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Superresolved polarization-enhanced second-harmonic generation for direct imaging of nanoscale changes in collagen architecture

Superresolved polarization-enhanced second-harmonic generation for direct imaging of nanoscale changes in collagen architecture
Superresolved polarization-enhanced second-harmonic generation for direct imaging of nanoscale changes in collagen architecture

Superresolution (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. Nonlinear 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 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 polarization 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 polarization-resolved SHG by observing anisotropy in signals. These new, to the best of our knowledge, findings allowed us to visualize 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 nondestructively 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
674-685
Johnson, Peter B.
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Karvounis, Artemios
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Johnson Singh, H.
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Brereton, Christopher J.
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Bourdakos, Konstantinos N.
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Lunn, Kerry
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Roberts, James J.W.
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Davies, Donna E.
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Muskens, Otto L.
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Jones, Mark G.
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Mahajan, Sumeet
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Johnson, Peter B.
3f6068ce-171e-4c2c-aca9-dc9b6a37413f
Karvounis, Artemios
878c12bb-c30e-46f4-8c56-86423b41cdba
Johnson Singh, H.
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Brereton, Christopher J.
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Bourdakos, Konstantinos N.
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Lunn, Kerry
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Roberts, James J.W.
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Davies, Donna E.
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Muskens, Otto L.
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Jones, Mark G.
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Mahajan, Sumeet
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Johnson, Peter B., Karvounis, Artemios, Johnson Singh, H., Brereton, Christopher J., Bourdakos, Konstantinos N., Lunn, Kerry, Roberts, James J.W., Davies, Donna E., Muskens, Otto L., Jones, Mark G. and Mahajan, Sumeet (2021) Superresolved polarization-enhanced second-harmonic generation for direct imaging of nanoscale changes in collagen architecture. Optica, 8 (5), 674-685. (doi:10.1364/OPTICA.411325).

Record type: Article

Abstract

Superresolution (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. Nonlinear 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 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 polarization 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 polarization-resolved SHG by observing anisotropy in signals. These new, to the best of our knowledge, findings allowed us to visualize 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 nondestructively 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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Accepted/In Press date: 16 March 2021
Published date: 13 May 2021
Additional Information: Funding. European Research Council (638258, NanoChemBioVision); Engineering and Physical Sciences Research Council (EP/N509747/1, EP/T020997/1); Leverhulme Trust (RPG-2018-251); Wellcome Trust (100638/Z/12/Z); NIHR Southampton Biomedical Research Centre

Identifiers

Local EPrints ID: 450864
URI: http://eprints.soton.ac.uk/id/eprint/450864
ISSN: 2334-2536
PURE UUID: 34b72e25-2f1f-44dd-bf05-9e5ac1fc24b5
ORCID for Peter B. Johnson: ORCID iD orcid.org/0000-0003-2306-4974
ORCID for Donna E. Davies: ORCID iD orcid.org/0000-0002-5117-2991
ORCID for Otto L. Muskens: ORCID iD orcid.org/0000-0003-0693-5504
ORCID for Mark G. Jones: ORCID iD orcid.org/0000-0001-6308-6014
ORCID for Sumeet Mahajan: ORCID iD orcid.org/0000-0001-8923-6666

Catalogue record

Date deposited: 17 Aug 2021 16:30
Last modified: 26 Nov 2021 02:54

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Contributors

Author: Artemios Karvounis
Author: H. Johnson Singh
Author: Christopher J. Brereton
Author: Konstantinos N. Bourdakos
Author: Kerry Lunn
Author: James J.W. Roberts
Author: Donna E. Davies ORCID iD
Author: Otto L. Muskens ORCID iD
Author: Mark G. Jones ORCID iD
Author: Sumeet Mahajan ORCID iD

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