Biology subjects

McConnell, G.

Publications and source records attributed to McConnell, G..

At least 19 recordsLinked to original sources

bioRxiv2026

A method for correcting background non-uniformity while preserving specimen-level information in Fourier ptychographic microscopy images

Fourier ptychographic microscopy (FPM) enables high resolution imaging across large fields of view, but reconstructed images often exhibit background non-uniformity that interferes with quantitative image analysis. Here, a post-reconstruction background correction approach is presented that identifies specimen-containing regions and corrects only the surrounding background, leaving specimen pixel intensities unchanged. The method was effective across amplitude and phase reconstructions of multiple biological specimen types with no loss in image resolution. The method also improved object detection compared with analysis of the uncorrected image data. FPM background correction reduced residual background non-uniformity to 0.61% with only 0.04% structural error, compared with 6.13% structural error for pseudo flat field correction. These results demonstrate that FPM background correction can support more accurate quantitative analysis of FPM image data while preserving specimen information and image resolution, enabling more reliable segmentation and measurement across large fields of view.

biophysics↗

bioRxiv2026

Cross-scale Imaging of Live Botanical Specimens using Fourier Ptychographic Microscopy

Light microscopic imaging of botanical specimens is typically performed using diffraction-limited methods which can image either a small field of view (FOV) with a shallow depth of field (DOF) at high resolution, or a large FOV and large DOF at low resolution. As such, many of the important small features, such as cells in the root cap of Arabidopsis, chloroplasts in algae and leaf guard cells, cannot be resolved in high numbers for meaningful statistical interpretation of results. Fourier ptychographic microscopy (FPM) is an accessible low-cost solution for large format bioimaging which makes use of multiangle illumination and iterative phase retrieval to recover high-resolution estimates of sample phase and amplitude. To date much FPM research has focused on optimising hardware and image reconstruction techniques with biological and biomedical applications limited primarily to mammalian cell cultures and histological tissue sections. We have, for the first time, applied FPM for label-free imaging of these large, living botanical specimens to investigate cellular and sub-cellular structures over multiple length scales simultaneously, with the aim of resolving structures that cannot be seen with diffraction-limited methods over a large field of view. With our FPM system we demonstrate imaging of living samples mounted in water with a resolution of 615 nm over a 3.6 mm diagonal FOV, producing a 9-fold improvement in resolution compared to conventional brightfield imaging. This allows for individual root cap cells of Arabidopsis thaliana to be resolved within the context or larger root structures, individual chloroplasts identified in large section of Spirogyra varians, and high-resolution colour imaging of Tradescantia zebrina stomata and chloroplasts across large leaf sections.

plant biology↗

bioRxiv2026

Multimodal imaging reveals surface degradation of voice prosthesis implants resulting from colonisation by polymicrobial biofilms

Total laryngectomy is performed for advanced laryngeal and hypopharyngeal cancers. Following the surgery, the insertion of indwelling silicone-based voice prosthesis (VP) implants is considered the gold standard for speech rehabilitation. However, early VP failure due to biofilm formation and invasion of the VP surface can lead to routine use of nystatin and the need for frequent valve changes. Despite these burdens, the composition and impact of colonising pathogens on VP integrity are poorly understood. We have developed a rapid pipeline which permitted the transfer, fixation, staining, and imaging of explanted VPs from patients within 90 minutes of sample collection. Using this pipeline, we demonstrate a multimodal reflection contrast and fluorescence confocal laser scanning microscopy technique to simultaneously map the VP surface and visualise the biofilms in situ, providing new insights into the role of pathogens on VP failure. Our results reveal that complex fungal and bacterial biofilms form polymicrobial biofilms on the VP surface. We demonstrate that biofilms not only reside on the VP surface but can invade the device, releasing silicone particles that are visible within the volume of the biofilms and are separate from the VP surface. Our findings highlight the complexity of the biofilms that form on speech valves; complex polymicrobial biofilms require more advanced treatment than what is conventionally used. Work should focus both on the patient factors that may influence this and the material properties of the valve.

microbiology↗

bioRxiv2026

Human buccal epithelial microplicae maintain a characteristic wavelength despite variable network topology

