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  1. Abstract

    Single-molecule localization microscopy (SMLM) breaks the optical diffraction limit by numerically localizing sparse fluorescence emitters to achieve super-resolution imaging. Spectroscopic SMLM or sSMLM further allows simultaneous spectroscopy and super-resolution imaging of fluorescence molecules. Hence, sSMLM can extract spectral features with single-molecule sensitivity, higher precision, and higher multiplexity than traditional multicolor microscopy modalities. These new capabilities enabled advanced multiplexed and functional cellular imaging applications. While sSMLM suffers from reduced spatial precision compared to conventional SMLM due to splitting photons to form spatial and spectral images, several methods have been reported to mitigate these weaknesses through innovative optical design and image processing techniques. This review summarizes the recent progress in sSMLM, its applications, and our perspective on future work.

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  2. Abstract

    The borondipyrromethene (BODIPY) chromophore is a versatile platform for the construction of photoresponsive dyes with unique properties. Specifically, its covalent connection to a photocleavable group can be exploited to engineer compounds with photoswitchable fluorescence. The resulting photoactivatable fluorophores can increase their emission intensity or shift their emission wavelengths in response to switching. Such changes permit the spatiotemporal control of fluorescence with optical stimulations and the implementation of imaging strategies that would be impossible to replicate with conventional fluorophores. Indeed, BODIPYs with photoactivatable fluorescence enable the selective highlighting of intracellular targets, the nanoscaled visualization of sub‐cellular components, the real‐time monitoring of dynamic events and the photochemical writing of optical barcodes.

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  3. Osiński, Marek ; Kanaras, Antonios G. (Ed.)
    Single-molecule localization microscopy (SMLM) strategies based on fluorescence photoactivation permit the imaging of live cells with subdiffraction resolution and the high-throughput tracking of individual biomolecules in their interior. They rely predominantly on genetically-encoded fluorescent proteins to label live cells selectively and allow the sequential single-molecule localization of sparse populations of photoactivated fluorophores. Synthetic counterparts to these photoresponsive proteins are limited to a few remarkable examples at the present stage, mostly because of the daunting challenges in engineering biocompatible molecular constructs with appropriate photochemical and photophysical properties for live-cell SMLM. Our laboratory developed a new family of synthetic photoactivatable fluorophores specifically designed for these imaging applications. They combine a borondipyrromethene (BODIPY) fluorophore and an ortho-nitrobenzyl (ONB) photocage in a single molecular skeleton. The photoinduced ONB cleavage extends electronic delocalization to shift bathochromically the BODIPY absorption and emission bands. As a result, these photochemical transformations can be exploited to switch fluorescence on in a spectral region compatible with bioimaging applications and allow the localization of the photochemical product at the single-molecule level. Furthermore, our compounds can be delivered and operated in the interior of live cells to enable the visualization of organelles with nanometer resolution and the intracellular tracking of single photoactivated molecules. 
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  4. Photoactivated localization microscopy (PALM) relies on fluorescence photoactivation and single-molecule localization to overcome optical diffraction and reconstruct images of biological samples with spatial resolution at the nanoscale. The implementation of this subdiffraction imaging method, however, requires fluorescent probes with photochemical and photophysical properties specifically engineered to enable the localization of single photoactivated molecules with nanometer precision. The synthetic versatility and outstanding photophysical properties of the borondipyrromethene (BODIPY) chromophore are ideally suited to satisfy these stringent requirements. Specifically, synthetic manipulations of the BODIPY scaffold can be invoked to install photolabile functional groups and photoactivate fluorescence under photochemical control. Additionally, targeting ligands can be incorporated in the resulting photoactivatable fluorophores (PAFs) to label selected subcellular components in live cells. Indeed, photoactivatable BODIPYs have already allowed the sub-diffraction imaging of diverse cellular substructures in live cells using PALM and can evolve into invaluable analytical probes for bioimaging applications. 
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  5. By manipulating the spectral dispersion of detected photons, spectroscopic single-molecule localization microscopy (sSMLM) permits concurrent high-throughput single-molecular spectroscopic analysis and imaging. Despite its promising potential, using discrete optical components and managing the delicate balance between spectral dispersion and spatial localization compromise its performance, including non-uniform spectral dispersion, high transmission loss of grating, high optical alignment demands, and reduced precision. We designed a dual-wedge prism (DWP)-based monolithic imaging spectrometer to overcome these challenges. We optimized the DWP for spectrally dispersing focused beam without deviation and with minimal wavefront error. We integrated all components into a compact assembly, minimizing total transmission loss and significantly reducing optical alignment requirements. We show the feasibility of DWP using ray-tracing and numerical simulations. We validated our numerical simulations by experimentally imaging individual nanospheres and confirmed that DWP-sSMLM achieved much improved spatial and spectral precisions of grating-based sSMLM. We also demonstrated DWP-sSMLM in 3D multi-color imaging of cells. 
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  6. null (Ed.)