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We report on a novel human retina imaging system based on Spatio-Temporal Optical Coherence Tomography (STOC-T). We present structural images and spatially-resolve functional responses of the retinal tissue to white light stimulation.

Fourier-domain full-field optical coherence tomography (FD-FF-OCT) is a fast interferometric imaging technique capable of volumetric sample imaging. However, half of the backscattered light from a sample is lost as it passes through a 50/50 beam splitter, which is at the heart of almost every interferometer. Here, it is demonstrated that this light could be extracted by spatially splitting the illumination pupil plane and detecting it with a separate camera. When a line camera is used to detect the recovered signal, it enables real-time axial imaging of the human cornea in vivo, which serves as a useful visual feedback for aligning a patient for imaging.

Fourier-domain full-field optical coherence tomography (FD-FF-OCT) has recently emerged as a fast alternative to point-scanning confocal OCT in eye imaging. However, when imaging the cornea with FD-FF-OCT, a spatially coherent laser can focus down on the retina to a spot that exceeds the maximum permissible exposure level. Here we demonstrate that a long multimode fiber with a small core can be used to reduce the spatial coherence of the laser and, thus, enable ultrafast in vivo volumetric imaging of the human cornea without causing risk to the retina.

Fourier-domain full-field optical coherence tomography (FD-FF-OCT) is currently the fastest volumetric imaging technique that is able to generate a single 3D data volume in less than 10 ms. FD-FF-OCT is based on a fast camera, a rapidly tunable laser source and a Fourier-domain signal detection. However, crosstalk corrupts images with speckle and therefore lowers image quality. Corneal imaging with FD-FF-OCT is also challenging since the laser focuses down on the retina causing safety concerns. We present spatially incoherent FD-FF-OCT system that reduces crosstalk noise in retina images and enables corneal imaging by reducing spatial coherence of the laser.

Optical coherence tomography (OCT) is a well-established modality providing cross-sectional images of the human retina. OCT, however, does not provide high-resolution en face images of the outer retinal layers due to eye aberrations and the tradeoff between imaging depth and transverse resolution. Recently, we demonstrated the spatiotemporal optical coherence (STOC) that provides high-resolution, aberration-free volumetric images of the retina and cornea with the voxel rate of 8 GHz. Here, we show that STOC can be used for angiography, and allows us to estimate Doppler broadening, enabling visualization of the choroidal blood vessels in the human retina in vivo.

We developed and applied spectral laser doppler holography (SLDH) to Fourier-Domain Full-Field Optical Coherence Tomography (FD-FF-OCT) enabling visualization and quantification of the human retinal blood flow at high speeds and high transverse resolution in vivo.

We present an optimized Fourier-domain full-field optical coherence tomography system for corneal in vivo imaging that uses a line camera for real-time cross-sectional preview and a multimode fiber for preventing retinal damage.

For many years electroretinography (ERG) has been used for obtaining information about the retinal physiological function. More recently, a new technique called optoretinography (ORG) has been developed. In one form of this technique, the physiological response of retinal photoreceptors to visible light, resulting in a nanometric photoreceptor optical path length change, is measured by phase-sensitive optical coherence tomography (OCT). To date, a limited number of studies with phase-based ORG measured the retinal response to a flickering light stimulation. In this work, we use a spatio-temporal optical coherence tomography (STOC-T) system to capture optoretinograms with a flickering stimulus over a 1.7 × 0.85 mm2 area of a light-adapted retina located between the fovea and the optic nerve. We show that we can detect statistically-significant differences in the photoreceptor optical path length (OPL) modulation amplitudes in response to different flicker frequencies and with better signal to noise ratios (SNRs) than for a dark-adapted eye. We also demonstrate the ability to spatially map such response to a patterned stimulus with light stripes flickering at different frequencies, highlighting the prospect of characterizing the spatially-resolved temporal-frequency response of the retina with ORG.

