USER-FRIENDLY SUPER-RESOLUTION METHODOLOGY FOR STUDYING NEURAL SYSTEMS

Our understanding of biological structures and functions at the nanoscale has been greatly advanced by optical super-resolution imaging techniques that overcome the classical diffraction limit. However, the widespread adoption of these methods in neuroscience remains limited due to complex and costly instrumentation, image reconstruction-related artifacts, high laser intensities, and restricted applicability to two-dimensional imaging and visible wavelengths.
We aim to realize in neural samples an alternative super-resolution approach that circumvents these limitations, coined Super-linear Excitation–Emission (SEE) microscopy, (D. Denkova et al., Nat. Comm, 2019). SEE microscopy enables three-dimensional super-resolution imaging on standard confocal microscopes, without hardware modifications or computational post-processing, making it readily accessible to existing bio-imaging facilities. The method exploits fluorescent probes with super-linear excitation–emission response, specifically upconversion nanoparticles, distinct from conventional fluorophores, exhibiting linear emission. Notably, spatial resolution improves spontaneously in all three dimensions as excitation power is reduced, minimizing phototoxicity. SEE microscopy operates at near-infrared wavelengths, a spectral regime of particular interest for neuroscience due to reduced light scattering, low auto-fluorescence and increased imaging depth. Our previous proof-of-principle results show that the 3D imaging resolution improves more than twice over the diffraction limit, when particles are endocytosed in neuronal cells (see Figure).
Given that similar upconversion particles are already used in deep-brain excitation and optogenetics (Chen et al., Science, 2018), SEE microscopy offers a promising route towards enabling super-resolution capability in deep-tissue imaging and optical control in neural systems. We actively seek collaborations to apply this methodology to biologically relevant neuroscience questions.