Multi-modal, label-free, polarisation-resolved super-resolution multiphoton microscopy

Abstract number
Presentation Form
Corresponding Email
[email protected]
Super-Resolution Microscopy
Peter Johnson (4, 6), Artemios Karvounis (2), H. Johnson Singh (3), Christopher J Brereton (1), Konstantinos Bourdakos (4, 6), Kerry Lunn (5), James JW Roberts (5), Donna E Davies (1, 6), Otto L Muskens (3), Mark G Jones (1, 6), Sumeet Mahajan (4, 6)
1. NIHR Southampton Biomedical Research Centre, Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, United Kingdom
2. Optoelectronics Research Centre and Centre for Photonic Metamaterials, University of Southampton, UK
3. Physics and Astronomy, Faculty of Engineering and Physical Sciences, University of Southampton, Southampton, UK
4. School of Chemistry, Faculty of Engineering and Physical Sciences, University of Southampton, Southampton, United Kingdom;
5. Synairgen Research Ltd, Southampton, United Kingdom
6. Institute for Life Sciences, University of Southampton, Southampton, United Kingdom

Multiphoton, Second Harmonic Generation, Two-photon excited autofluorescence, Super-resolution, photonic nanojet, polarisation

Abstract text

Summary: In this work the photonic nanojet (PNJ) phenomenon is exploited to achieve super-resolution imaging using endogenous contrast sources. Second harmonic generation (SHG) and two-photon excited autofluorescence (TPEAF) were used to image fibrillar collagen and elastin respectively. By manipulating the polarisation of the PNJ, polarisation resolved images were acquired and used to measure the structural disorder of the sample. It was observed that upon treatment with a cross linking inhibitor the degree of disorder within fibrillar collagen increased, furthermore the degree of disorder measured in elastin was similar to that of fibrillar collagen indicating an unexpectedly ordered structure.

Super-resolution (SR) optical microscopy has allowed the investigation of many biological structures below the diffraction limit, however, most of the established super-resolution techniques are hampered by the need for fluorescent labels. Multiphoton label-free techniques such as second harmonic generation (SHG) and two-photon excited autofluorescence (TPEAF) provide structurally and chemically selective contrast without the addition of exogenous labels, allowing observation of unperturbed biological systems. Attempts to achieve super-resolution imaging using these methods have seen limited success. 

High refractive index microspheres can be used to focus light into a jet with a waist narrower than the diffraction limit, so called, photonic nanojets (PNJs) (Figure 1). Unlike many of the common super-resolution techniques the PNJs do not rely on manipulating fluorescence emission to overcome the diffraction limit, as such they are amenable to multiphoton label-free imaging such as SHG and TPEAF. 

We use the photonic nanojet (PNJ) phenomenon to achieve super-resolution SHG (SR-SHG)1. 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 measurements 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 imaging to be used over a large area. Furthermore, PNJ imaging is independent of the contrast mechanism. This allowed us to apply the method to multimodal imaging with multiphoton techniques. We imaged elastin (via TPEAF) and collagen (via SHG) in lung cell spheroids (Figure 2). We observed unexpectedly high organisation of elastin molecules, similar to those is the highly organised fibrillar SHG. Thus, PNJ assisted multiphoton imaging was able to provide new biophysical insight and has implications for understanding the mechanisms of fibrotic disease progression and treatments. 

The ability to non-destructively image 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.


Johnson, P. et al. Super-resolved polarisation-enhanced second harmonic generation for direct imaging of nanoscale changes in collagen architecture. bioRxiv (2020). doi:10.1101/2020.02.07.934000