Biology subjects

Zhang, H. F.

Publications and source records attributed to Zhang, H. F..

29 records · Page 2Linked to original sources

bioRxiv2023

Quantify nanoscopic alterations associated with mitochondrial dysfunctions using three-dimensional single-molecule localization microscopy

The morphology of mitochondria provides insights into their functions. Three-dimensional (3D) single-molecule localization microscopy (SMLM) uniquely enables the analysis of mitochondrial morphological features individually, thanks to its high resolution. However, nearly all reported studies of mitochondrial morphology have been qualitative and without statistical analysis. We report a quantitative method to extract mitochondrial morphological metrics, including volume, aspect ratio, and local protein density, from 3D SMLM images, with single-mitochondrion sensitivity. We validated our approach using simulated ground-truth SMLM images of mitochondria. We further tested our morphological analysis on mitochondria that have been altered functionally and morphologically in controlled manners. This work sets the stage to analyze mitochondrial morphological alterations associated with disease progression quantitatively.

biophysics↗

bioRxiv2022

Longitudinal analysis of retinal ganglion cell damage at individual axon bundle level in mice using visible-light optical coherence tomography fibergraphy

We developed a new analytic tool based on visible-light optical coherence tomography fibergraphy (vis-OCTF) to longitudinally track individual axon bundle transformation following acute optic nerve crush injury (ONC) in mice. We analyzed four parameters: lateral bundle width, axial bundle height, cross-sectional area, and the shape of individual bundles. We showed that axon bundles became wider and thicker at 3-days post ONC. The bundle swelling at 3-days post-ONC has correlated with about 15% retinal ganglion cell (RGC) soma loss. At 6-days post-ONC, axon bundles showed a significant reduction in lateral width and cross-sectional area, followed by a reduction in bundle height at 9-days post-ONC. Bundle shrinking at 9-days post-ONC has correlated with about 68% RGC soma loss. Both experimental and simulated results suggested that the cross-sectional area of individual RGC axon bundles is more sensitive than the bundle width and height to indicate RGC soma loss. This study is the first to track and quantify individual RGC axon bundles in vivo following ONC injury and establish the correlation between the morphological changes of RGC axon bundles and RGC soma loss.

neuroscience↗

bioRxiv2022

Experimental Parameters-Based Monte-Carlo Simulation of Single-Molecule Localization Microscopy of Nuclear Pore Complex to Evaluate Clustering Algorithms

Single-molecule localization microscopy (SMLM) enables the detailed visualization of nuclear pore complexes (NPC) in vitro with sub-20 nm resolution. However, it is challenging to translate the localized coordinates in SMLM images to NPC functions because different algorithms to cluster localizations as individual NPCs can be biased without ground truth for validation. We developed a Monte-Carlo simulation to generate synthetic SMLM images of NPC and used the simulated NPC images as the ground truth to evaluate the performance of six clustering algorithms. We identified HDBSCAN as the optimal clustering algorithm for NPC counting and sizing. Furthermore, we compared the clustering results between the experimental and synthetic data for NUP133, a subunit in the NPC, and found them to be in good agreement.

bioinformatics↗

bioRxiv2022

Multiple forward scattering reduces the measured scattering coefficient of whole blood in visible-light optical coherence tomography

Optical properties of blood encode oxygen-dependent information. Noninvasive optical detection of these properties is increasingly desirable to extract biomarkers for tissue health. Recently, visible-light optical coherence tomography (vis-OCT) demonstrated retinal oxygen saturation (sO2) measurements using the depth-resolved spectrum of blood. Such measurements rely on differences between the absorption and scattering coefficients of oxygenated and deoxygenated blood. However, there is still broad disagreement, both theoretically and experimentally, on how vis-OCT measures bloods scattering coefficient. Incorrect assumptions of bloods optical properties can add additional uncertainties or biases into vis-OCTs sO2 model. Using Monte Carlo simulation of a retinal vessel, we determined that vis-OCT almost exclusively detects multiple-scattered photons in blood. Meanwhile, photons mostly forward scatter in blood within the visible spectral range, allowing photons to maintain ballistic paths and penetrate deeply, leading to a reduction in the measured scattering coefficient. We defined a scattering scaling factor (SSF) to account for such a reduction and found that SSF varied with measurement conditions, such as numerical aperture, depth resolution, and depth selection. We further experimentally validated SSF in ex vivo blood phantoms pre-set sO2 levels and in the human retina, both of which agreed well with our simulation.

bioengineering↗

bioRxiv2022

Differential roles of neural crest- and endothelial-derived FOXC2 in trabecular meshwork and Schlemms Canal in glaucomatous pathology

