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Super-resolved polarisation-enhanced second harmonic generation for direct imaging of nanoscale changes in collagen architecture

Super-resolved polarisation-enhanced second harmonic generation for direct imaging of nanoscale changes in collagen architecture
Super-resolved polarisation-enhanced second harmonic generation for direct imaging of nanoscale changes in collagen architecture
Super-resolution (SR) optical microscopy has allowed the investigation of many biological structures below the diffraction limit, however, most of the techniques are hampered by the need for fluorescent labels. Non-linear label-free techniques such as Second Harmonic Generation (SHG) provide structurally specific contrast without the addition of exogenous labels, allowing observation of unperturbed biological systems. We use the photonic nanojet (PNJ) phenomena to achieve super-resolution SHG (SR-SHG). A resolution of ~λ/6 with respect to the fundamental wavelength, that is, a ~2.3-fold improvement over conventional or diffraction-limited SHG under the same imaging conditions is achieved. Crucially we find that the polarisation properties of excitation are maintained in a PNJ. This is observed in experiment and simulations. This may have widespread implications to increase sensitivity by detection of polarisation-resolved SHG (p-SHG) by observing anisotropy in signals. These new findings allowed us to visualise biological SHG-active structures such as collagen at an unprecedented and previously unresolvable spatial scale. Moreover, we demonstrate that the use of an array of self-assembled high-index spheres overcomes the issue of a limited field of view for such a method, allowing PNJ-assisted SR-SHG to be used over a large area. Dysregulation of collagen at the nanoscale occurs in many diseases and is an underlying cause in diseases such as lung fibrosis. Here we demonstrate that pSR-SHG allows unprecedented observation of changes at the nanoscale that are invisible by conventional diffraction-limited SHG imaging. The ability to non-destructively image SHG-active biological structures without labels at the nanoscale with a relatively simple optical method heralds the promise of a new tool to understand biological phenomena and drive drug discovery.
2334-2536
Johnson, Peter
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Karvounis, Artemios
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Singh, H. Johnson
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Brereton, Christopher J
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Bourdakos, Konstantinos
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Lunn, Kelly
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Roberts, James J.W.
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Davies, Donna
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Muskens, Otto
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Jones, Mark
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Mahajan, Sumeet
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Johnson, Peter
01e8bf5b-9ab6-4403-8281-93879dfdf084
Karvounis, Artemios
878c12bb-c30e-46f4-8c56-86423b41cdba
Singh, H. Johnson
709a93d7-e379-4c3f-86e2-3dc39b11eeb4
Brereton, Christopher J
948ca4ea-b04c-4b7a-bfe4-f79f184d7e43
Bourdakos, Konstantinos
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Lunn, Kelly
ffbe28d0-aaa3-4a77-852a-bac0c13eda14
Roberts, James J.W.
459b5e29-66ed-42ea-b282-d6573e2d4f86
Davies, Donna
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Muskens, Otto
2284101a-f9ef-4d79-8951-a6cda5bfc7f9
Jones, Mark
a6fd492e-058e-4e84-a486-34c6035429c1
Mahajan, Sumeet
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Johnson, Peter, Karvounis, Artemios, Singh, H. Johnson, Brereton, Christopher J, Bourdakos, Konstantinos, Lunn, Kelly, Roberts, James J.W., Davies, Donna, Muskens, Otto, Jones, Mark and Mahajan, Sumeet (2021) Super-resolved polarisation-enhanced second harmonic generation for direct imaging of nanoscale changes in collagen architecture. Optica. (In Press)

Record type: Article

Abstract

Super-resolution (SR) optical microscopy has allowed the investigation of many biological structures below the diffraction limit, however, most of the techniques are hampered by the need for fluorescent labels. Non-linear label-free techniques such as Second Harmonic Generation (SHG) provide structurally specific contrast without the addition of exogenous labels, allowing observation of unperturbed biological systems. We use the photonic nanojet (PNJ) phenomena to achieve super-resolution SHG (SR-SHG). A resolution of ~λ/6 with respect to the fundamental wavelength, that is, a ~2.3-fold improvement over conventional or diffraction-limited SHG under the same imaging conditions is achieved. Crucially we find that the polarisation properties of excitation are maintained in a PNJ. This is observed in experiment and simulations. This may have widespread implications to increase sensitivity by detection of polarisation-resolved SHG (p-SHG) by observing anisotropy in signals. These new findings allowed us to visualise biological SHG-active structures such as collagen at an unprecedented and previously unresolvable spatial scale. Moreover, we demonstrate that the use of an array of self-assembled high-index spheres overcomes the issue of a limited field of view for such a method, allowing PNJ-assisted SR-SHG to be used over a large area. Dysregulation of collagen at the nanoscale occurs in many diseases and is an underlying cause in diseases such as lung fibrosis. Here we demonstrate that pSR-SHG allows unprecedented observation of changes at the nanoscale that are invisible by conventional diffraction-limited SHG imaging. The ability to non-destructively image SHG-active biological structures without labels at the nanoscale with a relatively simple optical method heralds the promise of a new tool to understand biological phenomena and drive drug discovery.

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SR-SHG MainText vOptica_vRevised_final - Accepted Manuscript
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Accepted/In Press date: 17 March 2021

Identifiers

Local EPrints ID: 447980
URI: http://eprints.soton.ac.uk/id/eprint/447980
ISSN: 2334-2536
PURE UUID: a30150d2-ba16-47a6-9c09-ed816e1e1682
ORCID for Donna Davies: ORCID iD orcid.org/0000-0002-5117-2991
ORCID for Otto Muskens: ORCID iD orcid.org/0000-0003-0693-5504
ORCID for Mark Jones: ORCID iD orcid.org/0000-0001-6308-6014
ORCID for Sumeet Mahajan: ORCID iD orcid.org/0000-0001-8923-6666

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Date deposited: 29 Mar 2021 16:36
Last modified: 30 Mar 2021 01:43

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Contributors

Author: Peter Johnson
Author: Artemios Karvounis
Author: H. Johnson Singh
Author: Christopher J Brereton
Author: Konstantinos Bourdakos
Author: Kelly Lunn
Author: James J.W. Roberts
Author: Donna Davies ORCID iD
Author: Otto Muskens ORCID iD
Author: Mark Jones ORCID iD
Author: Sumeet Mahajan ORCID iD

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