High-resolution ophthalmic imaging is imperative for detecting subtle changes of photoreceptor abnormality at the early stage of retinal diseases. However, optical resolution in retinal imaging is inherently limited by the low numerical aperture of the ocular optics. Virtually structured detection (VSD) has been demonstrated to break the diffraction limit of imaging systems by shifting the high-frequency components to the passing bandwidth of the imaging system. However, its implementation for human subjects remains a challenge due to the uncertain cut-off frequency of the modulation transfer function (MTF) required for VSD processing. This study demonstrates an objective method to derive the MTF from spectral profiles, enabling quantitative estimation of the optimal cut-off frequency. A custom-built line-scan scanning laser ophthalmoscopy was developed, and two-dimensional line-profile patterns were acquired at a 25 kHz frame rate. We found that the MTF profiles exhibited significant differences between subjects as well as view fields. VSD-based super-resolution images exhibited improved resolution and contrast to differentiate individual photoreceptors compared to the equivalent wide-field imaging. Besides, the motility process on the VSD image further improved the image quality as the photoreceptors revealed clear boundaries and more integrated shape, compared to that in the VSD image. We anticipate that the VSD-based imaging will provide a simple, low-cost, and phase-artifact-free strategy to achieve super-resolution retinal ophthalmoscopy.
KEYWORDS: Intrinsic optical signal imaging, Information operations, In vivo imaging, Signal attenuation, Optical coherence tomography, Retina, Physiology, Optical imaging, Intrinsic optical signal, Image segmentation
Early detection of photoreceptor dysfunction is essential for preventing vision loss due to retinal degenerative diseases, such as age-related macular degeneration (AMD) and inherited retinal degenerations (IRDs). Functional intrinsic optical signal (IOS) imaging promises a high-resolution method for objective optoretinography (ORG). Stimulus-evoked photoreceptor-IOS has been recently demonstrated in healthy animal and human retinas. The fast photoreceptor-IOS response was found to occur at the photoreceptor outer segment (OS) right after the onset of retinal stimulation. However, in vivo IOS response of photoreceptor dysfunctions is not yet validated, which is essential to measure the clinical usability of ORG measurement. In this study, we report in vivo IOS imaging of rod photoreceptor dysfunction in retinal degeneration 10 (rd10) mice. A custom-designed optical coherence tomography (OCT) was used for photoreceptor-IOS imaging. A significant attenuation of the photoreceptor-IOS was found in rd10 mice due to disorganized ultrastructure of the photoreceptor OSs, which appeared ahead of progressive rod cell death. Our experiments demonstrate that fast photoreceptor-IOS is highly sensitive to ultrastructural integrity of the photoreceptor OSs. We anticipate that quantitative imaging of fast photoreceptor-IOS will provide objective ORG measurement to advance the study and diagnosis of AMD, IRDs, and other retinal diseases that can cause photoreceptor dysfunctions.
KEYWORDS: In vivo imaging, Retina, Super resolution, Retinal scanning, Rods, Information operations, Line scan image sensors, Spatial resolution, Video, Physiology
Rod-dominated transient retinal phototropism (TRP) has been observed in freshly isolated retinas, promising a noninvasive biomarker for objective assessment of retinal physiology. However, in vivo mapping of TRP is challenging due to its subcellular signal magnitude and fast time course. We report here a virtually structured detection-based super-resolution ophthalmoscope to achieve subcellular spatial resolution and millisecond temporal resolution for in vivo imaging of TRP. Spatiotemporal properties of in vivo TRP were characterized corresponding to variable light intensity stimuli, confirming that TRP is tightly correlated with early stages of phototransduction.
Rod-dominated transient retinal phototropism (TRP) has been observed in freshly isolated retinas, promising a noninvasive biomarker for high resolution assessment of retinal physiology. However, in vivo mapping of TRP is challenging due to its fast time course and sub-cellular signal magnitude. By developing a line-scanning and virtually structured detection based super-resolution ophthalmoscope, we report here in vivo observation of TRP in frog retina. In vivo characterization of TRP time course and magnitude were implemented by using variable light stimulus intensities.
