Biology subjects

Foylan, S.

Publications and source records attributed to Foylan, S..

8 recordsLinked to original sources

bioRxiv2026

A rapid, low-cost approach to solid immersion lens fabrication for enhanced resolution in optical microscopy

Solid immersion lenses (SILs) enhance the spatial resolution of an optical microscope by increasing the effective numerical aperture (NA) without physical modification of the objective lens. However, SIL application remains limited by cost, fragility, and accessibility. We present a rapid, single-step fabrication process to create optical quality hemispherical SILs using consumer-grade UV-curable transparent resin which reduces material costs by over five orders of magnitude relative to commercial glass counterparts. Our method produced resin SILs within seconds which can be easily implemented into conventional microscopy setups for increasing the effective NA. Quantitative imaging of USAF resolution targets and histology muscle preparations demonstrated a resolution enhancement approaching theoretical limits and comparable performance to N-BK7 glass SILs. This enabled visualisation of features usually below the diffraction limit of low NA dry objectives at a fraction of the cost of otherwise required high-powered objective lenses. To demonstrate accessibility and translational potential, our workflow was taught in a practical tutorial of an international microscopy course, where non-expert participants successfully fabricated, characterised, and applied SILs within a single session, reporting high confidence in independent implementation. We established ultra-low-cost resin SILs as a practical, scalable option to enhance the spatial resolution of routine optical microscopes and as an accessible and cost-effective platform for optics education.

biophysics↗

bioRxiv2025

A large 3D-printed integrated lens-biprism element enhances contrast in transmission stereomicroscopy

Stereomicroscopes are routinely used across disciplines for material and surface characterisation due to their simplicity of use and minimal specimen preparation requirements. However, the stereomicroscope transillumination design is suboptimal, as a single incident beam at the specimen plane is shared and transmitted via two laterally offset detection axes. This flaw limits life science applications due to the transparent nature of samples which results in poor contrast images. We use a single additional element in the illumination path to correct the illumination uniformity across the field of view and, by doing so, enhance image contrast and facilitate detection of refractive structures in transparent biological specimens. We designed and fabricated an integrated lens-biprism element using low-cost, consumer-grade 3D printing methods and consumables. This 3D printed lens-biprism distributed diverging rays from a single incandescent light source into two parallel beams that converged at the specimen plane and transmitted through the respective left and right detection axes of a stereomicroscope. This improved transillumination setup increased the image contrast by up to 67.62% compared to the conventional stereomicroscope setup. We demonstrated the benefit of the lens-biprism element by visualising dynamic cellular events in live tissue and discerning refractive structures more easily in transparent specimens.

biophysics↗

bioRxiv2025

PerfectlyAverage: a classical open-source software method to determine the optimal averaging parameters in laser scanning fluorescence microscopy

Laser scanning fluorescence microscopy (LSFM) is a widely used imaging method, but image quality is often degraded by noise. Averaging techniques can enhance the signal-to-noise ratio (SNR), but while this can improve image quality, excessive frame accumulation can introduce photobleaching and may lead to unnecessarily long acquisition times. A classical software method called PerfectlyAverage is presented to determine the optimal number of frames for averaging in LSFM using SNR, photobleaching, and power spectral density (PSD) measurements. By assessing temporal intensity variations across frames, PerfectlyAverage identifies the point where additional averaging ceases to provide significant noise reduction. Experiments with fluorescently stained tissue paper and fibroblast cells validated the approach, demonstrating that up to a 4-fold reduction in averaging time may be possible. Data are also presented to suggest that frame and line averaging with the same number of averages may produce different results, and this may influence the optimal number of averages. PerfectlyAverage is open source, compatible with any LSFM data obtained using line or frame averaging during image acquisition, and it is aimed at improving imaging workflows while reducing the reliance on subjective criteria for choosing the number of averages.

cell biology↗

bioRxiv2024

Revealing the Ultrastructure of Live Candida albicans using Stimulated Emission Depletion Microscopy

Candida albicans, a commensal fungal pathogen, is a major cause of opportunistic infections in immunocompromised individuals. Understanding its cellular structures and pathogenic mechanisms is critical for developing targeted antifungal therapies. Stimulated emission depletion (STED) microscopy enables nanoscale visualization of cellular components, surpassing the diffraction limit of conventional light microscopy. In this study, we employed STED microscopy to investigate the ultrastructural organization of C. albicans in live specimens. We showed that dyes commonly used in STED microscopy of mammalian cells are ineffective for the study of C. albicans, and we showed the utility of Nile Red staining for visualising the organisation of dynamic cellular components, including tracking of lipid droplets, using time-lapse recording in experiments exceeding 12 hours. STED microscopy offered more than a two-fold improvement in resolution compared to confocal laser scanning microscopy applied to the same specimens with negligible photobleaching. This study demonstrates the utility of STED microscopy in advancing our understanding of C. albicans biology at the nanoscale, providing a platform for future investigations into fungal pathogenicity and antifungal development. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/625149v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@d46837org.highwire.dtl.DTLVardef@105f661org.highwire.dtl.DTLVardef@728d11org.highwire.dtl.DTLVardef@8b65ae_HPS_FORMAT_FIGEXP M_FIG C_FIG We present an optimised fluorescence staining method for super-resolution live-cell imaging of Candida albicans, using Stimulated Emission Depletion (STED) microscopy to resolve and track sub-cellular structures. We compare the performance of conventional confocal laser scanning microscopy (CLSM) to STED imaging, providing a three-fold resolution improvement beyond the diffraction limit. Finally, we perform live cell tracking to visualise and quantify the trajectories of multiple sub-diffraction limit-sized objects over a period of 12 hours, demonstrating the potential for live-cell STED imaging of Candida to visualise key processes involved in pathogenesis, drug resistance and infection.

