Advanced light-sheet and structured illumination microscopy techniques for neuroscience and disease diagnosis

Optical microscopy is a cornerstone of biomedical research. Advances in optical techniques enable specific, high resolution, sterile, and biologically compatible imaging. In particular, beam shaping has been used to tailor microscopy techniques to enhance microscope performance. The aim of this Thesis is to investigate the use of novel beam shaping techniques in emerging optical microscopy methods, and to apply these methods in biomedicine. To overcome the challenges associated with high resolution imaging of large specimens, the use of Airy beams and related techniques are applied to light-sheet microscopy. This approach increases the field-of-view that can be imaged at high resolution by over an order of magnitude compared to standard Gaussian beam based light-sheet microscopy, has reduced phototoxicity, and can be implemented with a low-cost optical system. Advanced implementations show promise for imaging at depth within turbid tissue, in particular for neuroscience. Super-resolution microscopy techniques enhance the spatial resolution of optical methods. Structured illumination microscopy is investigated as an alternative for electron microscopy in disease diagnosis, capable of visualising pathologically relevant features of kidney disease. Separately, compact optical manipulation methods are developed with the aim of adding functionality to super-resolution techniques

Multimode fibre based imaging for optically cleared samples

UK Engineering and Physical Sciences Research Council (EPSRC) (EP/P030017/1); European Union’s Horizon 2020 Marie Skłodowska-Curie Actions (MSCA) (707084)Optical clearing is emerging as a popular approach particularly for studies in neuroscience. However the use of corrosive clearing solutions typically requires sophisticated objectives or extreme care with optical components chosen for single- or multi-photon imaging. In contrast to the use of complex, custom-made microscope objectives, we show that the use of a corrected multimode fibre (MMF) offers a route that is resistant to corrosion, can be used in clearing media, is not constrained by the refractive index of the immersion medium and offers flexible working distances. Using a corrected MMF, we demonstrate fluorescence imaging of beads and stained neuroblastoma cells through optically cleared mouse brain tissue, as well as imaging in an extreme oxidative environment to show the versatility of our approach. Additionally, we perform Raman imaging of polystyrene beads in clearing media to demonstrate that this approach may be used for vibrational spectroscopy of optically cleared samples.Publisher PDFPeer reviewe

Understanding student perceptions and engagement for formative assessment: A study of interactive online quizzes

Formative activities are integral for student learning and students that engage with these are more likely to achieve success in summative assessments (Rust, O’Donovan and Price, 2005; Jordan, 2011). However, formative activities are often seen as optional and uptake by a cohort can be low. Typically, some module credit is given as an incentive to engage with these activities, but care must be taken in implementing this or the focus can shift from learning to counting marks (Jordan and Butcher, 2010). How should such activities be incentivised? To investigate this, we sought to determine student engagement habits and perceptions of the online quizzes on a 60 credit Level 2 core module of a distance learning undergraduate physics degree course. This module features online quizzes as a key formative activity for providing instantaneous feedback. However, the online quizzes do not contribute directly to the module assessment strategy. Instead, engagement with the online quizzes is encouraged indirectly by an item in formative tutor marked assignments in which students are asked to reflect on their performance in the quizzes. This is a mixed methods study, making use of learning analytics data and a student survey. Learning analytics is used for quantitative analysis of quiz use behaviours over 4 academic years (from 2018/19 – 2022/23). A survey of the 2022/23 student cohort is used for qualitative analysis of students’ perceptions of the online quizzes and their motivations to engage with these activities. We will present the findings of our study, showing how and why student’s make use of formative online quizzes when indirectly incentivised by a reflective item in another assessment. These will be comapred and contrasted with other incentivisation strategies (as reported in, e.g. (Gikandi, Morrow and Davis, 2011; Agnew, Kerr and Watt, 2021)). References: Agnew, S., Kerr, J. and Watt, R. (2021) ‘The effect on student behaviour and achievement of removing incentives to complete online formative assessments’, Australasian Journal of Educational Technology, 37(4), pp. 173–185. Gikandi, J.W., Morrow, D. and Davis, N.E. (2011) ‘Online formative assessment in higher education: A review of the literature’, Computers & Education, 57(4), pp. 2333–2351. Jordan, S. (2011) ‘Using interactive computer‐based assessment to support beginning distance learners of science’, Open Learning: The Journal of Open, Distance and e-Learning, 26(2), pp. 147–164. Jordan, S. and Butcher, P.G. (2010) ‘Using e-assessment to support distance learners of science’, in The GIREP-EPEC & PHEC 2009 International Conference, 17-21 Aug 2009, Leicester, UK. Rust, C., O’Donovan, B. and Price, M. (2005) ‘A social constructivist assessment process model: how the research literature shows us this could be best practice’, Assessment & Evaluation in Higher Education, 30(3), pp. 231–240

