Functional Fluorescence Microscopy Imaging: Quantitative Scanning-Free Confocal Fluorescence Microscopy for the Characterization of Fast Dynamic Processes in Live Cells.

Functional fluorescence microscopy imaging (fFMI), a time-resolved (21 µs/frame) confocal fluorescence microscopy imaging technique without scanning, is developed for quantitative characterization of fast reaction-transport processes in solution and in live cells. The method is based on massively parallel fluorescence correlation spectroscopy (FCS). Simultaneous excitation of fluorescent molecules in multiple spots in the focal plane is achieved using a diffractive optical element (DOE). Fluorescence from the DOE-generated 1024 illuminated spots is detected in a confocal arrangement by a matching matrix detector comprising 32 × 32 single-photon avalanche photodiodes (SPADs). Software for data acquisition and fast auto- and cross-correlation analysis by parallel signal processing using a graphic processing unit (GPU) allows temporal autocorrelation across all pixels in the image frame in 4 s and cross-correlation between first- and second-order neighbor pixels in 45 s. We present here this quantitative, time-resolved imaging method with single-molecule sensitivity and demonstrate its usefulness for mapping in live cell location-specific differences in the concentration and translational diffusion of molecules in different subcellular compartments. In particular, we show that molecules without a specific biological function, e.g., the enhanced green fluorescent protein (eGFP), exhibit uniform diffusion. In contrast, molecules that perform specialized biological functions and bind specifically to their molecular targets show location-specific differences in their concentration and diffusion, exemplified here for two transcription factor molecules, the glucocorticoid receptor (GR) before and after nuclear translocation and the Sex combs reduced (Scr) transcription factor in the salivary gland of Drosophila ex vivo.

Multispectral Depth-Resolved Fluorescence Lifetime Spectroscopy Using SPAD Array Detectors and Fiber Probes.

Single Photon Avalanche Diode (SPAD) arrays are increasingly exploited and have demonstrated potential in biochemical and biomedical research, both for imaging and single-point spectroscopy applications. In this study, we explore the application of SPADs together with fiber-optic-based delivery and collection geometry to realize fast and simultaneous single-point time-, spectral-, and depth-resolved fluorescence measurements at 375 nm excitation light. Spectral information is encoded across the columns of the array through grating-based dispersion, while depth information is encoded across the rows thanks to a linear arrangement of probe collecting fibers. The initial characterization and validation were realized against layered fluorescent agarose-based phantoms. To verify the practicality and feasibility of this approach in biological specimens, we measured the fluorescence signature of formalin-fixed rabbit aorta samples derived from an animal model of atherosclerosis. The initial results demonstrate that this detection configuration can report fluorescence spectral and lifetime contrast originating at different depths within the specimens. We believe that our optical scheme, based on SPAD array detectors and fiber-optic probes, constitute a powerful and versatile approach for the deployment of multidimensional fluorescence spectroscopy in clinical applications where information from deeper tissue layers is important for diagnosis.

A quantum-enhanced wide-field phase imager.

Quantum techniques can be used to enhance the signal-to-noise ratio in optical imaging. Leveraging the latest advances in single-photon avalanche diode array cameras and multiphoton detection techniques, here, we introduce a supersensitive phase imager, which uses space-polarization hyperentanglement to operate over a large field of view without the need of scanning operation. We show quantum-enhanced imaging of birefringent and nonbirefringent phase samples over large areas, with sensitivity improvements over equivalent classical measurements carried out with equal number of photons. The potential applicability is demonstrated by imaging a biomedical protein microarray sample. Our technology is inherently scalable to high-resolution images and represents an essential step toward practical quantum-enhanced imaging.

Sub-Rayleigh imaging via N-photon detection.

The Rayleigh diffraction bound sets the minimum separation for two point objects to be distinguishable in a conventional imaging system. We demonstrate sub-Rayleigh resolution by scanning a focused beam--in an arbitrary, object-covering pattern that is unknown to the imager--and using N-photon photodetection implemented with a single-photon avalanche detector array. Experiments show resolution improvement by a factor ∼(N-N(max))(½) beyond the Rayleigh bound, where N(max) is the maximum average detected photon number in the image, in good agreement with theory.

