Biology subjects

Miller, D. A.

Publications and source records attributed to Miller, D. A..

6 recordsLinked to original sources

bioRxiv2026

Spectroscopic DNA-PAINT for simultaneous multiplexed super-resolution microscopy

Simultaneous multiplexed super-resolution imaging remains a central challenge in single-molecule localization microscopy (SMLM), due to the limited number of spectrally distinguishable fluorophores and the trade-off between spatial and spectral precision. Here, we introduce spectroscopic DNA-PAINT (sDNA-PAINT), a framework that integrates DNA-PAINT with spectroscopic SMLM (sSMLM) to enable simultaneous multiplexed imaging with high spatial and spectral fidelity. Using DNA Origami Nanorulers, in vitro and fixed-cell imaging, we show that sDNA-PAINT conditions significantly improve spectral precision and photon budgets compared to conventional glass and antibody-conjugated conditions in sSMLM imaging. Across representative dyes from three fluorophore families (Rhodamine, Cyanine and Oxazine), narrow spectral centroid distributions are observed to enable reliable statistical discrimination at the single-molecule level, even for dyes with heavily overlapping ensemble spectra. In dual-target cellular imaging, sDNA-PAINT achieves accurate spatial reconstruction and high classification accuracy, demonstrating high-fidelity simultaneous multiplexing within a single acquisition. sDNA-PAINT provides a pathway toward high-throughput simultaneous multiplexed super-resolution interaction imaging.

bioengineering↗

bioRxiv2025

Development of a method for large-scale single-molecule analysis of tau proteoforms

Proteins exist as diverse proteoforms resulting from a combination of genetic variation, alternative splicing, and post-translational modifications. Current methods struggle to capture this complexity at the single-molecule level. Here we introduce Iterative Mapping of Proteoforms (IMaP), a method that enables the massively-parallel interrogation of millions to billions of single-protein molecules through iterative probing with fluorescently labeled antibodies. Using 12 site-specific antibodies, the method is capable of measuring 212 (4,096) potential proteoform groups. We used IMaP to measure proteoform group profiles of the tau protein, a key player in neurodegenerative diseases, using two pan anti-tau antibodies (Tau-13, Tau-216), three isoform-specific antibodies (Anti-0N, Anti-2N, Anti-4R), and seven phosphosite-specific antibodies (Anti-pT181, Anti-pS202+pT205, Anti-pT205, Anti-pS214, Anti-pT217, Anti-pT231, and Anti-pS396). The method demonstrates high sensitivity (detecting proteoforms at 0.1% abundance), high reproducibility (median CV <5.5%), and broad dynamic range (>3 orders of magnitude), outperforming conventional techniques in resolving closely related proteoform groups. We demonstrated that the method can be used on relevant biological samples by examining various neuronal models (iNeuron cells, organoids, MiBrains, and mouse brains) and human samples. This examination revealed 130 distinct tau proteoform groups with as many as six phosphorylation events. The non-random distribution of these phosphorylation events suggests ordered and site-specific modification processes rather than random, stochastic accumulation. Certain combinations of phosphorylation events were more abundant than others; for example, pT217 preferentially co-occurred with pT181. In validating the applicability of the assay to human disease samples, we noted a specific pattern of multiple phosphorylation events in an advanced Alzheimers disease patient that suggests a sequential pathway of pathological tau modification. Iterative Mapping of Proteoforms provides insights into proteoform complexity at the single-molecule level, with significant implications for understanding protein regulation in neurodegenerative diseases and beyond.

biochemistry↗

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↗

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↗

bioRxiv2019

Associated habitat and suitability modeling of goldenseal (Hydrastis canadensis L.) in Pennsylvania: explaining and predicting species distribution in a northern edge of range state

Goldenseal (Hydrastis canadensis L.) is a well-known perennial herb indigenous to forested areas in eastern North America. Owing to conservation concerns including wild harvesting for medicinal markets, habitat loss and degradation, and an overall patchy and often inexplicable absence in many regions, there is a need to better understand habitat factors that help determine the presence and distribution of goldenseal populations. In this study, flora and edaphic factors associated with goldenseal populations throughout Pennsylvania--a state near the northern edge of its range--were documented and analyzed to identify habitat indicators and provide possible in situ stewardship and farming (especially forest-based farming) guidance. Additionally, maximum entropy (Maxent) modeling was applied to better predict where suitable habitat might be encountered more broadly and explain species absence from regions of the state (and the northeastern states). Habitat study results identified rich, mesic, woodland sites as being suitable for goldenseal. The most prevalent overstory tree associates on such sites were tulip-poplar (Liriodendron tulipifera L.) and sugar maple (Acer saccharum Marshall), and the most common understory associates were spicebush (Lindera benzoin L.), Virginia creeper (Parthenocissus quinquefolia (L.) Planch.), Jack-in-the-pulpit (Arisaema triphyllum (L.) Schott), mayapple (Podophyllum peltatum L.), wood fern (Drypoteris marginalis (L.) A. Gray), and rattlesnake fern (Botrypus virginianus (L.) Michx.). Loam soils were the most common textural class (average sand, silt, and clay ratio of 50:30:20) with an average pH of 6.2 and high variation in macronutrients. While such sites are widespread in the state, Maxent modeling suggested the present distribution in Pennsylvania is largely restricted by winter temperatures and bedrock type. The latter of these, in turn, is correlated in part with land use legacy (e.g., clearing for farming or livestock grazing), especially in southeast portions of the state.

ecology↗