Biology subjects

Lees, R. M.

Publications and source records attributed to Lees, R. M..

6 recordsLinked to original sources

bioRxiv2025

Preserving Tissue Integrity Under the Beam: High-Energy, Low-Dose Synchrotron CT for in situ Imaging of Bovine Intervertebral Discs

In situ tomography enables non-destructive, time-lapse imaging of biological tissues under load, offering insights into structural and mechanical changes. However, repeated scans can expose samples to high radiation doses, potentially altering tissue properties. This study evaluated the feasibility of low-dose synchrotron computed tomography (sCT) for high-resolution, in situ imaging of intact bovine intervertebral discs (IVDs), and assessed the effects of repeated X-ray exposure on mechanical, microstructural, and molecular integrity. Intact oxtail IVD segments were imaged using propagation-based phase contrast sCT at 54 keV. Scan parameters were optimised to achieve high image quality within 66 seconds per scan, resulting in a total dose of ~30 kGy over six scans. Mechanical properties were assessed under cyclic loading, microstructural changes via digital volume correlation (DVC), and molecular alterations using Raman spectroscopy. High-resolution imaging of soft and calcified tissues was achieved. Changes in sample stiffness, hysteresis, or stress recovery between irradiated and control were not identified. DVC revealed no microstructural damage or strain accumulation in the calcified endplate. Raman spectroscopy indicated minimal changes in soft tissues, with bone showing slight increased collagen crosslinking and reduced mineralisation. Overall, this study demonstrates that high-energy, low-dose sCT enables repeated imaging of musculoskeletal tissues without compromising integrity, supporting its application in dynamic, time-lapse imaging studies. Importantly, larger, intact samples--such as whole bovine IVDs-- were imaged overcoming limitations of previous studies that relied on small animal models. This approach supports more physiologically relevant investigations of tissue mechanics and degeneration in complex systems.

bioengineering↗

bioRxiv2025

Desensitization of opsin responses during all-optical interrogation depends on imaging parameters

SignificanceThe combination of two-photon calcium imaging and two-photon optogenetic stimulation, termed all-optical interrogation, provides spatial and temporal precision when recording and manipulating neural circuit activity in vivo. All-optical experiments often use red-shifted opsins in combination with green fluorescent reporters of neuronal activity. However, their excitation spectra still partially overlap, meaning that the imaging laser can excite the opsin. Though some care has been taken in the past to understand the effects of this spectral overlap, further work is required to understand its impact on the findings of all-optical studies. AimWe aimed to investigate whether two-photon imaging of the green fluorescent calcium reporter GCaMP6s at 920 nm increases the rate of desensitization in neurons expressing the red-shifted opsin C1V1. ApproachWe systematically varied either the inter-stimulus interval or the duration of two-photon calcium imaging during two-photon optogenetic stimulation of mouse layer 2/3 barrel cortex or visual cortex neurons. ResultsWe found that two-photon imaging at 920 nm increases the desensitization of photostimulation responses across trials in C1V1-expressing neurons - an effect that is exacerbated at shorter inter-stimulus intervals. Reduced photostimulation responses are not limited to targeted cells, and are found across the entire field of view. Such network effects are less pronounced at shorter imaging doses. ConclusionsOur results provide methodological optimizations that enable opsin desensitization to be mitigated in all-optical experiments. This will reduce an external source of trial-by-trial variability in future all-optical experiments.

neuroscience↗

bioRxiv2024

Standardised Measurements for Monitoring and Comparing Multiphoton Microscope Systems

The goal of this protocol is to enable better characterisation of multiphoton microscopy hardware across a large user base. The scope of this protocol is purposefully limited to focus on hardware, touching on software and data analysis routines only where relevant. The intended audiences are scientists using and building multiphoton microscopes in their laboratories. The goal is that any scientist, not only those with optical expertise, can test whether their multiphoton microscope is performing well and producing consistent data over the lifetime of their system.

neuroscience↗

bioRxiv2023

Optical resolution is not the limiting factor for spatial precision of two-photon optogenetic photostimulation

Two-photon optogenetics combines nonlinear excitation with noninvasive activation of neurons to enable manipulation of neural circuits with a high degree of spatial precision. Combined with two-photon population calcium imaging, these approaches comprise a flexible platform for all-optical interrogation of neural circuits. However, a multitude of optical and biological factors dictate the exact precision of this approach in vivo, where it is most usefully applied. Here, we carefully assessed how the optical factors determine the spatial precision of activation. We found that optical resolution is not the limiting factor of the spatial precision of two-photon optogenetic photostimulation, and by doing so, reveal the key factors to improve to achieve maximal precision. Our results enable future work to focus on the optimal factors by providing key insight from controlled experiments in a manner not previously reported. This research can be applied to advance the state-of-the-art of all-optical interrogation, extending the toolkit for neuroscience research to achieve spatiotemporal precision at the crucial levels at which neural circuits operate.

neuroscience↗

bioRxiv2021

Perception and propagation of activity through the cortical hierarchy is determined by neural variability

The brains of higher organisms are composed of anatomically and functionally distinct regions performing specialised tasks; but regions do not operate in isolation. Orchestration of complex behaviours requires communication between brain regions, but how neural activity dynamics are organised to facilitate reliable transmission is not well understood. We studied this process directly by generating neural activity that propagates between brain regions and drives behaviour, allowing us to assess how populations of neurons in sensory cortex cooperate to transmit information. We achieved this by imaging two hierarchically organised and densely interconnected regions, the primary and secondary somatosensory cortex (S1 and S2) in mice while performing two-photon photostimulation of S1 neurons and assigning behavioural salience to the photostimulation. We found that the probability of perception is determined not only by the strength of the photostimulation signal, but also by the variability of S1 neural activity. Therefore, maximising the signal-to-noise ratio of the stimulus representation in cortex relative to the noise or variability in cortex is critical to facilitate activity propagation and perception. Further, we show that propagated, behaviourally salient activity elicits balanced, persistent, and generalised activation of the downstream region. Hence, our work adds to existing understanding of cortical function by identifying how population activity is formatted to ensure robust transmission of information, allowing specialised brain regions to communicate and coordinate behaviour.

neuroscience↗

bioRxiv2019

Presynaptic boutons that contain mitochondria are more stable

Addition and removal of presynaptic terminals reconfigures neuronal circuits of the mammalian neocortex, but little is known about how this presynaptic structural plasticity is controlled. Since mitochondria can regulate presynaptic function, we investigated whether the presence of axonal mitochondria relates to structural plasticity of presynaptic boutons in mouse neocortex. We found that the overall density of axonal mitochondria did not appear to influence loss and gain of boutons. However, positioning of mitochondria at individual presynaptic sites did relate to increased stability of those boutons. In line with this, synaptic localisation of mitochondria increased as boutons aged and showed differing patterns of localisation at en passant and terminaux boutons. These results suggest that mitochondria accumulate locally at boutons over time to increase bouton stability.

neuroscience↗