Biology subjects

Cadby, A. J.

Publications and source records attributed to Cadby, A. J..

7 recordsLinked to original sources

bioRxiv2026

Object Detection Techniques for Live Monitoring of Amoeba in Phase-Contrast Microscopic Images

In contemporary bio-imaging-based research, computer-based assessment is becoming crucial for the characterisation of biological structures, as it minimises the need for time-consuming human annotation, which is prone to human error. Furthermore, it allows for the use of optical techniques that use lower photon intensities, thereby reducing reliance on high-intensity excitation and mitigating adverse effects on their activities. This study details the development and evaluation of sophisticated deep-learning models for amoeba detection using phase-contrast imaging. Using a single-class annotated dataset comprising 88 images and 4,131 annotations, we developed nine object detection models based on Detectron 2 and six variants based on YOLO v10. The diversity of the dataset, acquired under varying setup parameters, facilitated a comprehensive evaluation of the strengths and limitations of each model. A comparative analysis of speed and accuracy was performed to identify the most efficient models for real-time detection, providing critical insights for future microscopic analyses.

biophysics↗

bioRxiv2024

Pulses of Class I PI3kinase activity identify the release and recapture of prey from neutrophil phagosomes

Class I PI3kinases coordinate the delivery of microbicidal effectors to the phagosome by forming the phosphoinositide lipid second messenger, phosphatidylinositol (3, 4, 5)-trisphosphate (PIP3). However, the dynamics of PIP3 in neutrophils during a bacterial infection are unknown. We have therefore developed an in vivo, live zebrafish infection model that enables visualisation of dynamic changes in Class 1 PI3kinases (PI3K) signalling on neutrophil phagosomes in real-time. We have identified that on approximately 12% of neutrophil phagosomes PHAkt-eGFP, a reporter for Class 1 PI3K signalling, re-recruits in pulsatile bursts. This phenomenon occurred on phagosomes containing structurally and morphologically distinct prey, including Staphylococcus aureus and Mycobacterium abscessus, and was dependent on the activity of the Class 1 PI3K isoform, PI3kinase {gamma}. Detailed imaging suggested that pulsing phagosomes represent neutrophils transiently reopening and reclosing phagosomes. This finding challenges the concept that phagosomes remain closed after prey engulfment and we propose that neutrophils occasionally use this alternative pathway of phagosome maturation to release phagosome contents and/or to restart phagosome maturation if digestion has stalled.

cell biology↗

bioRxiv2023

Geometry-preserving Expansion Microscopy microplates enable high fidelity nanoscale distortion mapping

Expansion microscopy (ExM) is a versatile super-resolution microscopy pipeline, leveraging nanoscale biomolecular cross- linking and osmotically driven swelling of hydrogels. In its current implementation, ExM remains a laborious and skill-intensive technique, involving manual handling of the hydrogels that can compromise the integrity of the gel matrix and diminish reproducibility. The lack of protocols to constrain the gel orientation during this process lends to challenges in tracking gel isotropy during or after the swelling. We have developed a bespoke microplate system capable of carrying out the entire ExM workflow within each well. The microplates enable in situ image acquisition and eliminate the need for direct physical handling of the hydrogels. The preservation of the gel geometry and orientation by the design of the microplate wells also enables convenient tracking of gel expansion, pre- and post-ExM image acquisition, and distortion mapping of every cell or region of interest. We demonstrate the utility of this approach with both single-colour and multiplexed ExM of HeLa cells cultured within the microplate wells to reveal nuclear and sub-plasmalemmal regions as distortion-prone structures.

biophysics↗

bioRxiv2022

An Open-source, Cost-efficient Excitation Module for Single-molecule Microscopy

In fluorescence microscopy, scientific-grade laser diodes are key components and contribute a considerable expense to the total cost of the microscope setup. Existing open-source laser excitation modules are mainly designed for super-resolution imaging and have not been fully characterised to meet the requirements of single-molecule spectroscopy measurements. In this paper we introduce an open-source, cost-efficient excitation module that can be used for confocal, accurate single-molecule FRET, fluorescence correlation spectroscopy (FCS) and low-power super-resolution techniques. The module delivers two diode lasers (520 nm and 638 nm) via a single-mode fibre, with stable power output (<1% deviation), which can be modulated on the microsecond timescale. Here, we benchmark these lasers against smFRET standards, and recover consistent diffusion coefficients from FCS measurements, thereby demonstrating their suitability for a range of single-molecule spectroscopic experiments, whilst reducing the overall costs of our open-source smFRET instrument (the smfBox) by ~50%, making these types of experiments even more accessible to the widest possible userbase.

