Chirped flicker optoretinography for in vivo characterization of human photoreceptors' frequency response to light.

Flicker electroretinography (ERG) has served as a valuable noninvasive objective tool for investigating retinal physiological function through the measurement of electrical signals originating from retinal neurons in response to temporally modulated light stimulation. Deficits in the response at certain frequencies can be used as effective biomarkers of cone-pathway dysfunction. In this Letter, we present the progress we made on its optical counterpart-photopic flicker optoretinography (f-ORG). Specifically, we focus on the measurement of the response of light-adapted retinal photoreceptors to a flicker stimulus with chirped frequency modulation. In contrast to measurements performed at discrete frequencies, this technique enables a significantly accelerated characterization of photoreceptor outer segment optical path length modulation amplitudes in the nanometer range as a function of stimulus frequency, enabling the acquisition of the characteristic frequency response in less than 2 sec.

Noninvasive two-photon optical biopsy of retinal fluorophores.

High-resolution imaging techniques capable of detecting identifiable endogenous fluorophores in the eye along with genetic testing will dramatically improve diagnostic capabilities in the ophthalmology clinic and accelerate the development of new treatments for blinding diseases. Two-photon excitation (TPE)-based imaging overcomes the filtering of ultraviolet light by the lens of the human eye and thus can be utilized to discover defects in vitamin A metabolism during the regeneration of the visual pigments required for the detection of light. Combining TPE with fluorescence lifetime imaging (FLIM) and spectral analyses offers the potential of detecting diseases of the retina at earlier stages before irreversible structural damage has occurred. The main barriers to realizing the benefits of TPE for imaging the human retina arise from concerns about the high light exposure typically needed for informative TPE imaging and the requirement to correlate the ensuing data with different states of health and disease. To overcome these hurdles, we improved TPE efficiency by controlling temporal properties of the excitation light and employed phasor analyses to FLIM and spectral data in mouse models of retinal diseases. Modeling of retinal photodamage revealed that plasma-mediated effects do not play a role and that melanin-related thermal effects are mitigated by reducing pulse repetition frequency. By using noninvasive TPE imaging we identified molecular components of individual granules in the retinal pigment epithelium and present their analytical characteristics.

Poster Session: Optoretinography-based frequency characterization of the retinal response to a chirped flickering light.

In this contribution, we present experimental results of in vivo characterization of the photoreceptor's response to a chirped flickering white light stimulating the retina. We acquire the ORG signal with Spatio-Temporal Optical Coherence Tomography (STOC-T) setup, which combines both temporal and coherence gating to overcome limitations present in Full Field Fourier Domain Optical Coherence Tomography. From the acquired volumes, we extract the changes in optical path length (OPL) between the inner and outer photoreceptor junction (ISOS) and the cone outer segment tips (COST). We perform the measurements for frequencies ranging from 5 Hz to 50 Hz. The chirped flickering facilitates significantly shorter data acquisition time. We present results of in vivo measurement from three volunteers. Our results show that we can measure OPL changes between ISOS and COST occurring in response to a chirped flickering stimulation in a reproducible manner and resolve the amplitude of the response in the function of flicker frequency.

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.

High-Throughput Monitoring of Bacterial Cell Density in Nanoliter Droplets: Label-Free Detection of Unmodified Gram-Positive and Gram-Negative Bacteria.

Droplet microfluidics disrupted analytical biology with the introduction of digital polymerase chain reaction and single-cell sequencing. The same technology may also bring important innovation in the analysis of bacteria, including antibiotic susceptibility testing at the single-cell level. Still, despite promising demonstrations, the lack of a high-throughput label-free method of detecting bacteria in nanoliter droplets prohibits analysis of the most interesting strains and widespread use of droplet technologies in analytical microbiology. We use a sensitive and fast measurement of scattered light from nanoliter droplets to demonstrate reliable detection of the proliferation of encapsulated bacteria. We verify the sensitivity of the method by simultaneous readout of fluorescent signals from bacteria expressing fluorescent proteins and demonstrate label-free readout on unlabeled Gram-negative and Gram-positive species. Our approach requires neither genetic modification of the cells nor the addition of chemical markers of metabolism. It is compatible with a wide range of bacterial species of clinical, research, and industrial interest, opening the microfluidic droplet technologies for adaptation in these fields.

