Subcellular Comparison of Visible-Light Optical Coherence Tomography and Electron Microscopy in the Mouse Outer Retina
Purpose/UNASSIGNED:We employed in vivo, 1.0-Âµm axial resolution visible-light optical coherence tomography (OCT) and ex vivo electron microscopy (EM) to investigate three subcellular features in the mouse outer retina: reflectivity oscillations inner to band 1 (study 1); hyperreflective band 2, attributed to the ellipsoid zone or inner segment/outer segment (IS/OS) junction (study 2); and the hyperreflective retinal pigment epithelium (RPE) within band 4 (study 3). Methods/UNASSIGNED:Pigmented (C57BL/6J, n = 10) and albino (BALB/cJ, n = 3) mice were imaged in vivo. Enucleated eyes were processed for light and electron microscopy. Using well-accepted reference surfaces, we compared micrometer-scale axial reflectivity of visible-light OCT with subcellular organization, as revealed by 9449 annotated EM organelles and features across four pigmented eyes. Results/UNASSIGNED:In study 1, outer nuclear layer reflectivity peaks coincided with valleys in heterochromatin clump density (-0.34 Â± 2.27 Âµm limits of agreement [LoA]). In study 2, band 2 depth on OCT and IS/OS junction depth on EM agreed (-0.57 Â± 0.76 Âµm LoA), with both having similar distributions. In study 3, RPE electron dense organelle distribution did not agree with reflectivity in C57BL/6J mice, with OCT measures of RPE thickness exceeding those of EM (2.09 Â± 0.89 Âµm LoA). Finally, RPE thickness increased with age in pigmented mice (slope = 0.056 Âµm/mo; P = 6.8 Ã— 10-7). Conclusions/UNASSIGNED:Visible-light OCT bands arise from subcellular organization, enabling new measurements in mice. Quantitative OCT-EM comparisons may be confounded by hydration level, particularly in the OS and RPE. Caution is warranted in generalizing results to other species.
Optical imaging and spectroscopy for the study of the human brain: status report
This report is the second part of a comprehensive two-part series aimed at reviewing an extensive and diverse toolkit of novel methods to explore brain health and function. While the first report focused on neurophotonic tools mostly applicable to animal studies, here, we highlight optical spectroscopy and imaging methods relevant to noninvasive human brain studies. We outline current state-of-the-art technologies and software advances, explore the most recent impact of these technologies on neuroscience and clinical applications, identify the areas where innovation is needed, and provide an outlook for the future directions.
Scanning interferometric near-infrared spectroscopy
In diffuse optics, quantitative assessment of the human brain is confounded by the skull and scalp. To better understand these superficial tissues, we advance interferometric near-infrared spectroscopy (iNIRS) to form images of the human superficial forehead blood flow index (BFI). We present a null source-collector (S-C) polarization splitting approach that enables galvanometer scanning and eliminates unwanted backscattered light. Images show an order-of-magnitude heterogeneity in superficial dynamics, implying an order-of-magnitude heterogeneity in brain specificity, depending on forehead location. Along the time-of-flight dimension, autocorrelation decay rates support a three-layer model with increasing BFI from the skull to the scalp to the brain. By accurately characterizing superficial tissues, this approach can help improve specificity for the human brain.
Parallel interferometric Diffusing Wave Spectroscopy (iDWS) with Time-of-Flight Discrimination
[S.l.] : Optica Publishing Group (formerly OSA), 2022
Neurophotonic tools for microscopic measurements and manipulation: status report
Proactive spectrometer matching for excess noise suppression in balanced visible light optical coherence tomography (OCT)
Supercontinuum sources for visible light spectral domain OCT (SDOCT) are noisy and often expensive. Balanced detection can reduce excess noise, but is rarely used in SDOCT. Here, we show that balanced detection can achieve effective excess noise cancellation across all depths if two linear array spectrometers are spectrally well-matched. We propose excess noise correlation matrices as tools to achieve such precise spectral matching. Using optomechanical adjustments, while monitoring noise correlations, we proactively match wavelength sampling of two different spectrometers to just a few picometers in wavelength, or 0.001% of the overall spectral range. We show that proactively-matched spectrometers can achieve an excess noise suppression of more than two orders-of-magnitude in balanced visible light OCT, outperforming simple retrospective software calibration of mismatched spectrometers. High noise suppression enables visible light OCT of the mouse retina at 70 kHz with 125 microwatts incident power, with an inexpensive, 30MHz repetition rate supercontinuum source. Averaged images resolve the retinal pigment epithelium in a highly pigmented mouse strain.
