Biology subjects

French, P. M. W.

Publications and source records attributed to French, P. M. W..

6 recordsLinked to original sources

bioRxiv2025

Assessing PARP trapping dynamics in ovarian cancer using a CRISPR-engineered FRET biosensor

Poly(ADP-ribose) polymerase inhibitors (PARPi) have revolutionised the treatment of ovarian high grade serous carcinoma (HGSC), especially those with defective homologous recombination. However, the emergence of resistance poses a critical challenge, as over 50% of patients relapse within three years. The mechanisms underlying changes in PARP trapping, a central aspect of PARPi efficacy, are not well understood due to limitations in current experimental methodologies. Existing techniques lack resolution and throughput, impeding efforts to study PARP trapping dynamics over time with single-cell resolution. Effective tools to study PARP trapping in live cells are urgently needed to elucidate resistance mechanisms and inform therapeutic strategies. To address this, we used CRISPR-Cas9 gene editing to dual-label endogenous PARP1 with EGFP and mCherryFP in OVCAR4 cells to develop a novel intramolecular FRET-based biosensor that enables real-time, single-cell visualization of PARP trapping dynamics in live cells. High-content fluorescence lifetime imaging microscopy (FLIM) revealed dose dependent PARP trapping upon exposure to PARP inhibitor and distinguished between the trapping efficiencies of four different PARPi (veliparib, olaparib, rucaparib, talazoparib). Moreover, we found reduced PARP trapping in PARPi-resistant models, both in vitro and in vivo, providing critical evidence for altered PARP trapping as a resistance mechanism and illustrating the potential of this FRET biosensor to interrogate resistance mechanisms quantitatively. This PARP trapping biosensor represents a transformative advance, enabling dynamic, high-resolution analysis of mechanisms underlying cancer drug resistance. It provides critical insights into the heterogeneity of PARPi resistance, with implications for developing more effective therapies and improving personalised treatment strategies for ovarian cancer patients. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=58 SRC="FIGDIR/small/642798v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@1c5ee67org.highwire.dtl.DTLVardef@1a6d101org.highwire.dtl.DTLVardef@e3a2bborg.highwire.dtl.DTLVardef@d0ba03_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

PolSpec: polarisation-based detection for spectral classification of optical signals

Spectrally resolved imaging is typically realised using bandpass filters, which are inefficient when they reject "out-of-band" photons or using angularly dispersive devices with at least one image dimension requiring scanning to acquire a full hyperspectral dataset and therefore sequential data acquisition, unless cascades of dichroic beamsplitters are employed, for which cost and experimental complexity scale with the number of spectral channels. Here we present a new approach, "PolSpec", to realise rapid and flexible widefield hyperspectral imaging with lower cost and complexity using polarisation optics instead of dielectric coatings or dispersive devices. PolSpec utilises Lyot filters that provide continuously varying transmission across the desired spectral range to generate orthogonal "spectral modulation vectors" that can represent specific spectral signatures with significantly lower data volumes than full spectral profiles. We demonstrate single-shot widefield hyperspectral imaging using a polarisation-resolving camera and rapid, electronically reconfigurable, more photon efficient, hyperspectral imaging using a liquid crystal variable retarder.

biophysics↗

bioRxiv2022

Robust, single-shot, optical autofocus system utilizing cylindrical lenses to provide high precision and long range of operation

We present a robust, "real-time" optical autofocus system for microscopy that provides high accuracy (<230 nm) and long range ([~]130 {micro}m) with a 1.4 numerical aperture oil immersion objective lens. This autofocus can operate in a closed loop, single-shot functionality over a range of {+/-}37.5 {micro}m and can also operate as a 2-step process up to {+/-}68 {micro}m. A real-time autofocus capability is useful for experiments with long image data acquisition times, including single molecule localization microscopy, that may be impacted by defocusing resulting from drift of components, e.g., due to changes in temperature or mechanical drift. It is also vital for automated slide scanning or multiwell plate imaging where the sample may not be in the same horizontal plane for every field of view during the image data acquisition. To realise high precision and long range, we implement orthogonal optical readouts using cylindrical lenses. We demonstrate the performance of this new optical autofocus system with automated multiwell plate imaging and single molecule localisation microscopy and illustrate the benefit of using a superluminescent diode as the autofocus light source.

bioengineering↗

bioRxiv2021

Single-Shot Optical Projection tomography for high-speed volumetric imaging

We present a single-shot adaptation of Optical Projection Tomography (OPT) for high-speed volumetric snapshot imaging of dynamic mesoscopic samples. Conventional OPT has been applied to in vivo imaging of animal models such as D. rerio but the sequential acquisition of projection images required for volumetric reconstruction typically requires samples to be immobilised during the acquisition of an OPT data set. We present a proof-of-principle system capable of single-shot imaging of a 1 mm diameter volume, demonstrating camera-limited rates of up to 62.5 volumes/second, which we have applied to 3D imaging of a freely-swimming zebrafish embryo. This is achieved by recording 8 projection views simultaneously on 4 low-cost CMOS cameras. With no stage required to rotate the sample, this single-shot OPT system can be implemented with a component cost of under {pound}5,000. The system design can be adapted to different sized fields of view and may be applied to a broad range of dynamic samples, including fluid dynamics.

biophysics↗

bioRxiv2021

Single-shot phase contrast microscopy using polarisation-resolved differential phase contrast

We present a robust, low-cost single-shot implementation of differential phase microscopy utilising a polarisation-sensitive camera to simultaneously acquire 4 images from which the phase gradients and quantitative phase image can be calculated. This polarisation-resolved differential phase contrast (pDPC) microscopy technique can be interleaved with single-shot imaging polarimetry.

biophysics↗

bioRxiv2021

Robust optical autofocus system utilizing neural networks trained for extended range and time-course and automated multiwell plate imaging including single molecule localization microscopy

We present a robust, long-range optical autofocus system for microscopy utilizing machine learning. This can be useful for experiments with long image data acquisition times that may be impacted by defocusing resulting from drift of components, e.g. due to changes in temperature or mechanical drift. It is also useful for automated slide scanning or multiwell plate imaging where the sample(s) to be imaged may not be in the same horizontal plane throughout the image data acquisition. To address the impact of (thermal or mechanical) fluctuations over time in the optical autofocus system itself, we utilise a convolutional neural network (CNN) that is trained over multiple days to account for such fluctuations. To address the trade-off between axial precision and range of the autofocus, we implement orthogonal optical readouts with separate CNN training data, thereby achieving an accuracy well within the 600 nm depth of field of our 1.3 numerical aperture objective lens over a defocus range of up to approximately +/- 100 m. We characterise the performance of this autofocus system and demonstrate its application to automated multiwell plate single molecule localisation microscopy.

bioengineering↗