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.

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.

Corneal tissue properties following scleral lens wear using Scheimpflug imaging.

PURPOSE: To investigate the effect of short-term scleral lens wear on the corneal stroma at a macroscopic (thickness) and microscopic (within tissue) level, including regional variations. METHODS: Fourteen young, healthy participants wore a rotationally symmetric, 16.5 mm diameter, scleral lens for 8 h. Scheimpflug images were captured before, and immediately after, lens wear, and also on a second day (without lens wear) to quantify natural corneal diurnal variations. After corneal segmentation, pixel intensities of the stromal tissue were statistically modelled using a Weibull probability density function from which parameters α and ß were derived. RESULTS: Both α and ß parameters increased significantly following scleral lens wear (by 5.7 ± 10% and 6.5 ± 6.5%, respectively, both p < 0.01). Corneal thickness also increased slightly following lens wear (mean increase 0.49 ± 1.77%, p = 0.01); however, the change in α and ß parameters did not correlate with the magnitude of corneal swelling. On the control day, small but significant corneal thinning was observed (-0.82 ± 1.1%, p = 0.03), while α and ß parameters remained stable. Both microparameters varied significantly across the cornea, with α decreasing (-15.4 ± 0.7%) and ß increasing towards the periphery (+4.4 ± 2.6%) (both p < 0.001). CONCLUSION: Corneal microparameters α and ß varied regionally across the cornea and displayed a statistically significant increase following short-term scleral lens wear, but remained stable between morning and evening measurements taken during a control day without lens wear. These corneal microparameters may be a useful metric to quantify subclinical corneal changes associated with low level hypoxia.

Human infrared vision is triggered by two-photon chromophore isomerization.

Vision relies on photoactivation of visual pigments in rod and cone photoreceptor cells of the retina. The human eye structure and the absorption spectra of pigments limit our visual perception of light. Our visual perception is most responsive to stimulating light in the 400- to 720-nm (visible) range. First, we demonstrate by psychophysical experiments that humans can perceive infrared laser emission as visible light. Moreover, we show that mammalian photoreceptors can be directly activated by near infrared light with a sensitivity that paradoxically increases at wavelengths above 900 nm, and display quadratic dependence on laser power, indicating a nonlinear optical process. Biochemical experiments with rhodopsin, cone visual pigments, and a chromophore model compound 11-cis-retinyl-propylamine Schiff base demonstrate the direct isomerization of visual chromophore by a two-photon chromophore isomerization. Indeed, quantum mechanics modeling indicates the feasibility of this mechanism. Together, these findings clearly show that human visual perception of near infrared light occurs by two-photon isomerization of visual pigments.

Differentiation of morphotic elements in human blood using optical coherence tomography and a microfluidic setup.

We demonstrate a novel optical method for the detection and differentiation between erythrocytes and leukocytes that uses amplitude and phase information provided by optical coherence tomography (OCT). Biological cells can introduce significant phase modulation with substantial scattering anisotropy and dominant forward-scattered light. Such physical properties may favor the use of a trans-illumination imaging technique. However, an epi-illumination mode may be more practical and robust in many applications. This study describes a new way of measuring the phase modulation introduced by flowing microobjects. The novel part of this invention is that it uses the backscattered signal from the substrate located below the flowing/moving objects. The identification of cells is based on phase-sensitive OCT signals. To differentiate single cells, a custom-designed microfluidic device with a highly scattering substrate is introduced. The microchannels are molded in polydimethylsiloxane (PDMS) mixed with titanium dioxide (TiO2) to ensure high scattering properties. The statistical parameters of the measured signal depend on the cells' features, such as their size, shape, and internal structure.

Blue-light Fourier-domain optical-coherence microscopy with linear k-sampling using second-harmonic generation.

