O bottleneck as only the final histogram is transferred. This can enable 50x higher acquisition rates (up to 10 billion counts per second), along with time-zoomable histogramming operation from 1.6ns to 205ns with 50ps resolution. A broad spectral range can be interrogated with the sensor (450-900nm). Overall, these sensors provide a unique combination of light sensing capabilities for use in high speed, sensitive, optical instrumentation in the time/wavelength domain. We test the sensor performance by observation of fluorescence resonance energy transfer (FRET) between FAM and TAMRA and between EGFP and RFP FRET standards.
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