Transient retinal phototropism (TRP) has been observed in rod photoreceptors activated by oblique visible light flashes. Time-lapse confocal microscopy and optical coherence tomography (OCT) revealed rod outer segment (ROS) movements as the physical source of TRP. However, the physiological source of TRP is still not well understood. In this study, concurrent TRP and electroretinogram (ERG) measurements disclosed a remarkably earlier onset time of the ROS movements (≤10 ms) than that (~38 ms) of the ERG a-wave. Furthermore, low sodium treatment reversibly blocked the photoreceptor ERG a-wave, which is known to reflect hyperpolarization of retinal photoreceptors, but preserved the TRP associated rod OS movements well. Our experimental results and theoretical analysis suggested that the physiological source of TRP might be attributed to early stages of phototransduction, before the hyperpolarization of retinal photoreceptors.
It is well established that major retinal diseases involve distortions of the retinal neural physiology and blood vascular
structures. However, the details of distortions in retinal neurovascular coupling associated with major eye diseases are
not well understood. In this study, a multi-modal optical coherence tomography (OCT) imaging system was developed
to enable concurrent imaging of retinal neural activity and vascular hemodynamics. Flicker light stimulation was applied
to mouse retinas to evoke retinal neural responses and hemodynamic changes. The OCT images were acquired
continuously during the pre-stimulation, light-stimulation, and post-stimulation phases. Stimulus-evoked intrinsic optical
signals (IOSs) and hemodynamic changes were observed over time in blood-free and blood regions, respectively. Rapid
IOSs change occurred almost immediately after stimulation. Both positive and negative signals were observed in
adjacent retinal areas. The hemodynamic changes showed time delays after stimulation. The signal magnitudes induced
by light stimulation were observed in blood regions and did not show significant changes in blood-free regions. These
differences may arise from different mechanisms in blood vessels and neural tissues in response to light stimulation.
These characteristics agreed well with our previous observations in mouse retinas. Further development of the multimodal
OCT may provide a new imaging method for studying how retinal structures and metabolic and neural functions
are affected by age-related macular degeneration (AMD), glaucoma, diabetic retinopathy (DR), and other diseases,
which promises novel noninvasive biomarkers for early disease detection and reliable treatment evaluations of eye
diseases.
KEYWORDS: Super resolution, In vivo imaging, Spatial frequencies, Retinal scanning, Image resolution, Retina, Eye, Spatial resolution, Signal to noise ratio, Microscopy
High resolution is important for sensitive detection of subtle distortions of retinal morphology at an early stage of eye diseases. We demonstrate virtually structured detection (VSD) as a feasible method to achieve in vivo super-resolution ophthalmoscopy. A line-scanning strategy was employed to achieve a super-resolution imaging speed up to 127 frames/s with a frame size of 512×512 pixels. The proof-of-concept experiment was performed on anesthetized frogs. VSD-based super-resolution images reveal individual photoreceptors and nerve fiber bundles unambiguously. Both image contrast and signal-to-noise ratio are significantly improved due to the VSD implementation.
Intrinsic optical signal (IOS) imaging promises a noninvasive method for advanced study and diagnosis of eye diseases. Before pursuing clinical applications, it is essential to understand anatomic and physiological sources of retinal IOSs and to establish the relationship between IOS distortions and eye diseases. The purpose of this study was designed to demonstrate the feasibility of in vivo IOS imaging of mouse models. A high spatiotemporal resolution spectral domain optical coherence tomography (SD-OCT) was employed for depth-resolved retinal imaging. A custom-designed animal holder equipped with ear bar and bite bar was used to minimize eye movements. Dynamic OCT imaging revealed rapid IOS from the photoreceptor’s outer segment immediately after the stimulation delivery, and slow IOS changes were observed from inner retinal layers. Comparative photoreceptor IOS and electroretinography recordings suggested that the fast photoreceptor IOS may be attributed to the early stage of phototransduction before the hyperpolarization of retinal photoreceptor.