We demonstrate Fourier-domain optical-coherence microscopy (OCM) method that uses blue light for high-resolution microscopic imaging. Spectrally broad bandwidth is obtained by means of second-harmonic generation of Ti:sapphire laser light on the nonlinear crystal. Angular scanning of the crystal performed by a resonant scanner results in second-harmonic generation for a broad range of frequencies producing blue light with central wavelength of 402 nm and bandwidth of 35 nm in one cycle. The axial resolution of the new setup is 3.5 µm in air, and the transverse resolution for Olympus 40× objective lens is 2.7 µm in X direction and 3.2 µm in Y direction. The developed technique enables registering spectral interferometric signal directly in k domain. Additionally, we present examples of imaging a biological specimen using the newly developed method.

Microscopic OCT imaging with focus extension by ultrahigh-speed acousto-optic tunable lens and stroboscopic illumination.

We develop high-resolution optical coherence tomography (OCT) system with high-speed acousto-optic tunable lens. Stroboscopic pulsed illumination is used for the first time to perform time-resolved OCT imaging with acousto-optic tunable focusing. The operation of ultrahigh-speed tunable acousto-optic lens is demonstrated theoretically and experimentally. Focal position tuning at MHz frequency range is experimentally shown in the imaging system leading to OCT images with extended depth of focus. Imaging with active optical elements is helpful for improvement of photon collection efficiency, depth of focus and enhancement of the image quality.

Quantitative optical inspection of contact lenses immersed in wet cell using swept source OCT.

We demonstrate swept source optical coherence tomography (OCT) imaging of contact lenses (CLs) in a wet cell and comprehensive quantitative characterization of CLs from volumetric OCT datasets. The approach is based on a technique developed for lens autopositioning and autoleveling enabled by lateral capillary interactions between the wet cell wall and the lens floating on the liquid surface. The demonstrated OCT imaging has enhanced contrast due to the application of a scattering medium and it improves visualization of both CL interfaces and edges. We also present precise and accurate three-dimensional metrology of soft and rigid CLs based on the OCT data. The accuracy and precision of the extracted lens parameters are compared with the manufacturer's specifications. The presented methodology facilitates industrial inspection methods of the CLs.

Extracellular carbonic anhydrase mediates hemorrhagic retinal and cerebral vascular permeability through prekallikrein activation.

Excessive retinal vascular permeability contributes to the pathogenesis of proliferative diabetic retinopathy and diabetic macular edema, leading causes of vision loss in working-age adults. Using mass spectroscopy-based proteomics, we detected 117 proteins in human vitreous and elevated levels of extracellular carbonic anhydrase-I (CA-I) in vitreous from individuals with diabetic retinopathy, suggesting that retinal hemorrhage and erythrocyte lysis contribute to the diabetic vitreous proteome. Intravitreous injection of CA-I in rats increased retinal vessel leakage and caused intraretinal edema. CA-I-induced alkalinization of vitreous increased kallikrein activity and its generation of factor XIIa, revealing a new pathway for contact system activation. CA-I-induced retinal edema was decreased by complement 1 inhibitor, neutralizing antibody to prekallikrein and bradykinin receptor antagonism. Subdural infusion of CA-I in rats induced cerebral vascular permeability, suggesting that extracellular CA-I could have broad relevance to neurovascular edema. Inhibition of extracellular CA-I and kallikrein-mediated innate inflammation could provide new therapeutic opportunities for the treatment of hemorrhage-induced retinal and cerebral edema.

Averaging techniques for OCT imaging.

State-of-the-art Fourier-domain optical coherence tomography (OCT) allows for the acquisition of up to millions of spectral fringes per second. This large amount of data can be used to improve the quality of structural tomograms after effective averaging. Here, we compare three OCT image improvement techniques: magnitude averaging, complex averaging, and spectral and time domain OCT (STdOCT). We evaluate the performance for images on both linear and logarithmic intensity scales and discuss their advantages and disadvantages. We propose the use of the STdOCT approach as it offers the best advantages. Applications to in vivo imaging and speckle reduction are presented.

Quantitative lateral and axial flow imaging with optical coherence microscopy and tomography.

