Structured Illumination Microscopy Improves Spot Detection Performance in Spatial Transcriptomics

Spatial biology is a rapidly growing research field that focuses on the transcriptomic or proteomic profiling of single cells within tissues with preserved spatial information. Imaging-based spatial transcriptomics uses epifluorescence microscopy, which has shown remarkable results for the identification of multiple targets in situ. Nonetheless, the number of genes that can be reliably visualized is limited by the diffraction of light. Here, we investigate the effect of structured illumination (SIM), a super-resolution microscopy approach, on the performance of single-gene transcript detection in spatial transcriptomics experiments. We performed direct mRNA-targeted hybridization in situ sequencing for multiple genes in mouse coronal brain tissue sections. We evaluated spot detection performance in widefield and confocal images versus those with SIM in combination with 20×, 25× and 60× objectives. In general, SIM increases the detection efficiency of gene transcript spots compared to widefield and confocal modes. For each case, the specific fold increase in localizations is dependent on gene transcript density and the numerical aperture of the objective used, which has been shown to play an important role, especially for densely clustered spots. Taken together, our results suggest that SIM has the capacity to improve spot detection and overall data quality in spatial transcriptomics

Extending resolution within a single imaging frame

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.Fil: Torres García, Esley. Universidad Nacional Autónoma de México; MéxicoFil: Pinto Cámara, Raúl. Universidad Nacional Autónoma de México; MéxicoFil: Linares, Alejandro. Universidad Nacional Autónoma de México; MéxicoFil: Martínez, Damián. Universidad Nacional Autónoma de México; MéxicoFil: Abonza, Víctor. Universidad Nacional Autónoma de México; MéxicoFil: Brito Alarcón, Eduardo. Universidad Nacional Autónoma de México; MéxicoFil: Calcines Cruz, Carlos. Universidad Nacional Autónoma de México; MéxicoFil: Valdés Galindo, Gustavo. Universidad Nacional Autónoma de México; MéxicoFil: Torres, David. Universidad Nacional Autónoma de México; MéxicoFil: Jabloñski, Martina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Torres Martínez, Héctor H.. Universidad Nacional Autónoma de México; MéxicoFil: Martínez, José L.. Universidad Nacional Autónoma de México; MéxicoFil: Hernández, Haydee O.. Universidad Nacional Autónoma de México; MéxicoFil: Ocelotl Oviedo, José P.. Universidad Nacional Autónoma de México; MéxicoFil: Garcés, Yasel. Universidad Nacional Autónoma de México; MéxicoFil: Barchi, Marco. University of Rome Tor Vergata; ItaliaFil: D'Antuono, Rocco. Crick Advanced Light Microscopy Facility; Reino UnidoFil: Bošković, Ana. European Molecular Biology Laboratory; AlemaniaFil: Dubrovsky, Joseph G.. Universidad Nacional Autónoma de México; MéxicoFil: Darszon, Alberto. Universidad Nacional Autónoma de México; MéxicoFil: Buffone, Mariano Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Rodríguez Morales, Roberto. No especifíca;Fil: Rendon Mancha, Juan Manuel. Universidad Autónoma del Estado de Morelos; MéxicoFil: Wood, Christopher D.. Universidad Autónoma del Estado de Morelos; MéxicoFil: Hernández García, Armando. Universidad Autónoma del Estado de Morelos; MéxicoFil: Krapf, Diego. University of Colorado; Estados UnidosFil: Crevenna, Álvaro H.. European Molecular Biology Laboratory; ItaliaFil: Guerrero, Adán. Universidad Autónoma del Estado de Morelos; Méxic

Extending resolution within a single imaging frame

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