Непрерывное профессиональное образование – залог подготовки квалифицированных кадров социальной сферы

В статье рассматривается непрерывное профессиональное образование как способ подготовки квалифицированных кадров социальной сфер

Widefield light sheet microscopy using an Airy beam combined with deep-learning super-resolution

Imaging across length scales and in depth has been an important pursuit of widefield optical imaging. This promises to reveal fine cellular detail within a widefield snapshot of a tissue sample. Current advances often sacrifice resolution through selective sub-sampling to provide a wide field of view in a reasonable time scale. We demonstrate a new avenue for recovering high-resolution images from sub-sampled data in light sheet microscopy using deep-learning super-resolution. We combine this with the use of a widefield Airy beam to achieve high-resolution imaging over extended fields of view and depths. We characterise our method on fluorescent beads as test targets. We then demonstrate improvements in imaging amyloid plaques in a cleared brain from a mouse model of Alzheimer’s disease, and in excised healthy and cancerous colon and breast tissues. This development can be widely applied in all forms of light sheet microscopy to provide a two-fold increase in the dynamic range of the imaged length scale. It has the potential to provide further insight into neuroscience, developmental biology, and histopathology

Light-sheet microscopy with attenuation-compensated propagation-invariant beams

Scattering and absorption limit the penetration of optical fields into tissue, but wavefront correction, often used to compensate for these effects, is incompatible with wide field-of-view imaging and complex to implement. We demonstrate a new approach for increased penetration in light-sheet imaging, namely attenuation-compensation of the light field. This tailors an exponential intensity increase along the illuminating propagation-invariant field, enabling the redistribution of intensity strategically within a sample. This powerful yet straightforward concept, combined with the self-healing of the propagation-invariant field, improves the signal-to-background ratio of Airy light-sheet microscopy up to five-fold and the contrast-to-noise ratio up to eight-fold in thick biological specimens across the field-of-view without any aberration-correction. This improvement is not limited to Airy beam light-sheet microscopy, but can also significantly increase the imaging capabilities of Bessel and lattice light-sheet microscopy techniques, paving the way for widespread uptake by the biomedical community.Comment: 15 pages, 5 figures, supplementary information (35 pages, 22 figures

Wavefront corrected light sheet microscopy in turbid media

This project was supported by the UK Engineering and Physical Sciences Research Council.Light sheet microscopy is a powerful method for three-dimensional imaging of large biological specimens. However, its imaging ability is greatly diminished by sample scattering and aberrations. Optical clearing, Bessel light modes, and background rejection have been employed in attempts to circumvent these deleterious effects. We present an in situ wavefront correction that offers a major advance by creating an “optimal” light sheet within a turbid sample. Crucially, we show that no tissue clearing or specialized sample preparation is required, and clear improvements in image quality and depth resolution are demonstrated both in Gaussian and Bessel beam-based light sheet modalities.Publisher PDFPeer reviewe

Probing neural tissue with airy light-sheet microscopy:Investigation of imaging performance at depth within turbid media

Funding: UK Engineering and Physical Sciences Research Council under grant EP/J01771X/1 (KD), the 'BRAINS' 600th anniversary appeal, and Dr. E. Killick; The Northwood Trust and The RS Macdonald Charitable Trust (JAT); Royal Society Leverhulme Trust Senior Fellowship (KD).Light-sheet microscopy (LSM) has received great interest for fluorescent imaging applications in biomedicine as it facilitates three-dimensional visualisation of large sample volumes with high spatiotemporal resolution whilst minimising irradiation of, and photo-damage to the specimen. Despite these advantages, LSM can only visualize superficial layers of turbid tissues, such as mammalian neural tissue. Propagation-invariant light modes have played a key role in the development of high-resolution LSM techniques as they overcome the natural divergence of a Gaussian beam, enabling uniform and thin light-sheets over large distances. Most notably, Bessel and Airy beam-based light-sheet imaging modalities have been demonstrated. In the single-photon excitation regime and in lightly scattering specimens, Airy-LSM has given competitive performance with advanced Bessel-LSM techniques. Airy and Bessel beams share the property of self-healing, the ability of the beam to regenerate its transverse beam profile after propagation around an obstacle. Bessel-LSM techniques have been shown to increase the penetration-depth of the illumination into turbid specimens but this effect has been understudied in biologically relevant tissues, particularly for Airy beams. It is expected that Airy-LSM will give a similar enhancement over Gaussian-LSM. In this paper, we report on the comparison of Airy-LSM and Gaussian-LSM imaging modalities within cleared and non-cleared mouse brain tissue. In particular, we examine image quality versus tissue depth by quantitative spatial Fourier analysis of neural structures in virally transduced fluorescent tissue sections, showing a three-fold enhancement at 50 μm depth into non-cleared tissue with Airy-LSM. Complimentary analysis is performed by resolution measurements in bead-injected tissue sections.Publisher PD