Chemical and structural imaging with nanoresolution under ambient conditions are of utmost importance for advancing our understanding of biological processes at the sub-cellular level, which is needed for achieving a thorough understanding of severe diseases such as cancers, neurodegenerative or autoimmune disorders. These perspectives motived over the past decades a consistent body of work in the field of nanoscale imaging, which led to the advent of a wide variety of techniques, each with its own strengths and limitations. Among these, fluorescence based super-resolution microscopy (SRM) techniques succeed in overcoming the resolution limits imposed by diffraction, reaching resolutions in the 30-70 nm range.
However, they still suffer a series of limitations, as they require very specialised fluorescent probes (whose presence can influence the metabolic behaviour and locomotion of the investigated cells) and rely on laser beam exposure levels that can lead to phototoxicity and photodamage. Microscopy techniques based on tip-enhancement effects can overcome a significant part of these limitations. Tip-enhanced Fluorescence Near-field Optical Microscopy (TEFSNOM) is one of these techniques, and its value relies on its underlying contrast mechanism that allows probing fluorescence in the near-field of an investigated sample at resolutions <10nm.
The potential of TEFSNOM is very high as this technique can be used to investigate at nanoscale resolution any type of fluorescent sample ranging from biological species (even through their autofluorescent properties) to nanostructured materials, with unsophisticated laser sources. However, TEFSNOM is still poorly used in life sciences because the detection suffers from a low signal to noise ratio due to a high background resulting in low acquisition rates (since high signal integration times are needed to produce meaningful TEFSNOM images).
In this project, we will demonstrate a novel imaging technique and associated data acquisition strategies, coined TEFPLASNOM, that exceeds current TEFSNOM approaches in terms of sensitivity and speed by one order of magnitude. This new technique will rely on exploiting plasmon resonance energy transfer to excite fluorescence instead of laser beam excitation with wavelengths falling in the absorption band of the fluorophore. Under this illumination scheme, the other fluorophores in the proximity of the fluorophore of interest (accounting for unwanted background in traditional TEFSNOM approaches) remain switched off, resulting in higher spatial sensitivity and contrast, and leading to unprecedented optical resolutions based on fluorescence, even using auto-fluorescence of biological samples. To achieve this goal the TEFPLASNOM project will leverage the synergy and skills of two partner teams with expertise in near-field optical microscopy (UPB) and in plasmonic materials for fluorescence enhancement (UNIPG).