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Label-free light sheet microscopy for 3D imaging of biological specimens

Label-free light sheet microscopy for 3D imaging of biological specimens
Label-free light sheet microscopy for 3D imaging of biological specimens
Light sheet microscopy (LSM) has emerged as one of most profound three dimensional (3D) imaging tools for life sciences applications in the past decade. This advance was awarded Nature’s Method of the Year in 2014, and has revolutionised imaging of live biological samples at high spatial and temporal resolution. Label-free imaging in microscopy is growing, but Light Sheet Microscopy has remained largely restricted to using fluorescence-based methods requiring exogenous labels for contrast. This thesis explores the development of three new scattering-based label-free imaging modalities for light sheet microscopy, and their applications in bioimaging.

A novel high-speed wavelength-scanning approach to vibrational imaging for LSM is explored first with Raman-LSM. This is compared to a spectral resolution model for the system and to experimental Raman microspectroscopy. Highly uniform nanosensors are used to characterise SERS-LSM for pH-sensitive imaging. Finally, steps are taken towards a biological application of pH-sensitive imaging with SERS measurements of MBA-modified intracellular nanoparticles.

An advance in nonlinear imaging for LSM is presented next, with the first results of label-free Second Harmonic Generation Light Sheet Microscopy from endogenous biomaterials. Orthogonal detection of SHG signals is verified and well-characterised with standard materials and two biological models, and this is then applied to imaging of large 3D cell cultures.

Finally, the first demonstration of Rayleigh Scattering Light Sheet microscopy is presented, using wavelength-and polarisation-resolved scattering response of targets for material-specific imaging, using the scattering response from well-characterised nanoparticles for proof-of-concept. Biological application for large and complex scattering environments follows, separating distinct scattering regions within 3D cell spheroids, matched to 2PF autofluorescence emissions.

These three label-free imaging methods offer a paradigm shift away from the fluorescence-based imaging modalities that dominate contemporary light sheet microscopy, and represent three practical alternative approaches for contrast generation with LSM.
University of Southampton
Hanrahan, Niall, Anthony Joseph
88556765-3d33-4496-8c12-f57763c76382
Hanrahan, Niall, Anthony Joseph
88556765-3d33-4496-8c12-f57763c76382
Mahajan, Sumeet
b131f40a-479e-4432-b662-19d60d4069e9

Hanrahan, Niall, Anthony Joseph (2021) Label-free light sheet microscopy for 3D imaging of biological specimens. University of Southampton, Doctoral Thesis, 251pp.

Record type: Thesis (Doctoral)

Abstract

Light sheet microscopy (LSM) has emerged as one of most profound three dimensional (3D) imaging tools for life sciences applications in the past decade. This advance was awarded Nature’s Method of the Year in 2014, and has revolutionised imaging of live biological samples at high spatial and temporal resolution. Label-free imaging in microscopy is growing, but Light Sheet Microscopy has remained largely restricted to using fluorescence-based methods requiring exogenous labels for contrast. This thesis explores the development of three new scattering-based label-free imaging modalities for light sheet microscopy, and their applications in bioimaging.

A novel high-speed wavelength-scanning approach to vibrational imaging for LSM is explored first with Raman-LSM. This is compared to a spectral resolution model for the system and to experimental Raman microspectroscopy. Highly uniform nanosensors are used to characterise SERS-LSM for pH-sensitive imaging. Finally, steps are taken towards a biological application of pH-sensitive imaging with SERS measurements of MBA-modified intracellular nanoparticles.

An advance in nonlinear imaging for LSM is presented next, with the first results of label-free Second Harmonic Generation Light Sheet Microscopy from endogenous biomaterials. Orthogonal detection of SHG signals is verified and well-characterised with standard materials and two biological models, and this is then applied to imaging of large 3D cell cultures.

Finally, the first demonstration of Rayleigh Scattering Light Sheet microscopy is presented, using wavelength-and polarisation-resolved scattering response of targets for material-specific imaging, using the scattering response from well-characterised nanoparticles for proof-of-concept. Biological application for large and complex scattering environments follows, separating distinct scattering regions within 3D cell spheroids, matched to 2PF autofluorescence emissions.

These three label-free imaging methods offer a paradigm shift away from the fluorescence-based imaging modalities that dominate contemporary light sheet microscopy, and represent three practical alternative approaches for contrast generation with LSM.

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More information

Published date: 2021

Identifiers

Local EPrints ID: 456710
URI: http://eprints.soton.ac.uk/id/eprint/456710
PURE UUID: 297aa84e-ad80-4f27-b015-f6a0389616e3
ORCID for Sumeet Mahajan: ORCID iD orcid.org/0000-0001-8923-6666

Catalogue record

Date deposited: 09 May 2022 17:20
Last modified: 17 Mar 2024 03:10

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Contributors

Author: Niall, Anthony Joseph Hanrahan
Thesis advisor: Sumeet Mahajan ORCID iD

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