Microplicae are ridge-like membrane projections that are prominent features of many epithelial surfaces, yet little is known about the principles governing their spatial organisation. Determining whether microplicae represent stochastic membrane folds or biologically organised surface architectures is essential for understanding their formation and functional roles. Here, differential interference contrast images of human buccal epithelial cells were analysed using quantitative image-processing approaches. Ridge networks were segmented and characterised using complementary measurements of characteristic wavelength, including medial-axis and nearest-neighbour Voronoi analyses, together with skeleton-based metrics describing network architecture. Analysis of n=100 buccal epithelial cells sampled from n=10 donors revealed a reproducible sub-micron characteristic wavelength. Mean medial-axis spacing was 0.511 {+/-} 0.042 {micro}m and mean Voronoi nearest-neighbour spacing was 0.588 {+/-} 0.057 {micro}m. Characteristic wavelength exhibited CV of between only 8.26% and 9.67% across the dataset. However, metrics describing network architecture, including ridge density, branching and connectivity, varied by up to 109%. Donor-level analysis reported the same overall trends, with conservation of the characteristic wavelength while network parameters had considerably greater variation. These findings identify a previously unrecognised organising principle of microplical architecture, suggesting that epithelial membrane organisation is regulated through conservation of an intrinsic geometric length scale while network topology remains comparatively free to remodel.

cell biology↗

bioRxiv2026

CASC: Content-Aware Compaction of Sparse Microscopy Images

Microscopy datasets are often spatially sparse, wherein relevant structures occupy only a small fraction of the total field of view (FOV), leaving large regions of background devoid of signal. This inherent inefficiency creates file sizes that are larger than needed, which increases the time needed for computational image data analysis and processing, and means unnecessarily large data volumes. In this work, a classical open-source method for content-aware spatial compaction of microscopy images (CASC) is reported that explicitly removes spatial redundancy by reorganising foreground objects into a new, smaller image. CASC combines adaptive intensity normalisation, statistical thresholding, morphological refinement, and connected-component analysis to isolate foreground structures. These structures are then extracted with contextual padding and repacked into a compact domain using a heuristic shelf-based spatial packing strategy. CASC intentionally destroys the spatial topology of the image but preserves pixel intensities exactly, retaining object-level information. The method achieves demonstrable reductions in image area and background content while maintaining high object-level preservation of biological structures, with a reduction in file size of more than 390-fold shown in real image datasets.

Cell Biology↗

bioRxiv2026

Open Fourier Ptychographic Microscopy (OpenFPM)

Fourier ptychographic microscopy (FPM) uses sequential multi-angle illumination and iterative phase retrieval to recover a high-resolution complex image from a series of low-resolution brightfield and darkfield images. We present OpenFPM, an open-source FPM platform in which conventional and optomechanical hardware is replaced with compact, low-cost 3D printed components. Illumination, sample and objective positioning, and camera triggering are controlled using a Python-based interface on a Raspberry Pi microcomputer. With a 10 x /0.25 NA objective lens and 636 nm illumination, OpenFPM experimentally achieves amplitude and phase reconstructions with an effective synthetic NA of 0.90 over a 1 mm field-of-view. This platform gives researchers accessible and affordable hardware for developing and testing LED-array microscopy techniques for a range of biomedical imaging applications.

biophysics↗

bioRxiv2026

Analysis of Biofilm Complexity in 3D (ABC3D): An open-source framework for quantitative fractal, textural, and statistical analysis of colony biofilm morphology in three dimensions

2.Quantitative image analysis is central to understanding microbial growth, morphology, and spatial organisation. However, conventional metrics such as mean intensity or object count often do not capture the complex structural heterogeneity and patterning characteristic of microbial colonies and biofilms. To address this limitation, Analysis of Biofilm Complexity in 3D (ABC3D), an open-source Python framework for automated extraction of fractal, textural, and statistical descriptors from volumetric microscopy images, is reported. ABC3D computes a set of parameters including fractal dimension, lacunarity, entropy, grey-level co-occurrence matrix features, and wavelet sub-band energies from three-dimensional (3D) image datasets. ABC3D is demonstrated in macrocolony biofilms formed by cell shape mutants of Escherichia coli, where it is shown that nutrient availability accounts for the majority of structural variance, while cell shape produces additional structural variation that differs between nutrient conditions. ABC3D provides researchers with an accessible, quantitative approach to assessing biofilm morphology in microscopy datasets. SummaryAn open-source, quantitative analysis pipeline is presented that integrates fractal, lacunarity, entropy, texture and wavelet descriptors to characterise colony biofilm architecture in three dimensions. Application to Escherichia coli cell shape mutants demonstrates that macrocolony biofilm architecture is best understood as a coordinated, multiscale phenotype rather than as an aggregate of independent structural metrics. 3. Impact statementBiofilm architecture is pivotal for microbial survival, antimicrobial tolerance, and ecological function but tools to quantify structural organisation in these cell communities remain limited. The commonest metrics describe bulk properties such as width, thickness, or cell number, but they do not capture multiscale spatial heterogeneity. Here, an open-source framework for Analysis of Biofilm Complexity in 3 Dimensions (ABC3D) is reported. This software integrates measurements of fractal geometry, lacunarity, entropy, texture statistics, and wavelet energy. ABC3D is demonstrated in Escherichia coli macrocolony biofilms, where it is shown that nutrient environment has a leading role in determining colony architecture in E. coli biofilms, while cell shape has a lesser but still significant influence on structural variation. The ABC3D pipeline can be applied to any microbial communities imaged by confocal microscopy and other volumetric imaging methods and has the potential to give a deeper understanding of how cells organise in biofilms. 4. Data summaryFull code for ABC3D and data analysis is available at https://github.com/gailmcconnell/ABC3D. Image data are available upon request. The author confirms all supporting data, code and protocols have been provided within the article or through supplementary data files.

