Biology subjects

Lightley, J.

Publications and source records attributed to Lightley, J..

5 recordsLinked to original sources

bioRxiv2026

Quantitative evaluation of LED based optical autofocus module

We report an improved version of the open-source optical autofocus module ("openAF") for light microscopy using a light emitting diode (LED), together with a method to independently quantify the performance of optical autofocus systems using 2D autocorrelation analysis of astigmatic imaging of fluorescent nanobeads. We apply the latter for both the LED-based and the previous super luminescent diode (SLD) based implementations of the openAF optical autofocus approach used in conjunction with a 100x 1.4 NA oil-immersion objective lens. The new approach accounts for power variations in the autofocus light source and we demonstrate that the convenient LED-based system can provide axial stability with a standard deviation <10 nm over at least 45 minutes when switched on from cold, during which the LED power varies as it reaches thermal equilibrium.

bioengineering↗

bioRxiv2025

VP-CLEM-Kit: An accessible pipeline for visual proteomics using super resolution volume correlative light and electron microscopy (SR-vCLEM)

Visual proteomics (VP) aims to allow researchers to visualise, measure and analyse proteins in the context of cell and tissue structure in health and disease. VP is becoming a reality through technological advances across several domains, including in situ structural biology and correlative light and electron microscopy (CLEM). However, widespread adoption remains limited due to the complexity and cost of the various VP approaches reported to date. Here we present the VP-CLEM-Kit, a disruptive cost-effective pipeline for super resolution volume CLEM (SR-vCLEM) that can be implemented with minimal advanced electron microscopy expertise and equipment, making it accessible to light microscopy facilities and research labs. SR-vCLEM is based on in-resin fluorescence (IRF), where fluorophores are preserved through processing into resin. The easyIRF protocol reported here reduces the requirement for complex costly sample preparation equipment and toxic chemicals compared to standard IRF protocols. easyIRF blocks are cut into ultrathin sections that are imaged using tomoSTORM, a new modular and cost-effective openFrame-based light microscope controlled by the open-source software package Micro-Manager that provides serial single molecule localisation microscopy in array tomography format. Sections are then post-stained and imaged using a tabletop scanning electron microscope controlled by open-source SBEMimage software to run in array tomography format. We demonstrate the potential of the VP-CLEM-Kit by imaging organelle reporters in human cell lines and stem cell derived neurons, fluorescently labelled protein in neurons, and immunolabelled cells in human kidney biopsy tissue from transplant patients. The VP-CLEM-Kit delivers a [~]5-fold improvement in resolution at a [~]7-fold lower cost, and thus provides new technical capability as well as a blueprint for more equitable access to advanced imaging workflows.

cell biology↗

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 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↗