Spatiotemporal optical coherence (STOC) imaging is a new technique for suppressing coherent crosstalk noise in Fourier-domain full-field optical coherence tomography (FD-FF-OCT). In STOC imaging, the timevarying inhomogeneous phase masks modulate the incident light to alter the interferometric signal. Resulting interference images are then processed as in standard FD-FF-OCT and averaged incoherently or coherently to produce crosstalk-free volumetric OCT images of the sample. Here, we show that coherent averaging is suitable when phase modulation is performed for both interferometer arms simultaneously. We explain the advantages of coherent over incoherent averaging. Specifically, we show that modulated signal, after coherent averaging, preserves lateral phase stability. This enables computational phase correction to compensate for geometrical aberrations. Ultimately, we employ it to correct for aberrations present in the image of the photoreceptor layer of the human retina that reveals otherwise invisible photoreceptor mosaic., 4 pages, 5 figures

Spatiotemporal optical coherence (STOC) imaging is a novel technique for suppressing coherent cross-talk noise in full-field swept-source OCT (FF-SS-OCT). In STOC, we use time-varying phase masks to modulate light incident on the sample. The modulated signals are averaged incoherently or coherently to yield cross-talk-free 3D images of the sample. We show that coherent averaging is only possible under specific hardware configuration. We explain this theoretically and confirm experimentally by imaging USAF chart covered by diffusers and the rat skin ex vivo. Finally, we present human forearm skin imaging in vivo. In all cases, STOC imaging reveals otherwise invisible sample features.

We apply spatiotemporal optical coherence (STOC) manipulation with digital aberration correction to achieve aberration-free, crosstalk-free, high-speed, high-resolution Fourier-domain full-field optical coherence tomography (FD-FF-OCT). We demonstrate the results of human in vivo retinal and corneal imaging.

We present a crosstalk-free Fourier-domain full-field optical coherence tomography system that uses a fast deformable membrane for spatial coherence reduction. It enables the visualization of the choroids and helps to better delineate the retinal layers.

We present spatially incoherent Fourier-domain full-field optical coherence tomography system that reduces crosstalk noise in retina images and enables corneal imaging. It is achieved by using a fast deformable membrane to reduce spatial coherence.

We apply coherent averaging and digital aberration correction to spatiotemporal optical coherence (STOC) imaging to achieve aberration-free, crosstalk-free volumetric imaging. We demonstrate results of ex vivo samples and human in vivo retinal and corneal imaging.

Fourier-domain full-field optical coherence tomography (FD-FF-OCT) is an emerging tool for high-speed eye imaging. However, cross-talk formation in images limits the imaging depth. To this end, we have recently shown that reducing spatial coherence with a fast deformable membrane can suppress the noise but over a limited axial range and with substantial data processing. Here, we demonstrate that a multimode fiber with carefully chosen parameters enables cross-talk-free imaging over a long axial range and without significant artifacts. We also show that it can be used to image the human retina and choroid in vivo with exceptional contrast.

The emergence of Full Field-Optical Coherence Tomography (FF-OCT) for fingerprint imaging has shown it's ability in addressing and solving the drawbacks of traditional fingerprinting solutions such as spoofing attacks, low accuracy for abraded fingerprint. With the availability of multiple internal fingerprints (from subsurface captured at different depths), it is also essential to consider the aspects of ideal biometrics where the privacy of the fingerprint data is preserved. In this work, we propose a new framework for fingerprint template protection, highly customized to FF-OCT by exploring the interplay between subsurface. As a first of it's kind work attempting template protection for FF-OCT fingerprints, we explore deeply learnt features to derive first level of template for subsurface fingerprint image. We further propose to intertwine subsurface level templates to provide better and robust templates. With the set of extensive experiments on a FF-OCT fingerprint database of 200 unique fingerprints with a total of 2400 images, we demonstrate reliable biometric performance resulting in EER of 5.69% for unprotected template at first layer (subsurface) of fingerprint in FF-OCT, an EER of 5.86% for the protected templates at same layer and EER of 5.08% with the final protected templates with proposed intertwining of subsurface fingerprint. Further, through the security analysis, we also validate the strength of the proposed approach with near ideal unlinkability.