microbiology↗

bioRxiv2024

Obtaining super-resolved images at the mesoscale through Super-Resolution Radial Fluctuations

Super-resolution microscopy overcomes the diffraction limit of light to achieve higher spatial resolutions than are typically available when using light microscopy techniques. However, these methods are usually restricted to imaging a very small field of view (FOV). Here, we have applied one of these super-resolution techniques, Super-Resolution Radial Fluctuations (SRRF) in conjunction with the Mesolens, which has the unusual combination of a low-magnification and high numerical aperture, to obtain super-resolved images over a FOV of 4.4 mm x 3.0 mm. We assessed the accuracy of these SRRF images through error maps calculated using a secondary analysis method, Super-resolution Quantitative Image Rating and Reporting of Error Locations (SQUIRREL). We demonstrate it is possible to achieve images with a resolution of 446.3 {+/-} 10.9 nm, providing a [~]1.6-fold improvement in spatial resolution over a uniquely large field, with consistent structural agreement between raw data and SRRF processed images. MotivationCurrent super-resolution imaging techniques allow for a greater understanding of cellular structures however they are often complex or only have the ability to image a few cells at once. This small field of view may not represent the behaviour across the entire sample and the manual selection of which restricted ROI to use may introduce bias. Currently, this is often circumvented by stitching and tiling methods which stitch many small ROI together, however this can result in artefacts across an image which poses an issue when analysing data. To combat this, we have used the Mesolens alongside Super-Resolution Radial Fluctuations analysis, to obtain super-resolved images over a field of view of 4.4 mm x 3.0 mm with minimal error.

biophysics↗

bioRxiv2023

Printing, Characterising, and Assessing Transparent 3D Printed Lenses for Optical Imaging

High-quality lens production has involved subtractive manufacturing methods for centuries. These methods demand specialist equipment and expertise that often render custom high-grade glass optics inaccessible. We aimed to develop a low-cost, accessible, and reproducible method to manufacture high-quality three-dimensional (3D) printed lenses using consumer-grade technology. Various planoconvex lenses were produced using a consumer-grade 3D printer and low-cost spin coating setup, and printed lenses were compared to commercial glass counterparts. A range of mechanical and optical methods are introduced to determine the surface quality and curvature of 3D printed lenses. Amongst others, high-resolution interference reflection microscopy methods were used to reconstruct the convex surface of printed lenses and quantify their radius of curvature. The optical throughput and performance of 3D printed lenses were assessed using optical transmissivity measurements and classical beam characterisation methods. We determined that 3D printed lenses had comparable curvature and performance to commercial glass lenses. Finally, we demonstrated the application of 3D printed lenses for brightfield transmission microscopy, resolving sub-cellular structures over a 2.3 mm field-of-view. The high reproducibility and comparable performance of 3D printed lenses present great opportunities for additive manufacturing of bespoke optics for low-cost rapid prototyping and improved accessibility to high-quality optics in low-resource settings.

biophysics↗

bioRxiv2023

Standing wave mesoscopy

Standing wave (SW) microscopy is a method that uses an interference pattern to excite fluorescence from labelled cellular structures and produces high-resolution images of three-dimensional objects in a two-dimensional dataset. SW microscopy is performed with high magnification, high numerical aperture objective lenses, and while this results in high resolution images, the field of view is very small. Here we report upscaling of this interference imaging method from the microscale to the mesoscale using the Mesolens, which has the unusual combination of a low magnification and high numerical aperture. With this method, we produce SW images within a field of view of 4.4 mm x 3.0 mm that can readily accommodate over 16,000 cells in a single dataset. We demonstrate the method using both single-wavelength excitation and the multi-wavelength SW method TartanSW. We show application of the method for imaging of fixed and living cells specimens, with the first application of SW imaging to study cells under flow conditions.

cell biology↗

bioRxiv2022

MesoTIRF: a Total Internal Reflection Fluorescence illuminator for axial super-resolution membrane imaging at the mesoscale

Total Internal Reflection Fluorescence (TIRF) illumination bypasses the axial diffraction limit of light by using an evanescent field to excite fluorophores close to a sample substrate. TIRF illumination significantly improves image contrast, allowing researchers to study membrane structure and dynamics with localized reductions in photobleaching. However, a significant limitation of most TIRF microscopes is the relatively small field of view (FOV). TIRF objectives require a high numerical aperture (NA) to generate the evanescent wave. Such lenses invariably have a high magnification and result in a [~] 50 {micro}m diameter imaging field, requiring many subsequent images for accurate statistical analysis. Waveguide and prism-based TIRF systems are, in principle, compatible with lower magnification lenses to widen the FOV but these have a correspondingly low NA and lateral resolution. To overcome these limitations, we present a prism-based TIRF illuminator for the Mesolens - a specialist objective lens with the unusual combination of low magnification and high NA. This new imaging mode - MesoTIRF - enables TIRF imaging across a 4.4 mm x 3.0 mm FOV. We demonstrate evanescent wave illumination of cell specimens, and show the multi-wavelength capability of the modality across more than 700 cells in a single image. MesoTIRF images have up to a 6-fold improvement in signal-to-background ratio compared to widefield epi-fluorescence illumination, and we illustrate the benefit of this improved contrast for the detection and quantification of focal adhesions in fixed cells. Fluorescence intensities and resolvable structural detail do not vary considerably in homogeneity across the MesoTIRF FOV.

biophysics↗