biophysics↗

bioRxiv2020

An open-source experimental framework for automation of cell biology experiments

Modern data analysis methods, such as optimisation algorithms or machine and deep learning, have been successfully applied to a number of biological, biotechnological and medical questions. For these methods to be efficient, a large number of high quality experiments need to be conducted, which requires a high degree of automation. Here we report an open-source hardware that allows for automatic high-throughput generation of large amounts of cell biology data. The hardware consists of an automatic XY-stage for moving a multiwell plate containing growing cells; a perfusion manifold allowing application of up to 8 different solutions; and a small epifluorescent microscope. It is extremely cheap (approximately {pound}400 without and {pound}2500 with a fluorescent microscope) and is easily customizable for individual experimental needs. We demonstrate the usability of this platform with high-throughput Ca2+ imaging and large-scale labelling experiments. Key points- We present an open source framework for automation of cell biology experiments - The framework consists of an XY platform, application of up to 8 solutions and a small epifluorescent microscope with autofocusing - Very cheap ({pound}400 without a fluorescent microscope and {pound}2500 with a fluorescent microscope), customisable, - Can be used in a variety of biological applications such as imaging of fluorescent reporters, optimisation of treatment conditions and fluorescent labelling

cell biology↗

bioRxiv2018

Sperm gatekeeping: 3D imaging reveals a constricted entrance to zebra finch sperm storage tubules

Females across many internally fertilising taxa store sperm, often in specialised storage organs in their reproductive tracts. In birds, several hundred sperm storage tubules exist in the utero-vaginal junction of the oviduct and there is growing evidence that sperm storage in these tubules is selective. The mechanisms underlying female sperm storage in birds remain unknown due to our limited ability to make three dimensional, live observations inside the large, muscular avian oviduct. Here, we describe a new application of fluorescence selective plane illumination microscopy to optically section oviduct tissue from zebra finch Taeniopygia guttata females label-free, by harnessing tissue autofluorescence. Our data provide the first description of the 3D structure of sperm storage organs in any vertebrate and reveal the presence of gate-like constricted openings that may play a role in sperm selection.\n\nStatement of SignificanceFemale birds can store sperm in microscopic tubular structures in their reproductive tract for up to several months, depending on species. Studying these sperm storage tubules has been a major challenge due to the muscular and opaque nature of reproductive tracts in birds. We have developed a new method for imaging live reproductive tract tissue using selective plane illumination microscopy, a fluorescence microscopy technique. From these images, we could extract three-dimensional measurements of sperm storage tubules and found these structures to have a gate-like constriction, providing evidence that females can actively select sperm at storage and ultimately influence the paternity of her offspring. Understanding these reproductive adaptations can help improve captive breeding programs and similar conservation strategies.

evolutionary biology↗

bioRxiv2018

A Multimodal Adaptive Super-Resolution and Confocal Microscope

Existing optical microscopy techniques compromise between resolution, photodamage, speed of acquisition and imaging in to deep samples. This often confines a technique to a certain biological system or process. We present a versatile imaging system which can switch between imaging modalities with sub millisecond transition times to adapt to the needs of a wide range of sample types. The imaging modalities provide the minimally invasive but low-resolution epi-fluorescence though increasing invasive but higher resolution confocal and structured illumination until the highest resolution is achieved through the most intrusive, localisation microscopy. The ability of the system to overcome the limitations of conventional single mode microscopy is demonstrated by several biological investigations. The ideas presented in this work allow researchers to move away from the model of a single imaging modality to study a specific process and instead follow those processes using the most suitable method available during the lifetime of the investigation.

biophysics↗