microbiology↗

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

Low-Cost 3D Printed Optics for Super-Resolution Multifocal Structured Illumination Microscopy

We present a low-cost 3D printing method of fabricating optical quality lenslet arrays for integration in a multifocal structured illumination microscope (mSIM), achieving fluorescence imaging below the optical diffraction limit. We detail the design and manufacturing processes to produce high-quality 3D printed optics, showing their comparable surface roughness of 30 {+/-} 2.5 nm for the 3D printed elements compared to 37 {+/-} 1.4 nm for commercial glass optics. A 3D printed lenslet array with a honeycomb geometry and 1.2 mm lenslet diameter was compared to a high-end glass commercial lenslet array with 250 {micro}m lenslet diameter and a lower cost commercial lenslet array with a 1.2 mm by 1.6 mm lenslet footprint. The imaging performance of the different optics was benchmarked using a custom mSIM setup by quantifying the beam profile homogeneity and the experimental lateral resolution. The mSIM setup incorporating the different microlens arrays was tested using a commercial bovine pulmonary artery endothelial cell specimen, highlighting an achievable resolution enhancement from 229 nm {+/-} 11 nm with widefield illumination to 137 {+/-} 11 nm using the high-end commercial microlens array and 134 nm {+/-} 9 nm using the 3D printed honeycomb lenslet array. Advantages of improved background rejection through the custom lenslet geometry are discussed, highlighting the super-resolution microscope performance achievable through custom low-cost 3D printed optics.

biophysics↗

bioRxiv2025

Extracting biological structure and heterogeneity from the nano to the macro scale

Fluorescence microscopy is an essential tool in biology. It has revealed great variability at multiple scales, in macromolecular complexes, cells, and organisms. Understanding this variability will reveal the mechanisms by which genetically or biochemically identical systems adopt different biological states. Achieving this requires the ability to extract both the underlying biological structure and how it varies across the population. Currently the field lacks general techniques to deal with arbitrary structures and different types of variability. Here we present SQUASSH, a new convolutional neural network-based approach to freely fit structural models to fluorescence microscopy data that simultaneously quantifies variability to reveal correlations, dynamics, and systematic distortions. SQUASSH is highly versatile: it accommodates diverse imaging modalities at length scales from nm to mm. This approach opens up applications such as imaging nanoscale macromolecular structures, revealing patterns in shape changes from organelle to tissue scale, and characterizing systems biology of dynamical processes.

cell biology↗

bioRxiv2025

Assessing the role of collagen to improve understanding of disease pathophysiology in patients with acute recurrent tonsillitis

Acute recurrent tonsillitis (ART) and obstructive sleep apnoea (OSA) are the most common indications for tonsillectomy worldwide however the disease pathophysiology is poorly understood. Acute recurrent tonsillitis causes repeated episodes of inflammation within the tonsils. Repeated episodes of inflammation can lead to scarring of tissue due to an overproduction of extracellular matrix proteins such as collagen, which in return can cause decreased immunological function. In this study we assess collagen prevalence within ART and OSA tonsils with the aim of better understanding the underlying disease pathophysiology. By performing label-free second harmonic generation (SHG) imaging of tonsil tissue from patients with ART and OSA it is possible to enable measurement of the relative abundance of immature and mature type I collagen within ex vivo paediatric palatine tonsils. We have demonstrated that there is a significant difference in the mean area fraction of immature type I collagen with a higher relative fraction in patients with ART compared to those with OSA (p = 0.0059). However, there was no statistically significant difference in the mean area fraction of mature type I collagen in patients with ART and OSA (p = 0.61). Spatial analysis demonstrated that there was a significantly fraction in tonsillar tissue from patients with ART of immature type I collagen in the epithelia dominant regions (p = 0.0074) and the interior regions (p = 0.0023). Our results provide new information regarding these two common paediatric diseases, giving us an improved insight into the disease pathophysiology.

