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Register a new userSub-100 nm 3-D fluorescence lifetime imaging using time correlated single photon counting detection and multifocal multiphoton excitation
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Suhas Kumar
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We propose and experimentally demonstrate a high-efficiency single-pixel imaging (SPI) scheme by integrating time-correlated single-photon counting (TCSPC) with time-division multiplexing to acquire full-color images at extremely low light level. This SPI scheme uses a digital micromirror device to modulate a sequence of laser pulses with preset delays to achieve three-color structured illumination, then employs a photomultiplier tube into the TCSPC module to achieve photon-counting detection. By exploiting the time-resolved capabilities of TCSPC, we demodulate the spectrum-image-encoded signals, and then reconstruct high-quality full-color images in a single-round of measurement. Based on this scheme, the strategies such as single-step measurement, high-speed projection, and undersampling can further improve the imaging efficiency.
We demonstrated a laser depth imaging system based on the time-correlated single-photon counting technique, which was incorporated with a low-jitter superconducting nanowire single-photon detector (SNSPD), operated at the wavelength of 1550 nm. A sub-picosecond time-bin width was chosen for photon counting, resulting in a discrete noise of less than one/two counts for each time bin under indoor/outdoor daylight conditions, with a collection time of 50 ms. Because of the low-jitter SNSPD, the target signal histogram was significantly distinguishable, even for a fairly low retro-reflected photon flux. The depth information was determined directly by the highest bin counts, instead of using any data fitting combined with complex algorithms. Millimeter resolution depth imaging of a low-signature object was obtained, and more accurate data than that produced by the traditional Gaussian fitting method was generated. Combined with the intensity of the return photons, three-dimensional reconstruction overlaid with reflectivity data was realized.
Fluorescence Lifetime Imaging Microscopy (FLIM) using multiphoton excitation techniques is now finding an important place in quantitative imaging of protein-protein interactions and intracellular physiology. We review here the recent developments in multiphoton FLIM methods and also present a description of a novel multiphoton FLIM system using a streak camera that was developed in our laboratory. We provide an example of a typical application of the system in which we measure the fluorescence resonance energy transfer between a donor/acceptor pair of fluorescent proteins within a cellular specimen.
We report the development and detailed calibration of a multiphoton fluorescence lifetime imaging system (FLIM) using a streak camera. The present system is versatile with high spatial (0.2 micron) and temporal (50 psec) resolution and allows rapid data acquisition and reliable and reproducible lifetime determinations. The system was calibrated with standard fluorescent dyes and the lifetime values obtained were in very good agreement with values reported in literature for these dyes. We also demonstrate the applicability of the system to FLIM studies in cellular specimens including stained pollen grains and fibroblast cells expressing green fluorescent protein. The lifetime values obtained matched well with those reported earlier by other groups for these same specimens. Potential applications of the present system include the measurement of intracellular physiology and Fluorescence Resonance Energy Transfer (FRET) imaging which are discussed in the context of live cell imaging.
Cathodoluminescence (CL) imaging spectroscopy is an important technique to understand resonant behavior of optical nanoantennas. We report high-resolution CL spectroscopy of triangular gold nanoantennas designed with near-vacuum effective index and very small metal-substrate interface. This design helped in addressing issues related to background luminescence and shifting of dipole modes beyond visible spectrum. Spatial and spectral investigations of various plasmonic modes are reported. Out-of-plane dipole modes excited with vertically illuminated electron beam showed high-contrast tip illumination in panchromatic imaging. By tilting the nanostructures during fabrication, in-plane dipole modes of antennas were excited. Finite-difference time-domain simulations for electron and optical excitations of different modes showed excellent agreement with experimental results. Our approach of efficiently exciting antenna modes by using low index substrates is confirmed both with experiments and numerical simulations. This should provide further insights into better understanding of optical antennas for various applications.
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