Impaired development and maintenance of the Schlemms Canal (SC) is associated with perturbed aqueous humor outflow regulation and glaucoma progression. Key molecular mechanisms, such as ANGPT/TIE2, PROX1, and VEGF-C/VEGFR-3 regulate SC development and maintenance, but mechanisms of paracrine signaling from neighboring tissues, including the trabecular meshwork (TM) are poorly understood. Here, we show Foxc2 is critical within the neural crest (NC)-derived TM and SC endothelium for development of the aqueous humor outflow pathway. In mice, NC- specific deletion of Foxc2 results in abnormal anterior eye segment development, including impaired SC morphogenesis and functional maintenance, loss of SC identity, and impaired maintenance of intraocular pressure (IOP). Visible light optical coherence tomography angiography analysis also demonstrated functional impairment of the SC in response to changes in IOP in NC-Foxc2-/- mice, suggesting increased TM stiffness. Utilization of single-cell RNA-sequencing (scRNA-seq) analysis then identified that this phenotype is predominately characterized by transcriptional changes associated with extracellular matrix organization and stiffness in TM-associated cell clusters, including increased matrix metalloproteinase (MMP) expression, which can generate soluble TIE2 that acts as an ANGPT trap. As FOXC2 is also critically involved in development of the lymphatic vasculature in other tissues, we also show that endothelial-specific deletion of Foxc2 resulted in impaired SC morphogenesis due to loss of TIE2 expression, which was rescued by deletion of the TIE2 phosphatase VE-PTP. Thus, NC-Foxc2 is critical for development of the TM, and both NC- and endothelial-Foxc2 are key for maintenance of SC identity and its morphogenesis.

developmental biology↗

bioRxiv2021

Balanced-detection visible-light optical coherence tomography

Increases in speed and sensitivity enabled rapid clinical adoption of optical coherence tomography (OCT) in ophthalmology. Recently visible-light OCT (vis-OCT) achieved ultrahigh axial resolution, improved tissue contrast, and new functional imaging capabilities, demonstrating the potential to improve clincal care further. However, limited speed and sensitivity caused by the high relative intensity noise (RIN) in supercontinuum lasers impeded the clinical adoption of vis-OCT. To overcome these limitations, we developed balanced-detection vis-OCT (BD-vis-OCT), which uses two calibrated spectrometers to cancel noises common to sample and reference arms, including RIN. We analyzed the RIN to achieve a robust pixel-to-pixel calibration between the two spectrometers and showed that BD-vis-OCT enhanced system sensitivity by up to 22.2 dB. We imaged healthy volunteers at an A-line rate of 125 kHz and a field-of-view as large as 10 mm x 4 mm. We found that BD-vis-OCT revealed retinal anatomical features previously obscured by the noise floor.

bioengineering↗

bioRxiv2021

Adaptive spectroscopic visible-light optical coherence tomography for human retinal oximetry

Alterations in the retinal oxygen saturation (sO2) and oxygen consumption are associated with nearly all blinding diseases. A technology that can accurately measure retinal sO2 has the potential to improve ophthalmology care significantly. Recently, visible-light optical coherence tomography (vis-OCT) showed great promise for noninvasive, depth-resolved measurement of retinal sO2 as well as ultra-high resolution anatomical imaging. We discovered that spectral contaminants (SC), if not correctly removed, could lead to incorrect vis-OCT sO2 measurements. There are two main types of SCs associated with vis-OCT systems and eye conditions, respectively. Their negative influence on sO2 accuracy is amplified in human eyes due to stringent laser power requirements, eye motions, and varying eye anatomies. We developed an adaptive spectroscopic vis-OCT (Ads-vis-OCT) method to iteratively remove both types of SCs. We validated Ads-vis-OCT in ex vivo bovine blood samples against a blood-gas analyzer. We further validated Ads-vis-OCT in 125 unique retinal vessels from 18 healthy subjects against pulse-oximeter readings, setting the stage for clinical adoption of vis-OCT.

bioengineering↗

bioRxiv2021

In Vivo Sublayer Analysis Of Human Retinal Inner Plexiform Layer Obtained By Visible-Light Optical Coherence Tomography

