Biology subjects

Rooney, L. M.

Publications and source records attributed to Rooney, L. M..

At least 19 recordsLinked to original sources

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

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

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

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↗

bioRxiv2023

Fractal complexity of Escherichia coli nutrient transport channels is influenced by cell shape and growth environment

Recent mesoscopic characterisation of nutrient-transporting channels in E. coli has allowed the identification and measurement of individual channels in whole mature biofilms. However, their complexity under different physiological and environmental conditions remains unknown. Analysis of confocal micrographs of biofilms formed by cell shape mutants of E. coli shows that channels have a high fractal complexity, regardless of cell phenotype or growth medium. In particular, biofilms formed by the mutant strain {Delta}ompR, which has a wide-cell phenotype, have a higher fractal dimension when grown on rich medium than when grown on minimal medium, with channel complexity affected by glucose and agar concentration in the medium. Osmotic stress leads to a dramatic reduction in {Delta}ompR cell size, but has a limited effect on channel morphology. This work shows that fractal image analysis is a powerful tool to quantify the effect of phenotypic mutations and growth environment on the morphological complexity of internal E. coli biofilm structures. If applied to a wider range of mutant strains, this approach could help elucidate the genetic determinants of channel formation in E. coli biofilms.

microbiology↗

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

Low-cost 3D printed lenses for brightfield and fluorescence microscopy

We present the fabrication and implementation of low-cost optical quality 3D printed lenses, and their application as microscope objectives with different prescriptions. The imaging performance of the 3D printed lenses was benchmarked against commercially available optics including a 20 mm focal length 12.7 mm diameter NBK-7 plano-convex lens used as a low magnification objective, and a separate high magnification objective featuring three 6 mm diameter NBK-7 lenses with different positive and negative focal lengths. We describe the design and manufacturing processes to produce high-quality 3D printed lenses. We tested their surface quality using a stylus profilometer, showing that they conform to that of commercial glass counterpart lenses. The 3D printed lenses were used as microscope objectives in both brightfield and epi-fluorescence imaging of specimens including onion, cyanobacteria, and variegated Hosta leaves, demonstrating a sub-cellular resolution performance obtained with low-cost 3D printed optical elements within brightfield and fluorescence microscopy.

biophysics↗

bioRxiv2023

Construction and Characterisation of a Structured, Tuneable, and Transparent 3D Culture Platform for Soil Bacteria

2.We have developed a tuneable workflow for the study of soil microbes in an imitative 3D soil environment that is compatible with routine and advanced optical imaging, is chemically customisable, and is reliably refractive index matched based on the metabolic profile of the study organism. We demonstrate our transparent soil pipeline with two representative soil organisms, Bacillus subtilis and Streptomyces coelicolor, and visualise their colonisation behaviours using fluorescence microscopy and mesoscopy. This spatially structured, 3D approach to microbial culture has the potential to further study the behaviour of other difficult-to-culture bacteria in conditions matching their native environment and could be expanded to study microbial interactions, such as interaction, competition, and warfare. 3. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/546105v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1123425org.highwire.dtl.DTLVardef@1d6cc3org.highwire.dtl.DTLVardef@1cf950aorg.highwire.dtl.DTLVardef@141dc0b_HPS_FORMAT_FIGEXP M_FIG C_FIG A step-by-step method for creating a tailored 3D culture medium for study of soil microbes. The complete workflow can be split into three parts: Growth and observation, metabolic profiling to provide a stable refractive index matching solution, and production of the 3D soil environment. The 3D culture scaffold was created by cryomilling Nafion resin pellets and size filtration. Chemical processing altered the surface chemistry of Nafion particles and facilitated nutrient binding by titration of a defined liquid culture medium. Metabolic profiling determined non-metabolisable sugars and provided an inert refractive index matching substrate, which was added to the final nutrient titration. Inoculation and growth of the test strain allowed for downstream assessment of colonisation behaviours and community dynamics in situ by, for example, optical microscopy.

microbiology↗

bioRxiv2023

A simple computational method to increase axial contrast in multi-wavelength interference microscopy

Multi-wavelength standing wave (SW) microscopy and interference reflection microscopy (IRM) are powerful techniques that use optical interference to study topographical structure. However, the use of more than two wavelengths to image the complex cell surface results in complicated topographical maps and it can be difficult to resolve the three-dimensional contours. We present a simple image processing method to reduce the thickness and spacing of antinodal fringes in multi-wavelength interference microscopy by up to a factor of two to produce clearer and more precise topographical maps of cellular structures. We first demonstrate this improvement using model non-biological specimens, and we subsequently demonstrate the benefit of our method for reducing the ambiguity of surface topography and revealing obscured features in live and fixed cell specimens.

biophysics↗

bioRxiv2021

Intra-colony channel morphology in Escherichia coli biofilms is governed by nutrient availability and substrate stiffness

Nutrient-transporting channels have been recently discovered in mature Escherichia coli biofilms, however the relationship between intra-colony channel structure and the surrounding environmental conditions is poorly understood. Using a combination of fluorescence mesoscopy and a purpose-designed open-source quantitative image analysis pipeline, we show that growth substrate composition and nutrient availability have a profound effect on the morphology of intra-colony channels in mature E. coli biofilms. Under all nutrient conditions, intra-colony channel width was observed to increase non-linearly with radial distance from the centre of the biofilm. Notably, the channels were around 25% wider at the centre of glucose-limited biofilms compared to ammonium-limited biofilms. Channel density also differed in colonies grown on rich and minimal media, with the former creating a network of tightly packed channels and the latter leading to well-separated, wider channels with defined edges. Our approach paves the way for measurement of internal patterns in a wide range of biofilms, offering the potential for new insights into infection and pathogenicity.

microbiology↗

bioRxiv2019

Functional channels in mature E. coli colonies

Biofilms are important in medicine, industry and the natural environment, however their structure is largely unexplored across multiple spatial scales. We have studied the architecture of mature Escherichia coli macro-colony biofilms by means of the Mesolens, an optical system which uniquely allows simultaneous imaging of individual bacteria and over one hundred cubic millimetres of its biofilm milieu. Our chief finding is the presence of intra-colony channels on the order of 10 m in diameter in E. coli biofilms. These channels have a characteristic structure and reform after total mechanical disaggregation of the colony. We present evidence that the channels transport particles and function to assist the absorption of nutrients. These channels potentially offer a new route for the delivery of dispersal agents or antimicrobial drugs to biofilms, ultimately lowering their impact on public health and industry.

microbiology↗