Programmable high-speed confocal reflectance microscopy enabled by a digital micromirror device.

The digital micromirror device (DMD) has been used to achieve parallel scanning in confocal microscopy significantly increasing acquisition speed. However, for confocal reflectance imaging, such an approach is limited to mostly surface imaging due to strong backreflections coming from the DMD that can dominate the signal recorded on a camera. Here, we report on an optical configuration that uses separate areas of DMD to generate multiple spots and pinholes and thereby prevents backreflections from the DMD from reaching the camera. We thus demonstrate confocal imaging of weakly reflecting objects, such as a pollen grain sample.

Time-domain full-field optical coherence tomography with a digital defocus correction.

Time-domain full-field optical coherence tomography (TD-FF-OCT) is an interferometric technique capable of acquiring high-resolution images deep within the biomedical tissue, utilizing a spatially and temporally incoherent light source. However, optical aberrations, such as sample defocus, can degrade the image quality, thereby limiting the achievable imaging depth. Here we demonstrate that the sample defocus within a highly scattering medium can be digitally corrected over a wide defocus range if the optical path lengths in the sample and reference arms are matched. We showcase the application of digital defocus correction on both reflective and scattering samples, effectively compensating digitally for up to 1 mm of defocus.

Time-domain full-field optical coherence tomography with digital confocal line scanning.

Full-field optical coherence tomography (FF-OCT) is a camera-based interferometric microscopy technique that can image deep in tissue with high spatial resolution. However, the absence of confocal gating leads to suboptimal imaging depth. Here, we implement digital confocal line scanning in time-domain FF-OCT by exploiting the row-by-row detection feature of a rolling-shutter camera. A digital micromirror device (DMD) is used in conjunction with the camera to produce synchronized line illumination. An improvement in the SNR by an order of magnitude is demonstrated on a sample of a US Air Force (USAF) target mounted behind a scattering layer.

Multimode fiber as a tool to reduce cross talk in Fourier-domain full-field optical coherence tomography.

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.

Fourier-domain full-field optical coherence tomography with real-time axial imaging.

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.

Multimode fiber enables control of spatial coherence in Fourier-domain full-field optical coherence tomography for in vivo corneal 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.

Light-efficient beamsplitter for Fourier-domain full-field optical coherence tomography.

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.

Computational aberration correction in spatiotemporal optical coherence (STOC) imaging.

Spatiotemporal optical coherence (STOC) imaging is a new technique for suppressing coherent cross talk noise in Fourier-domain full-field optical coherence tomography (FD-FF-OCT). In STOC imaging, the time-varying 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 cross-talk-free volumetric optical coherence tomography (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, enabling 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 mosaics.

Dark-field full-field optical coherence tomography.

Full-field optical coherence tomography (FF-OCT) provides en face images from deep in the tissue with high spatial resolution. Specular reflections, however, may reduce image contrast as it can be much stronger than the backscattered signal from a specimen. To this end, we demonstrate dark-field FF-OCT (d-FF-OCT) that can block specular reflections by the help of an opaque disk in the pupil-conjugated plane. The reference mirror is replaced by a blazed grating, which eliminates a walk-off between the sample and the reference beams on a camera that otherwise limits the imaging field-of-view (FOV). We show that d-FF-OCT can suppress specular reflections efficiently from the glass-specimen interface by at least two orders of magnitude and yield higher contrast images compared to the conventional FF-OCT.

Light-adapted flicker optoretinograms captured with a spatio-temporal optical coherence-tomography (STOC-T) system.

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.

Spatio-temporal optical coherence tomography provides full thickness imaging of the chorioretinal complex.

Despite the rapid development of optical imaging methods, high-resolution in vivo imaging with penetration into deeper tissue layers is still a major challenge. Optical coherence tomography (OCT) has been used successfully for non-invasive human retinal volumetric imaging in vivo, advancing the detection, diagnosis, and monitoring of various retinal diseases. However, there are important limitations of volumetric OCT imaging, especially coherent noise and the limited axial range over which high resolution images can be acquired. The limited range prevents simultaneous measurement of the retina and choroid with adequate lateral resolution. In this article, we address these limitations with a technique that we term spatio-temporal optical coherence tomography (STOC-T), which uses light with controlled spatial and temporal coherence and advanced signal processing methods. STOC-T enabled the acquisition of high-contrast and high-resolution coronal projection images of the retina and choroid at arbitrary depths.

Microclusters of inhibitory killer immunoglobulin-like receptor signaling at natural killer cell immunological synapses.