Multi-exposure interferometric diffusing wave spectroscopy
We present multi-exposure interferometric diffusing wave spectroscopy (MiDWS), which measures brain blood flow index (BFI) continuously and non-invasively. MiDWS employs interferometry to detect low light levels, probing the optical field autocorrelation indirectly by varying the sensor exposure time. Here MiDWS is compared with conventional interferometric diffusing wave spectroscopy and speckle contrast optical spectroscopy in phantoms. Notably, the MiDWS approach enables the use of low frame rate, two-dimensional complementary metal-oxide semiconductor cameras in a short exposure time regime, where detector noise greatly exceeds the sample photon count. Finally, we show that MiDWS can monitor the BFI simultaneously at two source-collector separations (1 and 3 cm) on the adult human head on a single camera, enabling the use of superficial signal regression techniques to improve brain specificity.
In vivo imaging of inner plexiform layer (IPL) stratification in the human retina with visible light Optical Coherence Tomography (OCT) [Meeting Abstract]
Purpose : Employing visible light OCT, we report a stereotyped reflectivity pattern of the inner plexiform layer (IPL) that parallels IPL stratification. We characterize this pattern noninvasively in adult human subjects without ocular pathology. Methods : Subjects were imaged by a visible light OCT prototype instrument at UC Davis with 1 micron axial resolution. A total of 15 eyes of 15 subjects were analyzed. The inner retinal layer boundaries were demarcated. At each transverse position, the IPL intensity was interpolated onto an IPL thickness percentage abscissa axis. Images were partitioned into transverse segments of 450 microns (1.5 degrees) and IPL intensities were averaged across each segment (Figure 1A-B). To detect salient features of intensity profiles, a 14 order polynomial fit was performed on the intensity profile within the IPL (Figure 1C). The polynomial fit provided access to features such as stratum location and intensity (Figure 1D). Results : Figure 2A shows subject-by-subject fitting of stratum S5 intensity versus IPL thickness with mixed effects and fixed effects models. The fixed slopes are all greater than zero, pointing to an increase in S5 prominence with IPL thickness (Figure 2B). Conclusions : The proposed method reveals IPL organization in living human subjects, potentially enabling studies of stratification during development and in diseases
Visible Light Optical Coherence Tomography (OCT) Quantifies Subcellular Contributions to Outer Retinal Band 4
Purpose:To use visible light optical coherence tomography (OCT) to investigate subcellular reflectivity contributions to the outermost (4th) of the retinal hyperreflective bands visualized by current clinical near-infrared (NIR) OCT. Methods:Visible light OCT, with 1.0 Âµm axial resolution, was performed in 28 eyes of 19 human subjects (21-57 years old) without history of ocular pathology. Two foveal and three extrafoveal hyperreflective zones were consistently depicted within band 4 in all eyes. The two outermost hyperreflective bands, occasionally visualized by NIR OCT, were presumed to be the retinal pigment epithelium (RPE) and Bruch's membrane (BM). RPE thickness, BM thickness, and RPE interior reflectivity were quantified topographically across the macula. Results:A method for correcting RPE multiple scattering tails was found to both improve the Gaussian goodness-of-fit for the BM intensity profile and reduce the coefficient of variation of BM thickness in vivo. No major topographical differences in macular BM thickness were noted. RPE thickness decreased with increasing eccentricity. Visible light OCT signal intensity in the RPE was weighted to the apical side and attenuated more across the RPE in the fovea than peripherally. Conclusions:Morphometry of the presumed RPE and BM bands is consistent with known anatomy. Weighting of RPE reflectivity toward the apical side suggests that melanosomes are the predominant contributors to RPE backscattering and signal attenuation in young eyes. Translational Relevance:By enabling morphometric analysis of the RPE and BM, visible light OCT deciphers the main reflectivity contributions to outer retinal band 4, commonly visualized by commercial OCT systems.
Approaches to improve brain specificity and accuracy with interferometric diffusing wave spectroscopy
[S.l.] : The Optical Society, 2021