Conventional microscopy is limited by the workings of light waves. When light hits a surface it is diffracted or bent, meaning that it can only be focused on a finite point. For the most powerful optical microscopes, this point has a diameter around half of visible light’s wavelength, under 200 nanometres. The European Research Council supported NanoChemBioVision project developed two methods to achieve ultra-high resolution beyond this diffraction limit. “Both methods uniquely achieve super-resolution without fluorescent labels which stain, affect or genetically modify samples,” explains project coordinator Sumeet Mahajan from the University of Southampton, the project host. “The biomedical applications could significantly improve public health. For example, these methods could image viral particles, including SARS-CoV-2, offering insights into its chemical make-up and structure, assisting efforts to tackle the disease.” So far one patent has been granted, with another pending.
Subcellular label-free detail
Over the last 20 years, there has been an explosion in biological imaging following the discovery of the green fluorescent protein and the invention of optical fluorescent super-resolution methods. Life scientists can now image cellular features in unprecedented detail. However, these fluorescent labels impair the imaging of some functions and features. Vibrational imaging offers one label-free solution, while also providing additional chemical and structural information. All molecules vibrate in specific ways, providing molecular ‘fingerprints’. NanoChemBioVision used infrared spectroscopy and Raman spectroscopy to read these fingerprints. The team also investigated another label-free technique of harmonic generation and two-photon fluorescence, where two photons are combined to provide optical imaging of specific structures and molecules. NanoChemBioVision applied these label-free techniques to both super-oscillation and photonic nanojet methods. The first method shapes the propagating beam of light by modulating it in intensity and time to obtain a very small light spot for imaging. The second exploits the effect of microspheres on a beam of light. The microspheres, made of a denser material than the medium, focus the light down to a nanoscale spot. Several simulations applied super-oscillation in standard single and multiphoton microscopes to set the optimum parameters for light modulation. This results in a 30 % to 70 % improvement in the diffraction-limited resolution. For example, super-oscillations combining with two-photon fluorescence imaged gold nanoparticles, achieving resolution beyond their diffraction limit. The photonic nanojet-assisted second harmonic generation technique was applied to biological samples such as collagen fibres in mouse tissue resulting in unprecedented detail. A resolution of under 125 nanometres was demonstrated, 2.3 times better than diffraction-limited imaging. This technique also diagnosed pulmonary diseases by imaging samples of idiopathic lung fibrosis. Collagen changes were observed at the nanoscale, invisible to diffraction-limited imaging. It clearly differentiated between samples treated with drugs or growth factors and healthy samples. “This offers new capabilities for super-resolved imaging of tissue and cells which, as well as being label-free, also simultaneously acquires chemical and structural information not possible before,” says Mahajan. The team are now working with partners at the Optoelectronics Research Centre in Southampton to develop methods to reduce the costs and size of the laser systems required for multiphoton imaging. They are also increasing the sensitivity of the system and expanding its capabilities for a versatile range of tissues, cells, diseases and conditions. Additionally, they are increasing the penetration depth of the techniques to enable ultra-high resolution imaging of deeper tissue layers.
NanoChemBioVision, microscopes, microscopy, imaging, photons, nano, COVID, resolution, oscillation, diffraction, label-free, light