Photolabeling of intracellular molecules is an invaluable approach to studying various dynamic processes in living cells with high spatiotemporal precision. Among fluorescent proteins, photoconvertible mechanisms and their products are in the visible spectrum (400–650 nm), limiting their in vivo and multiplexed applications. Here we report the phenomenon of near-infrared to far-red photoconversion in the miRFP family of near infrared fluorescent proteins engineered from bacterial phytochromes. This photoconversion is induced by near-infrared light through a non-linear process, further allowing optical sectioning. Photoconverted miRFP species emit fluorescence at 650 nm enabling photolabeling entirely performed in the near-infrared range. We use miRFPs as photoconvertible fluorescent probes to track organelles in live cells and in vivo, both with conventional and super-resolution microscopy. The spectral properties of miRFPs complement those of GFP-like photoconvertible proteins, allowing strategies for photoconversion and spectral multiplexed applications.

Photobleaching is a general hurdle of fluorescence-based techniques especially in high-resolution microscopy that relies on prolonged and complex illumination. Strategies to reduce photobleaching require chemical modifications of the cell medium, which often compromise physiological cellular conditions. Here, we outline an all-optical strategy to minimize photobleaching in reversibly switching fluorescent proteins (RSFPs), a class of probes used in super-resolution and protein-multiplexing imaging techniques. By identifying the photobleaching pathways, we develop imaging schemes to increase the number of on-off photoswitching cycles, either modulating the on-switching light or co-irradiating the RSFPs with light at longer wavelengths with respect to fluorescence excitation. We apply the optimized imaging scheme to achieve imaging multiplexing at high-spatiotemporal resolutions and to record longer time-lapse imaging of sub-cellular structures with both confocal microscopy and parallelized RESOLFT nanoscopy.

Single Layer Graphene (SLG) has emerged as a critically important nanomaterial due to its unique optical and electrical properties and has become a potential candidate for biomedical applications, biosensors, and tissue engineering. Due to its intrinsic 2D nature, SLG is an ideal surface for the development of large-area biosensors and, due to its biocompatibility, can be easily exploited as a substrate for cell growth. The cellular response to SLG has been addressed in different studies with high cellular affinity for graphene often detected. Still, little is known about the molecular mechanism that drives/regulates the cellular adhesion and migration on SLG and SLG-coated interfaces with respect to other substrates. Within this scenario, we used quantitative super-resolution microscopy based on single-molecule localization to study the molecular distribution of adhesion proteins at the nanoscale level in cells growing on SLG and glass. In order to reveal the molecular mechanisms underlying the higher affinity of biological samples on SLG, we exploited stochastic optical reconstruction microscopy (STORM) imaging and cluster analysis, quantifying the super-resolution localization of the adhesion protein vinculin in neurons and clearly highlighting substrate-related correlations. Additionally, a comparison with an epithelial cell line (Chinese Hamster Ovary) revealed a cell dependent mechanism of interaction with SLG.

Photo-bleaching is a general hurdle of fluorescence-based techniques that becomes even more severe in high- resolution microscopy that relies on prolonged, focused and complex illumination sequences. Strategies to reduce photo-bleaching require chemical modifications of the cell media, which often stave off physiological cellular conditions. Here, we outline an all-optical strategy to minimize photo-bleaching in reversibly switching fluorescent proteins (RSFPs), a class of probes used in several super-resolution and protein-multiplexing imaging techniques. By identifying the photobleaching pathways, we developed novel imaging schemes to increase the number of ON- OFF photo-switching cycles based on a designed modulation of the on-switching light or a co-irradiation with red- shifted light. By rationalizing the photo-cycle, we expand multiplexing strategies with RSFPs to high- spatiotemporal resolutions while maintaining the accuracy and recording longer time-lapse imaging of sub-cellular structures with both confocal microscopy and parallelized RESOLFT nanoscopy.

Reversibly switchable fluorescent proteins (RSFPs) transition many times between dark and fluorescent states under minimal light doses. The photoswitching can happen at different speed, contrast and length, and it is often challenging for users to select the optimal imaging scheme to generate images with high contrast and spatial resolution. Here, we experimentally investigate the photophysical properties of different RSFPs under imaging conditions, together with an in silico exploration of their role in nanoscale image formation. We developed open‐source software that uses measured parameters such as brightness, switching speed, photoswitching fatigue, labelling densities, noise and illumination type to generate the related RESOLFT (reversible saturable/switchable optical fluorescence transition) super‐resolution image. This tool can be used to select optimal imaging schemes for known RSFPs and to guide the rational development of new proteins.

Synaptic neurotransmission is a critical hallmark of brain activity and one of the first processes to be affected in neural diseases. Monitoring this process, and in particular synaptic vesicle recycling, in living cells has been instrumental in unraveling mechanisms responsible for neurotransmitter release. However, currently available reporters suffer from major limitations such large probe size or lack of suitability for human neurons, hampering the understanding of human synaptic pathophysiology. Here we describe the NbLumSyt1 toolkit, a panel of nanobody-based affinity probes targeting the luminal domain of the synaptic vesicle protein Synaptotagmin 1 (Syt1). These new tools enable quantitative, non-invasive imaging and functional interrogation of synaptic transmission in human neurons, with unprecedented precision, versatility and cost efficiency, in technologies ranging from fixed-and live-cell super-resolution imaging to electron microscopy and mass spectrometry. Overall, NbLumSyt1 nanobinders provide a valuable platform for human synaptic physiology and pathophysiology, benefiting fundamental neuroscience and translational efforts to study and develop treatments for brain-related disorders.