We report on a system that is able to significantly reduce cross-talk and speckle noise in Fourier domain full-field optical coherence tomography. It is achieved by fast phase modulation of a laser wavefront and angular compounding.

Fourier-domain full-field optical coherence tomography (FD-FF-OCT) is currently the fastest volumetric imaging technique that is able to generate a single 3-D volume of retina in less than 9 ms, corresponding to a voxel rate of 7.8 GHz. FD-FF-OCT is based on a fast camera, a rapidly tunable laser source, and Fourier-domain signal detection. However, crosstalk appearing due to multiply scattered light corrupts images with the speckle pattern, and therefore, lowers image quality. Here, for the first time, we report on a system that can acquire essentially crosstalk-free volumes of the retina by using a fast deformable membrane. It enables the visualization of choroids and a clear delineation of the retinal layers that is not possible with conventional FD-FF-OCT.

Any full-field optical coherence tomography (FF-OCT) system wastes almost 75% of light, including 50% of the OCT signal, because it uses a 50/50 beamsplitter (BS) in the standard implementation. Here, a design of a light-efficient BS is presented that loses almost no light when implemented in Fourier-domain FF-OCT. It is based on pupil engineering and a small highly asymmetric BS. The presented signal-to-noise ratio (SNR) analysis demonstrates almost four times improvement over the conventional design. In addition, it is shown that the light-efficient BS can be used to suppress specular reflections from a sample and, thus, further improve the SNR.

Full-field optical coherence tomography (FF-OCT) can rapidly acquire 2D en face OCT images through a scattering medium. However, the standard interferometer configurations waste almost 75% of light. In addition, specular reflections can saturate the detector, limiting FF-OCT performance. Here, we report on a high-throughput dark-field (HTDF) FF-OCT configuration that efficiently uses the available light budget and allows suppressing specular reflections. Specifically, we demonstrate that images acquired with the HTDF FF-OCT system have 3.5 times higher signal-to-noise ratio when compared to our previously developed regular FF-OCT system.

Optical coherence tomography (OCT) has become a useful skin imaging tool in recent years. It can be used to image skin structure with great detail and deep below the surface. However, there is an unmet need for an inexpensive device that would be able to acquire high-quality en face images in short time. To this end, we report on a cost-effective full-field optical coherent tomography (FF-OCT) system comprised of a silicon camera with high full well capacity and a powerful near-infrared LED light source. The system, for example, is able to record 1.7 cm × 1.7 cm en face images in 0.12 sec with the spatial sampling rate of 2116 dpi and the sensitivity of 93 dB. We show that the FF-OCT system can be used for biometrics purposes by imaging internal fingerprints and sweat ducts with a good contrast and in short-enough time. Blood flow can also be sensed in dermis that, for example, can be used for liveness detection. The developed instrument could also be used in other en face deep-tissue imaging applications because of its high sensitivity and speed. One particular application is passive elastography that can map the stiffness of biological tissues. We will also present FF-OCT configurations that help improving the imaging depth through rejection of specular reflections and better use of a light budget.

Cellular resolution imaging of biological structures has always been a challenge due to strong scattering that limits the achievable transverse resolution or imaging penetration depth. Recently, a major advancement toward high-resolution and volumetric imaging was achieved by implementing a parallel detection (i.e., full field) into Fourier-domain optical coherence tomography. The drawback of using parallel detection is that scattered light can travel laterally and get mapped improperly at a camera creating optical crosstalk, which severely impairs the interpretation of subcellular images and limits its use in medical diagnostics. In this work, we demonstrate for what we believe is the first time how to efficiently reduce crosstalk and enable microscopic quality volumetric reconstructions of the scattering tissue-like human skin in vivo, all within less than a half of a second. To minimize crosstalk, we implemented a very fast deformable membrane that introduces random phase illumination. Additionally, the sample is illuminated under variable angles to reduce the contrast of speckles by incoherent summation of the crosstalk-free volumes. Introducing crosstalk and speckle-free OCT will advance imaging prospects closer to the ideal of a noninvasive optical biopsy.

