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1. PSSR2: a user-friendly Python package for democratizing deep learning-based point-scanning super-resolution microscopy

   https://doi.org/10.1186/s44330-024-00020-5  

   Stites, Hayden_C; Manor, Uri (January 2025, BMC Methods)

   Abstract BackgroundTo address the limitations of large-scale high quality microscopy image acquisition, PSSR (Point-Scanning Super-Resolution) was introduced to enhance easily acquired low quality microscopy data to a higher quality using deep learning-based methods. However, while PSSR was released as open-source, it was difficult for users to implement into their workflows due to an outdated codebase, limiting its usage by prospective users. Additionally, while the data enhancements provided by PSSR were significant, there was still potential for further improvement. MethodsTo overcome this, we introduce PSSR2, a redesigned implementation of PSSR workflows and methods built to put state-of-the-art technology into the hands of the general microscopy and biology research community. PSSR2 enables user-friendly implementation of super-resolution workflows for simultaneous super-resolution and denoising of undersampled microscopy data, especially through its integrated Command Line Interface and Napari plugin. PSSR2 improves and expands upon previously established PSSR algorithms, mainly through improvements in the semi-synthetic data generation (“crappification”) and training processes. ResultsIn benchmarking PSSR2 on a test dataset of paired high and low resolution electron microscopy images, PSSR2 super-resolves high-resolution images from low-resolution images to a significantly higher accuracy than PSSR. The super-resolved images are also more visually representative of real-world high-resolution images. DiscussionThe improvements in PSSR2, in providing higher quality images, should improve the performance of downstream analyses. We note that for accurate super-resolution, PSSR2 models should only be applied to super-resolve data sufficiently similar to training data and should be validated against real-world ground truth data. 

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2. A Photoactivatable BODIPY Probe for Localization‐Based Super‐Resolution Cellular Imaging

   https://doi.org/10.1002/anie.201805827  

   Wijesooriya, Chamari S.; Peterson, Julie A.; Shrestha, Pradeep; Gehrmann, Elizabeth J.; Winter, Arthur H.; Smith, Emily A. (September 2018, Angewandte Chemie International Edition)

   Abstract The synthesis and application of a photoactivatable boron‐alkylated BODIPY probe for localization‐based super‐resolution microscopy is reported. Photoactivation and excitation of the probe is achieved by a previously unknown boron‐photodealkylation reaction with a single low‐power visible laser and without requiring the addition of reducing agents or oxygen scavengers in the imaging buffer. These features lead to a versatile probe for localization‐based microscopy of biological systems. The probe can be easily linked to nucleophile‐containing molecules to target specific cellular organelles. By attaching paclitaxel to the photoactivatable BODIPY, in vitro and in vivo super‐resolution imaging of microtubules is demonstrated. This is the first example of single‐molecule localization‐based super‐resolution microscopy using a visible‐light‐activated BODIPY compound as a fluorescent probe. 

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3. Synthesis and Characterization of Dye-Doped Au@SiO ₂ Core-Shell Nanoparticles for Super-Resolution Fluorescence Microscopy

   https://doi.org/10.1177/00037028221121357  

   Thompson, Shelby; Jorns, Mychele; Pappas, Dimitri (October 2022, Applied Spectroscopy)

   Dye-doped nanoparticles have been investigated as bright, fluorescent probes for localization-based super-resolution microscopy. Nanoparticle size is important in super-resolution microscopy to get an accurate size of the object of interest from image analysis. Due to their self-blinking behavior and metal-enhanced fluorescence (MEF), Ag@SiO₂and Au@Ag@SiO₂nanoparticles have shown promise as probes for localization-based super-resolution microscopy. Here, several noble metal-based dye-doped core-shell nanoparticles have been investigated as self-blinking nanomaterial probes. It was observed that both the gold- and silver-plated nanoparticle cores exhibit weak luminescence under certain conditions due to the surface plasmon resonance bands produced by each metal, and the gold cores exhibit blinking behavior which enhances the blinking and fluorescence of the dye-doped nanoparticle. However, the silver-plated nanoparticle cores, while weakly luminescent, did not exhibit any blinking; the dye-doped nanoparticle exhibited the same behavior as the core fluorescent, but did not blink. Because of the blinking behavior, stochastic optical reconstruction microscopy (STORM) super-resolution analysis was able to be performed with performed on the gold core nanoparticles. A preliminary study on the use of these nanoparticles for localization-based super-resolution showed that these nanoparticles are suitable for use in STORM super resolution. Resolution enhancement was two times better than the diffraction limited images, with core sizes reduced to 15 nm using the hybrid Au–Ag cores. 

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4. Extending resolution within a single imaging frame

   https://doi.org/10.1038/s41467-022-34693-9  

   Torres-García, Esley; Pinto-Cámara, Raúl; Linares, Alejandro; Martínez, Damián; Abonza, Víctor; Brito-Alarcón, Eduardo; Calcines-Cruz, Carlos; Valdés-Galindo, Gustavo; Torres, David; Jabloñski, Martina; et al (December 2022, Nature Communications)

   Abstract The resolution of fluorescence microscopy images is limited by the physical properties of light. In the last decade, numerous super-resolution microscopy (SRM) approaches have been proposed to deal with such hindrance. Here we present Mean-Shift Super Resolution (MSSR), a new SRM algorithm based on the Mean Shift theory, which extends spatial resolution of single fluorescence images beyond the diffraction limit of light. MSSR works on low and high fluorophore densities, is not limited by the architecture of the optical setup and is applicable to single images as well as temporal series. The theoretical limit of spatial resolution, based on optimized real-world imaging conditions and analysis of temporal image stacks, has been measured to be 40 nm. Furthermore, MSSR has denoising capabilities that outperform other SRM approaches. Along with its wide accessibility, MSSR is a powerful, flexible, and generic tool for multidimensional and live cell imaging applications. 

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5. Spectroscopic single-molecule localization microscopy: applications and prospective

   https://doi.org/10.1186/s40580-023-00363-9  

   Brenner, Benjamin; Sun, Cheng; Raymo, Françisco M.; Zhang, Hao F. (March 2023, Nano Convergence)

   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. Graphical Abstract 

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