Rod-dominated transient retinal phototropism (TRP) has been recently observed in freshly isolated mouse and frog retinas. Comparative confocal microscopy and optical coherence tomography revealed that the TRP was predominantly elicited from the rod outer segment (OS). However, the biophysical mechanism of rod OS dynamics is still unknown. Mouse and frog retinal slices, which displayed a cross-section of retinal photoreceptors and other functional layers, were used to test the effect of light stimulation on rod OSs. Time-lapse microscopy revealed stimulus-evoked conformational changes of rod OSs. In the center of the stimulated region, the length of the rod OS shrunk, while in the peripheral region, the rod OS swung toward the center region. Our experimental observation and theoretical analysis suggest that the TRP may reflect unbalanced rod disc-shape changes due to localized visible light stimulation.
Radiance is sensitive to the variations of tissue optical parameters, such as absorption coefficient μa, scattering coefficient μs, and anisotropy factor g. Therefore, similar to fluence, radiance can be used for tissue characterization. Compared with fluence, radiance has the advantage of offering the direction information of light intensity. Taking such advantage, the optical parameters can be determined by rotating the detector through 360 deg with only a single optode pair. Instead of the translation mode used in the fluence-based technologies, the Rotation mode has less invasiveness in the clinical diagnosis. This paper explores a new method to obtain the optical properties by measuring the distribution of light intensity in liquid phantom with only a single optode pair and the detector rotation through 360 deg. The angular radiance and distance-dependent radiance are verified by comparing experimental measurement data with Monte Carlo (MC) simulation for the short source-detector separations and diffusion approximation for the large source-detector separations. Detecting angular radiance with only a single optode pair under a certain source-detection separation will present a way for prostate diagnose and light dose calculation during the photon dynamic therapy (PDT).
Techniques of time-correlated single-photon counting (TCSPC) have been widely used in diffuse optical tomography (DOT) and diffuse fluorescence tomography (DFT). While a multi-channel TCSPC-based DOT/DFT system can be conveniently constructed using independent modules, the state-of-the-art TCSPC technique has extended its multidimensional function by facilitating a compact and cost-effective design of the multi-channel as well as multi-wavelength data-acquisition. We herein present a revised multi-channel TCSPC system that is based the multidimensional function of the TCSPC device. We also design a series of DOT and DFT experiments to validate effectiveness of the system.
Traditionally, volume based finite element method (FEM) or finite difference method (FDM) are applied to the forward
problem of the time-domain diffuse fluorescence tomography (DFT), this paper presents a new numerical method for
solving the problem: the boundary element method (BEM). Using BEM forward solver is explored as an alternative to
the FEM or FDM solution methodology for the elliptic equations used to model the generation and transport of
fluorescent light in highly scattering media. In contrast to the FEM or FDM, the boundary integral method requires only
representation of the surface meshes, thus requires many fewer nodes and elements than the FEM and FDM. By using
BEM forward solver for time-domain DFT, we can simultaneously reconstruct both fluorescent yield and lifetime images.
The results have demonstrated that the BEM is suitable for solving the forward problem of time-domain DFT.
A combined time-domain diffuse fluorescence and optical tomographic system is proposed based on the multi-channel
time-correlated single-photon counting (TCSPC) technique, aiming at enhancing the reliability of breast diffuse optical
tomography. The system equipped with two pulsed laser diodes at wavelengths of 780 nm and 830 nm that are specific
to the maximal excitation and emission of the FDA-approved ICG dye, and works with a 4-channel TCSPC module to
acquire the temporal distributions of the light re-emissions 32 boundary sites of tissues in a tandem serial-to-parallel
mode. The performance and efficacy of the system are investigated with phantom experiments for diffuse optical
tomography (DOT), as well as fluorescence-guided DOT.
In optical tumor detection region, there has been recently a considerable interest in simultaneously reconstructing yield
and lifetime distributions of fluorescent imaging agents inside a pathologic tissue, since combined monitoring of these
two parameters provides a potential means of in vivo interrogating quantitative and environmental information of
specific molecules as well as accessing interactions among them. This paper describes the structure of a multi-channel
time-correlated single photon counting (TCSPC) system for early breast tumor detection and how we use it to reconstruct
the distribution of fluorescent parameters. By using a normalized Born appropriation algorithm, the proposed
examination scheme in a transmission mode is experimentally validated to achieve simultaneous reconstruction of the
fluorescent yield and lifetime distributions with reasonable accuracy. The performance of the instrument will be proved
by using two targets be of different fluorescent agents embedded in solid phantom for image reconstruction.
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