PurposeGrowing evidence suggests, in glaucoma, the dendritic degeneration of subpopulation of the retinal ganglion cells (RGCs) may precede RGCs soma death. Since different RGCs synapse in different IPL sublayers, visualization of the lamellar structure of the IPL could enable both clinical and fundamental advances in glaucoma understanding and management. In this pilot study, we investigated whether visible-light optical coherence tomography (vis-OCT) could detect the difference in the inner plexiform layer (IPL) sublayers thicknesses between small cohorts of healthy and glaucomatous subjects. MethodWe investigated vis-OCT retinal images from nine healthy and five glaucomatous subjects. Four of the healthy subjects were scanned three times each in two separate visits, and five healthy and five glaucoma subjects were scanned three times during a single visit. Raster speckle-reduction scans (3 by 3 by 1.2 mm^3: horizontal; vertical; axial directions with 8192x8x1024 samplings, respectively) of the superior macular were acquired. IPL sublayers were then manually segmented using averaged A-line profiles. ResultsThe mean ages of glaucoma and healthy subjects are 59.6 +/- 13.4 and 45.4 +/- 14.4 years (p =0.02, Wilcoxon rank-sum test), respectively. The visual field mean deviation (MD) are -26.4 to -7.7 dB in glaucoma patient and -1.6 to 1.1 dB in healthy subjects (p =0.002). The mean circumpapillary retinal nerve fiber layer (RNFL) thicknesses are 59.6 +/- 9.1 m in glaucoma and 99.2 +/- 16.2 m in healthy subjects (p=0.004). Median coefficients of variation (CVs) of intra-session repeatability for the entire IPL and three sublayers are 3.1%, 5.6%, 6.9%, and 5.6% in healthy subjects and 1.8%, 6.0%, 7.7%, and 6.2% in glaucoma patients, respectively. The mean entire IPL thicknesses are 36.2 +/- 1.5 m in glaucomatous and 40.1 +/- 1.7 micrometer in healthy eyes (p=0.003, Mixed-effects model). We found that the middle sublayer thickness was responsible for the majority of the difference (14.2 +/- 1.8 m in glaucomatous and 17.5 +/- 1.4 in healthy eyes, p<0.01). ConclusionsIPL sublayer analysis revealed that the middle sublayer could be responsible for the majority of IPL thinning in glaucoma. Vis-OCT quantified IPL sublayers with good repeatability in both glaucoma and healthy subjects. Visualization of the IPL sublayers may enable the investigation of lamella-specific changes in the IPL in glaucoma and may help elucidate the response of different types of RGCs to the disease.

bioengineering↗

bioRxiv2020

Intrinsic spectrally-dependent background in spectroscopic visible-light optical coherence tomography

Visible-light optical coherence tomography (vis-OCT) enabled new spectroscopic applications, such as retinal oximetry, as a result of increased optical absorption and scattering contacts in biological tissue and improved axial resolution. Besides extracting tissue properties from back-scattered light, spectroscopic analyses must consider spectral alterations induced by image reconstruction itself. We investigated an intrinsic spectral bias in the background noise floor, which is hereby referred to as the spectrally-dependent background (SDBG). We developed an analytical model to predict the SDBG-induced bias and validated this model using numerically simulated and experimentally acquired data. We found that SDBG systemically altered the measured spectra of blood in human retinal vessels in vis-OCT, as compared to literature data. We provided solutions to quantify and compensate for SDBG in retinal oximetry. This work is particularly significant for clinical applications of vis-OCT.

bioengineering↗

bioRxiv2020

RainbowSTORM: An open-source ImageJ plugin for spectroscopic single-molecule localization microscopy (sSMLM) data analysis and image reconstruction

SummarySpectroscopic single-molecule localization microscopy (sSMLM) simultaneously captures the spatial locations and full spectra of stochastically emitting fluorescent single molecules. It provides an optical platform to develop new multi-molecular and functional imaging capabilities. While several open-source software suites provide sub-diffraction localization of fluorescent molecules, software suites for spectroscopic analysis of sSMLM data remain unavailable. RainbowSTORM is an open-source, user-friendly ImageJ/FIJI plugin for end-to-end spectroscopic analysis and visualization for sSMLM images. RainbowSTORM allows users to calibrate, preview, and quantitatively analyze emission spectra acquired using different reported sSMLM system designs and fluorescent labels. AvailabilityRainbowSTORM is a java plugin for ImageJ (https://imagej.net)/FIJI (http://fiji.sc) freely available through: https://github.com/FOIL-NU/RainbowSTORM. RainbowSTORM has been tested with Windows and Mac operating systems and ImageJ/FIJI version 1.52. Supplementary informationSupplementary data are available online.

bioinformatics↗

bioRxiv2019

Symmetrically-dispersed spectroscopic single-molecule localization microscopy

Spectroscopic single-molecule localization microscopy (sSMLM) achieved simultaneously imaging and spectral analysis of single molecules for the first time. Current sSMLM fundamentally suffers from reduced photon budget because of dividing photons from individual stochastic emission into spatial and spectral channels. Therefore, both spatial localization and spectral analysis only use a portion of the total photons, leading to reduced precisions in both channels. To improve the spatial and spectral precisions, we present symmetrically-dispersed sSMLM or SDsSMLM to fully utilize all photons from individual stochastic emissions in both spatial and spectral channels. SDsSMLM achieved 10-nm spatial and 0.8-nm spectral precisions at a total photon budget of 1000. Comparing with existing sSMLM using a 1:3 splitting ratio between spatial and spectral channels, SDsSMLM improved the spatial and spectral precisions by 42% and 10%, respectively, under the same photon budget. We also demonstrated multi-color imaging in fixed cells and three-dimensional single-particle tracking using SDsSMLM.

biophysics↗