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↗

bioRxiv2025

A simple silicone elastomer colonisation model highlights complexities of Candida albicans and Staphylococcus aureus interactions in biofilm formation.

Healthcare-associated infections (HAIs) significantly contribute to the burden of antimicrobial resistance (AMR). A major factor in HAIs is the colonisation of indwelling medical devices by biofilm-forming opportunistic pathogens such as Candida albicans and Staphylococcus aureus. These organisms frequently co-infect, resulting in synergistic interactions with enhanced virulence and resistance to treatment. C. albicans and S. aureus readily form dual-species biofilms on silicone elastomers, a commonly used medical device material, yet the colonisation phenotypes of these organisms on such surfaces remains poorly understood. We developed a simple, optically tractable model to mimic the colonisation of indwelling medical devices to investigate C. albicans and S. aureus biofilm formation. The system utilises discs of a silicone elastomer embedded in agar, reflecting device-associated conditions and enabling high-resolution imaging of biofilms formed by C. albicans and S. aureus co-culture. Initial results using the silicone elastomer colonisation model reveal robust biofilm formation. These biofilms exhibited morphological differences between dual species biofilms formed by S. aureus co-cultures with either yeast- or hyphal-form C. albicans, indicating the impact of differing C. albicans cell morphotypes in biofilm-associated medical device colonization on silicone elastomers. Quantification of biofilm formation by crystal violet staining provided further validation of the system. These findings underscore the importance of developing tools for biofilm study which more closely resemble the infectious microenvironment, with our work detailing such a system which can be employed in further study to improve strategies against device-related HAIs.

microbiology↗

bioRxiv2024

A fully 3D-printed optical microscope for low-cost histological imaging

We present the manufacture and characterisation of a fully 3D printed, low-cost optical microscope using both a 3D printed chassis and 3D printed illumination and imaging optics. The required commercial components, consisting of a basic camera for image acquisition and light emitting diode controlled by a Raspberry Pi for illumination, are integrated into the 3D printed microscope with the full design shown for ease of replication. Our 3D printed microscope uses a single 3D printed objective lens with a 2.9x magnification and a numerical aperture of 0.07. To benchmark the imaging performance of the system, we used standard test targets and histological specimens, namely, a Giemsa-stained blood smear sample and a thin section of mouse kidney stained with Haemotoxylin and Eosin. We demonstrated that sub-cellular resolution was obtained, and we corroborated this by imaging individual red blood cells and intricate anatomical details of the stained mouse kidney section. All of this was achieved using entirely 3D printed hardware and optics, at a fraction of the cost of a commercial brightfield microscope, while presenting remarkable potential for customisation and increased accessibility for diagnostic imaging applications.

biophysics↗

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↗

bioRxiv2024

Oxygen Microenvironments in E. coli Biofilm Nutrient Transport Channels: Insights from Complementary Sensing Approaches

Chemical gradients and the emergence of distinct microenvironments in biofilms are vital to the stratification, maturation and overall function of microbial communities. These gradients have been well characterised throughout the biofilm mass but the microenvironment of recently discovered nutrient transporting channels in Escherichia coli biofilms remains unexplored. This study employs three different oxygen sensing approaches to provide a robust quantitative overview of the oxygen gradients and microenvironments throughout the biofilm transport channel networks formed by E. coli macrocolony biofilms. Oxygen nanosensing combined with confocal laser scanning microscopy established that the oxygen concentration changes along the length of biofilm transport channels. Electrochemical sensing provided precise quantification of the oxygen profile in the transport channels, showing similar anoxic profiles compared with the adjacent cells. Anoxic biosensing corroborated these approaches, providing an overview of the oxygen utilisation throughout the biomass. The discovery that transport channels maintain oxygen gradients contradicts the previous literature that channels are completely open to the environment along the apical surface of the biofilm. We provide a potential mechanism for the sustenance of channel microenvironments via orthogonal visualisations of biofilm thin sections showing thin layers of actively growing cells. This complete overview of the oxygen environment in biofilm transport channels primes future studies aiming to exploit these emergent structures for new bioremediation approaches.

microbiology↗