Optical coherence tomography (OCT) and optical coherence microscopy (OCM) allow the acquisition of quantitative three-dimensional axial flow by estimating the Doppler shift caused by moving scatterers. Measuring the velocity of red blood cells is currently the principal application of these methods. In many biological tissues, blood flow is often perpendicular to the optical axis, creating the need for a quantitative measurement of lateral flow. Previous work has shown that lateral flow can be measured from the Doppler bandwidth, albeit only for simplified optical systems. In this work, we present a generalized model to analyze the influence of relevant OCT/OCM system parameters such as light source spectrum, numerical aperture and beam geometry on the Doppler spectrum. Our analysis results in a general framework relating the mean and variance of the Doppler frequency to the axial and lateral flow velocity components. Based on this model, we present an optimized acquisition protocol and algorithm to reconstruct quantitative measurements of lateral and axial flow from the Doppler spectrum for any given OCT/OCM system. To validate this approach, Doppler spectrum analysis is employed to quantitatively measure flow in a capillary with both extended focus OCM and OCT.

Assessment of the flow velocity of blood cells in a microfluidic device using joint spectral and time domain optical coherence tomography.

Although Doppler optical coherence tomography techniques have enabled the imaging of blood flow in mid-sized vessels in biological tissues, the generation of velocity maps of capillary networks remains a challenge. To better understand the origin and information content of the Doppler signal from small vessels and limitations of such measurements, we used joint spectral and time domain optical coherence tomography to monitor the flow in a model, semitransparent microchannel device. The results obtained for Intralipid, whole blood, as well as separated red blood cells indicate that the technique is suitable to record velocity profiles in vitro, in a range of microchannel configurations.

Control of the optical field coherence by spatiotemporal light modulation.

The novel spatiotemporal optical coherence manipulation technique, which allows one to tailor the second-order coherence properties of a light beam, is introduced. With the use of an interferometric setup we show that the basic measure of the contrast of interference fringes, i.e., Michelson's visibility, can be controlled across the interference pattern by modulating the phase of the spectral degree of coherence.

From mouse to human: Accessing the biochemistry of vision in vivo by two-photon excitation.

The eye is an ideal organ for imaging by a multi-photon excitation approach, because ocular tissues such as the sclera, cornea, lens and neurosensory retina, are highly transparent to infrared (IR) light. The interface between the retina and the retinal pigment epithelium (RPE) is especially informative, because it reflects the health of the visual (retinoid) cycle and its changes in response to external stress, genetic manipulations, and drug treatments. Vitamin A-derived retinoids, like retinyl esters, are natural fluorophores that respond to multi-photon excitation with near IR light, bypassing the filter-like properties of the cornea, lens, and macular pigments. Also, during natural aging some retinoids form bisretinoids, like diretinoid-pyridiniumethanolamine (A2E), that are highly fluorescent. These bisretinoids appear to be elevated concurrently with aging. Vitamin A-derived retinoids and bisretinoidss are detected by two-photon ophthalmoscopy (2PO), using a new class of light sources with adjustable spatial, temporal, and spectral properties. Furthermore, the two-photon (2P) absorption of IR light by the visual pigments in rod and cone photoreceptors can initiate visual transduction by cis-trans isomerization of retinal, enabling parallel functional studies. Recently we overcame concerns about safety, data interpretation and complexity of the 2P-based instrumentation, the major roadblocks toward advancing this modality to the clinic. These imaging and retina-function assessment advancements have enabled us to conduct the first 2P studies with humans.

Laser pulse train parameters determine the brightness of a two-photon stimulus.

This report presents the results of measurements of the two-photon vision threshold for various pulse trains. We employed three pulsed near-infrared lasers and pulse stretchers to obtain variations of the pulse duty cycle parameter over three orders of magnitude. We proposed and extensively described a mathematical model that combines the laser parameters with the visual threshold value. The presented methodology enables one to predict the visual threshold value for a two-photon stimulus for a healthy subject while using a laser source of known parameters. Our findings would be of value to laser engineers and the community interested in nonlinear visual perception.