Biology subjects

Lee, W. M.

Publications and source records attributed to Lee, W. M..

4 recordsLinked to original sources

bioRxiv2025

Combining Multi-site FRAP and HILO-TIRF microscopy using a Spatial Light Modulator

Fluorescence Recovery After Photobleaching (FRAP) has remained a powerful tool to probe intracellular dynamics. FRAP relies on two aspects: (1) localised excitation resulting in photobleaching, and (2) fluorescence recovery of the bleached volume which provides insight into kinetics. Existing FRAP systems are limited by a trade-off between time and spatial multiplexing due to galvanometric scanning methods, precluding the study of multiple independent positions simultaneously as well as advanced widefield imaging modes. Hence, they may not capture the dynamic and non-isotropic environments in biological studies. In this paper, we utilise phase profiles corresponding to an array of fresnel lenses and diffractive masks to switch between simultaneous FRAP of independent spatial positions whilst maintaining epifluorescence, highly inclined laminated optical sheet (HILO), and total internal reflection fluorescence (TIRF) microscopy modalities. Our approach bridges high contrast fluorescence imaging (HILO and TIRF) and a multi-position FRAP technique using a single spatial light modulator. As such, this technique enables high contrast bleaching and screening across volumes, which we envision will be of value to areas such as single particle tracking and single molecule imaging where dynamic photobleaching is necessary to measure fast events across the field of view in a single versatile instrument.

biophysics↗

bioRxiv2025

Using rotational integration of oblique interferometric scattering (RO-iSCAT) to track axial spatiotemporal responses of membrane protrusions

Despite the crucial importance of dynamic membrane protrusions for understanding phagocytosis, cellular communication and mechanobiology, current imaging modalities struggle to quantitatively track their real-time, 3D spatiotemporal dynamics with sufficient molecular specificity and minimal perturbation. Many membrane protrusions studies still utilize confocal microscopy where its axial resolution and high phototoxicity remains a key limiting factor for live axial imaging. We discovered that multiple rotational oblique interference scattering (RO-iSCAT) leverages off-axis illumination to induce a larger lateral shift in out-of-focus iSCAT signals compared to in-focus signals. This phenomenon provides a foundation to generate speckle-free widefield interferometric signals with a 10-fold signal to noise ratio improvement, eliminating the need for any background subtraction. RO-iSCAT enables real-time, label-free, and minimally invasive imaging of diverse membrane protrusions within complex co-cultures. RO-iSCAT enables nanoscale-sensitive tracking of membrane protrusion dynamics along the axial direction. This allows for the construction of dynamic axial variance maps, facilitating quantitative measurements of membrane protrusion formation at tens to hundreds of nanometer displacements, without requiring 3D volumetric imaging. RO-iSCAT empowers real time quantitatively dissection of the axial spatiotemporal complexities of membrane protrusions and unlock future insights into fundamental processes like cell migration, durotaxis, and intercellular communication. Key pointsO_LIDiscovered that multiple integrated rotational oblique interference scattering (RO-iSCAT) generates speckle-free widefield interferometric signals with a 10-fold signal to noise ratio improvement, eliminating the need for any background subtraction. C_LIO_LIRemoved need for 3D volumetric imaging to quantified axial motion of membrane protrusion forming tethers, trails and bridge with within [~] tens of nanometer accuracy. C_LIO_LIEnabled classification of membrane protrusions that, despite possessing identical chemical compositions, are differentiated by their interactions, thus offering a qualitative comparison of membrane protrusions at the nanoscale in living cells. C_LI Summary Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/644841v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@b183a4org.highwire.dtl.DTLVardef@39a944org.highwire.dtl.DTLVardef@197f019org.highwire.dtl.DTLVardef@1438b3_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Imaging throughput of compact handheld microscopes for quantitative single cell studies

High-throughput live-cell imaging within incubator environments often necessitates a compromise between optical resolution and instrument/computational complexity. In this work, we demonstrate that controlled defocusing, a default function in all optical microscopes, can be utilized as a primary contrast mechanism for large-scale cell analysis (7000 cells per field of view). Our results suggest that even at a low numerical aperture (NA [~] 0.01) and using just epi-illumination, defocused images can generate a uniform negative contrast across the cell body. This negative contrast improves automated cell segmentation efficiency over a wide field of view compared to in-focus imaging. We further evaluated the accessibility of this defocus imaging approach for both 2D and 3D cultures by implementing an automated cell segmentation protocol on a commercial off-the-shelf digital Universal Serial Bus (USB) microscope. The compact form factor of the digital USB microscope facilitates minimal pixel sampling, enabling high-throughput single-cell detection and continuous tracking across a large adherent cell population over several days. Our assessments of the utility of defocus imaging for 2D and 3D tissue cultures were further supported by monitoring the negative contrast changes during the migration and dissociation of 3D tissue spheroids. Our results show that negative contrast profiles from defocus images enable the quantification of cell proliferation, division, migration, and cell-to-cluster dissociation within standard culture environments. This defocusing methodology offers a scalable approach to extensive high-content screening through simplified instrumentation controls. Key pointsO_LIDefocused images of adherent cells using low NA optics homogenizes intracellular intensity hotspots, creating uniform negative contrast for whole cell segmentation; similar to shadow imaging in fluorescence. C_LIO_LIInverting look-up-table (LUT) combined with defocus imaging on large population of adherent cells in standard culture flask taken by standard digital USB microscopes allow for rapid, routine tracking of thousands of cells per field of view. C_LIO_LINegative contrast image profiles from defocusing enable the quantification cell proliferation, division, migration for 2D monolayer and 3D tissue spheroid cultures. C_LI

biophysics↗

bioRxiv2022

Combined scattering, interferometry and fluorescence oblique illumination for live cell nanoscale imaging

To determine the molecular and/or mechanical basis of cell migration using live cell imaging tools, it is necessary to correlate multiple 3D spatiotemporal events simultaneously. Fluorescence nanoscopy and label free nanoscale imaging can complement each other by providing both molecular specificity and structural dynamics of sub-cellular structure. In doing so, a combined imaging system would permit quantitative 3D spatial temporal detail of individual cellular components. In this paper, we empirically determined a series of optimal azimuthal scanning angles and rotating beam to achieve simultaneous and label-free nanoscale and fluorescence imaging. Label-free nanoscale imaging here refers to interferometric, brightfield (BF) and darkfield (DF) rotating coherence scattering (ROCS) microscopy, while fluorescence refers to high inclined Laminated Oblique (HiLO) and total internal reflection fluorescence (TIRF) imaging. The combined capabilities of interferometric, scattering and fluorescence imaging enables (1) the identification of molecular targets (substrate or organelle), (2) quantification of 3D cell morphodynamics, and (3) tracking of intracellular organelles in 3D. This combined imaging tool was then used to characterize migrating platelets and adherent endothelial cells, both critical to the process of infection and wound healing. The combined imaging results of over [~]1000 platelets, suggested that serum albumin (bovine) was necessary for platelets to migrate and scavenge fibrin/fibrinogen. Furthermore, we determine new asynchronous membrane fluctuations between the leading and rear edge of a migrating platelet. We further demonstrated that interferometric imaging permitted the quantification of mitochondria dynamics on lung microvascular cells (HMVEC). Our data suggests that axial displacement of mitochondria is minimized when it is closer to the nucleus or the leading edge of a cell membrane that exhibits retrograde motion. Taken together, this combined imaging platform has proven to quantify multiple spatial temporal events of a migrating cell, that will undoubtedly open ways to new quantitative correlative nanoscale live cell imaging.

biophysics↗