We report the supramolecular organization of killer Ig-like receptor (KIR) phosphorylation using a technique applicable to imaging phosphorylation of any green fluorescent protein-tagged receptor at an intercellular contact or immune synapse. Specifically, we use fluorescence lifetime imaging (FLIM) to report Förster resonance energy transfer (FRET) between GFP-tagged KIR2DL1 and a Cy3-tagged generic anti-phosphotyrosine monoclonal antibody. Visualization of KIR phosphorylation in natural killer (NK) cells contacting target cells expressing cognate major histocompatibility complex class I proteins revealed that inhibitory signaling is spatially restricted to the immune synapse. This explains how NK cells respond appropriately when simultaneously surveying susceptible and resistant target cells. More surprising, phosphorylated KIR was confined to microclusters within the aggregate of KIR, contrary to an expected homogeneous distribution of KIR signaling across the immune synapse. Also, yellow fluorescent protein-tagged Lck, a kinase important for KIR phosphorylation, accumulated in a multifocal distribution at inhibitory synapses. Spatial confinement of receptor phosphorylation within the immune synapse may be critical to how activating and inhibitory signals are integrated in NK cells.

In vivo imaging of the human cornea with high-speed and high-resolution Fourier-domain full-field optical coherence tomography.

Corneal evaluation in ophthalmology necessitates cellular-resolution and fast imaging techniques that allow for accurate diagnoses. Currently, the fastest volumetric imaging technique is Fourier-domain full-field optical coherence tomography (FD-FF-OCT), which uses a fast camera and a rapidly tunable laser source. Here, we demonstrate high-resolution, high-speed, non-contact corneal volumetric imaging in vivo with FD-FF-OCT that can acquire a single 3D volume with a voxel rate of 7.8 GHz. The spatial coherence of the laser source was suppressed to prevent it from focusing on a spot on the retina, and therefore, exceeding the maximum permissible exposure (MPE). The inherently volumetric nature of FD-FF-OCT data enabled flattening of curved corneal layers. The acquired FD-FF-OCT images revealed corneal cellular structures, such as epithelium, stroma and endothelium, as well as subbasal and mid-stromal nerves.

Crosstalk-free volumetric in vivo imaging of a human retina with Fourier-domain full-field optical coherence tomography.

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.

Fast subsurface fingerprint imaging with full-field optical coherence tomography system equipped with a silicon camera.

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 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×1.7 cmen 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 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.

Fingerprint imaging from the inside of a finger with full-field optical coherence tomography.

Imaging below fingertip surface might be a useful alternative to the traditional fingerprint sensing since the internal finger features are more reliable than the external ones. One of the most promising subsurface imaging technique is optical coherence tomography (OCT), which, however, has to acquire 3-D data even when a single en face image is required. This makes OCT inherently slow for en face imaging and produce unnecessary large data sets. Here we demonstrate that full-field optical coherence tomography (FF-OCT) can be used to produce en face images of sweat pores and internal fingerprints, which can be used for the identification purposes.

Dual-modality fluorescence and full-field optical coherence microscopy for biomedical imaging applications.

Full-field optical coherence microscopy (FFOCM) is a high-resolution interferometric technique that is particularly attractive for biomedical imaging. Here we show that combining it with structured illumination fluorescence microscopy on one platform can increase its versatility since it enables co-localized registration of optically sectioned reflectance and fluorescence images. To demonstrate the potential of this dual modality, a fixed and labeled mouse retina was imaged. Results showed that both techniques can provide complementary information and therefore the system could potentially be useful for biomedical imaging applications.

Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging.

We demonstrate stimulated emission depletion (STED) microscopy implemented in a laser scanning confocal microscope using excitation light derived from supercontinuum generation in a microstructured optical fiber. Images with resolution improvement beyond the far-field diffraction limit in both the lateral and axial directions were acquired by scanning overlapped excitation and depletion beams in two dimensions using the flying spot scanner of a commercially available laser scanning confocal microscope. The spatial properties of the depletion beam were controlled holographically using a programmable spatial light modulator, which can rapidly change between different STED imaging modes and also compensate for aberrations in the optical path. STED fluorescence lifetime imaging microscopy is demonstrated through the use of time-correlated single photon counting.

Fluorescence lifetime imaging to detect actomyosin states in mammalian muscle sarcomeres.

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.

Excitation-resolved hyperspectral fluorescence lifetime imaging using a UV-extended supercontinuum source.

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.