Imaging internal fingerprints instead of surface fingerprints by the traditional means might be more reliable since the internal fingerprints are less affected by the surface properties and skin damages. The most promising imaging technique that is able to image deep into biological tissue, and thus reach the layer of the internal fingerprints, is optical coherence tomography (OCT). Full-field optical coherence tomography (FF-OCT) is particularly well suited for the internal fingerprint imaging because it is able to record en face OCT images. Here we report a compact FF-OCT system operating in the visible region of spectrum that is able to produce en face images of sweat pores and internal fingerprints, which in turn can be used for the identification purposes.

Background Noninvasive methods are needed to improve the diagnosis of enteric neuropathies. Full-field optical coherence microscopy (FFOCM) is a novel optical microscopy modality that can acquire 1 μm resolution images of tissue. The objective of this research was to demonstrate FFOCM imaging for the characterization of the enteric nervous system (ENS). Methods Normal mice and EdnrB-/- mice, a model of Hirschsprung’s disease (HD), were imaged in three-dimensions ex vivo using FFOCM through the entire thickness and length of the gut. Quantitative analysis of myenteric ganglia was performed on FFOCM images obtained from whole-mount tissues and compared with immunohistochemistry imaged by confocal microscopy. Key Results Full-field optical coherence microscopy enabled visualization of the full thickness gut wall from serosa to mucosa. Images of the myenteric plexus were successfully acquired from the stomach, duodenum, colon, and rectum. Quantification of ganglionic neuronal counts on FFOCM images revealed strong interobserver agreement and identical values to those obtained by immunofluorescence microscopy. In EdnrB-/- mice, FFOCM analysis revealed a significant decrease in ganglia density along the colorectum and a significantly lower density of ganglia in all colorectal segments compared with normal mice. Conclusions & Inferences Full-field optical coherence microscopy enables optical microscopic imaging of the ENS within the bowel wall along the entire intestine. FFOCM is able to differentiate ganglionic from aganglionic colon in a mouse model of HD, and can provide quantitative assessment of ganglionic density. With further refinements that enable bowel wall imaging in vivo, this technology has the potential to revolutionize the characterization of the ENS and the diagnosis of enteric neuropathies.

Images recorded below the surface of a finger can have more details and be of higher quality than the conventional surface fingerprint images. This is particularly true when the quality of the surface fingerprints is compromised by, for example, moisture or surface damage. However, there is an unmet need for an inexpensive fingerprint sensor that is able to acquire high-quality images deep below the surface in short time. To this end, we report on a cost-effective full-field optical coherent tomography (FF-OCT) system comprised of a silicon camera and a powerful near-infrared LED light source. The system, for example, is able to record 1.7 cm x 1.7 cm en face images in 0.12 s with the spatial sampling rate of 2116 dots per inch and the sensitivity of 93 dB. We show that the system can be used to image internal fingerprints and sweat ducts with a good contrast. Finally, to demonstrate its biometric performance, we acquired subsurface fingerprint images from 240 individual fingers and estimated the equal-error-rate to be ~0.8 %. The developed instrument could also be used in other en face deep-tissue imaging applications because of its high sensitivity, such as in vivo skin imaging., 21 pages and 5 figures