Real-time multispectral fluorescence lifetime imaging using Single Photon Avalanche Diode arrays.

Autofluorescence spectroscopy has emerged in recent years as a powerful tool to report label-free contrast between normal and diseased tissues, both in vivo and ex vivo. We report the development of an instrument employing Single Photon Avalanche Diode (SPAD) arrays to realize real-time multispectral autofluorescence lifetime imaging at a macroscopic scale using handheld single-point fibre optic probes, under bright background conditions. At the detection end, the fluorescence signal is passed through a transmission grating and both spectral and temporal information are encoded in the SPAD array. This configuration allows interrogation in the spectral range of interest in real time. Spatial information is provided by an external camera together with a guiding beam that provides a visual reference that is tracked in real-time. Through fast image processing and data analysis, fluorescence lifetime maps are augmented on white light images to provide feedback of the measurements in real-time. We validate and demonstrate the practicality of this technique in the reference fluorophores and in articular cartilage samples mimicking the degradation that occurs in osteoarthritis. Our results demonstrate that SPADs together with fibre probes can offer means to report autofluorescence spectral and lifetime contrast in real-time and thus are suitable candidates for in situ tissue diagnostics.

Variable-load quenching circuit for single-photon avalanche diodes.

We present a compact 50 microm x 100 microm cell for single-photon detection, based on a new circuitry monolithically integrated together with a 20 microm-diameter CMOS Single-Photon Avalanche Diode (SPAD). The detector quenching relies on a novel mechanism based on starving the avalanche current till quenching through a variable-load (VLQC, Variable- Load Quenching Circuit). Fabricated in a standard 0.35 microm CMOS technology, the topology allows a SPAD bias voltage higher than the chip supply voltage to be used. Moreover it preserves the advantages of active quenching circuits, in terms of hold-off capability (from 40 ns to 2 micros) and fast reset (

Time-resolved double-slit interference pattern measurement with entangled photons.

The double-slit experiment strikingly demonstrates the wave-particle duality of quantum objects. In this famous experiment, particles pass one-by-one through a pair of slits and are detected on a distant screen. A distinct wave-like pattern emerges after many discrete particle impacts as if each particle is passing through both slits and interfering with itself. Here we present a temporally- and spatially-resolved measurement of the double-slit interference pattern using single photons. We send single photons through a birefringent double-slit apparatus and use a linear array of single-photon detectors to observe the developing interference pattern. The analysis of the buildup allows us to compare quantum mechanics and the corpuscular model, which aims to explain the mystery of single-particle interference. Finally, we send one photon from an entangled pair through our double-slit setup and show the dependence of the resulting interference pattern on the twin photon's measured state. Our results provide new insight into the dynamics of the buildup process in the double-slit experiment, and can be used as a valuable resource in quantum information applications.

Low-noise low-jitter 32-pixels CMOS single-photon avalanche diodes array for single-photon counting from 300 nm to 900 nm.

We developed a single-photon counting multichannel detection system, based on a monolithic linear array of 32 CMOS SPADs (Complementary Metal-Oxide-Semiconductor Single-Photon Avalanche Diodes). All channels achieve a timing resolution of 100 ps (full-width at half maximum) and a photon detection efficiency of 50% at 400 nm. Dark count rate is very low even at room temperature, being about 125 counts/s for 50 µm active area diameter SPADs. Detection performance and microelectronic compactness of this CMOS SPAD array make it the best candidate for ultra-compact time-resolved spectrometers with single-photon sensitivity from 300 nm to 900 nm.

Ultra high-throughput single molecule spectroscopy with a 1024 pixel SPAD.

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.

Complete single-photon counting and timing module in a microchip.

A complete module for single-photon counting and timing is demonstrated in a single chip. Features comparable with or better than commercially available macroscopic modules are obtained by integration of an active-quenching and active-reset circuit in complementary metal-oxide semiconductor technology together with a single-photon avalanche diode (SPAD). The integrated SPAD has a 12-microm-diameter sensitive area and operates with an overvoltage above breakdown adjustable up to 20 V. With a 5-V overvoltage the photon detection efficiency peaks above 40% around 500 nm, and the dark-counting rate is lower than 600 counts/s at room temperature. The overall counting dead time is 33 ns.