We present a stimulated emission depletion (STED) fluorescence lifetime imaging (FLIM) microscope, excited by a microstructured optical fibre supercontinuum source that is pumped by a femtosecond Ti:Sapphire-laser, which is also used for depletion. Implemented using a piezo-scanning stage on a laser scanning confocal fluorescence microscope system with FLIM realised using time correlated single photon counting (TCSPC), this provides convenient switching between confocal and STED-FLIM with spatial resolution down to below 60 nm. We will present our design considerations to make a robust instrument for biological applications including a comparison between fixed phase plate and spatial light modulator (SLM) approaches to shape the STED beam and the correlation of STED and confocal FLIM microscopy. Following our previous application of FLIM-FRET to study intercellular signalling at the immunological synapse (IS), we are employing STED microscopy to characterize the spatial distribution of cellular molecules with subdiffraction resolution at the IS. In particular, we are imaging cytoskeletal structure at the Natural Killer cell activated immune synapse. We will also present our progress towards multilabel STED microscopy to determine how relative spatial molecular organization, previously undetectable by conventional microscopy techniques, is important for NK cell cytotoxic function. Keywords: STED, Stimulated Emission Depletion Microscopy, Natural Killer (NK) cell, Fluorescence lifetime imaging, FLIM, Super resolution microscopy.

Publisher Summary This chapter describes the applications of multidimensional fluorescence imaging (MDFI) instrumentation. The chapter discusses fluorescence lifetime imaging (FLIM), with an emphasis on rapid wide-field time-gated imaging, including application to molecular biology, FLIM of tissue autofluorescence, and highspeed optically sectioned FLIM for live cell imaging. Optical sectioning is important to enhance image contrast and to minimize unwanted mixing of signals from axially separate fluorophores. The chapter discusses the extension to spectral FLIM, including (emission resolved) hyperspectral FLIM, implemented using line-scanning microscopy, and excitation-resolved imaging and FLIM utilizing supercontinuum generation to provide excitation throughout the visible spectrum. The combination of polarization-resolved and time resolved fluorescence imaging is described, mapping both lifetime and rotational correlation time as illustrated by an application to microfluidic devices. This chapter demonstrates that FLIM and MDFI can add significant value to microscopy, endoscopy, and assay technology. By resolving the fluorescence signal with respect to multiple dimensions including excitation and emission wavelength, lifetime, and polarization, it may be possible to enhance the fluorescence read-out for an experiment or assay, for example, in terms of improving the separation of multiple fluorophores or imaging variations in the local molecular environment.

We present a time-gated, optically sectioned, hyperspectral fluorescence lifetime imaging (FLIM) microscope incorporating a tunable supercontinuum excitation source extending into the UV. The system is capable of resolving the excitation spectrum, emission spectrum, and fluorescence decays in an optically sectioned image.

We describe the characterisation of a hyperspectral fluorescence lifetime imaging microscope that exploits high-speed time-gated imaging technology and a tunable continuum source for 6-D fluorescence imaging. This line-scanning confocal microscope can record the full spectral-temporal (i.e. excitation-emission-lifetime) fluorescence matrix at each pixel in a three dimensional (x-y-z) sample. This instrument has been applied to biological samples including model membranes and live cells labelled with the phase-sensitive membrane dye di-4-ANEPPDHQ, for which significant variation of lifetime with emission wavelength is observed.

We investigated the use of fluorescence lifetime imaging microscopy (FLIM) of a fluorescently labeled ATP analog (3′-O-{N-[3-(7-diethylaminocoumarin-3-carboxamido)propyl]carbamoyl}ATP) to probe in permeabilized muscle fibers the changes in the environment of the nucleotide binding pocket caused by interaction with actin. Spatial averaging of FLIM data of muscle sarcomeres reduces photon noise, permitting detailed analysis of the fluorescence decay profiles. FLIM reveals that the lifetime of the nucleotide, in its ADP form because of the low concentration of nucleotide present, changes depending on whether the nucleotide is free in solution or bound to myosin, and on whether the myosin is bound to actin in an actomyosin complex. Characterization of the fluorescence decays by a multiexponential function allowed us to resolve the lifetimes and amplitudes of each of these populations, namely, the fluorophore bound to myosin, bound to actin, in an actomyosin complex, and free in the filament lattice. This novel application of FLIM to muscle fibers shows that with spatial averaging, detailed information about the nature of nucleotide complexes can be derived.