Light-field flow cytometry for high-resolution, volumetric and multiparametric 3D single-cell analysis.

Imaging flow cytometry (IFC) combines flow cytometry and fluorescence microscopy to enable high-throughput, multiparametric single-cell analysis with rich spatial details. However, current IFC techniques remain limited in their ability to reveal subcellular information with a high 3D resolution, throughput, sensitivity, and instrumental simplicity. In this study, we introduce a light-field flow cytometer (LFC), an IFC system capable of high-content, single-shot, and multi-color acquisition of up to 5,750 cells per second with a near-diffraction-limited resolution of 400-600 nm in all three dimensions. The LFC system integrates optical, microfluidic, and computational strategies to facilitate the volumetric visualization of various 3D subcellular characteristics through convenient access to commonly used epi-fluorescence platforms. We demonstrate the effectiveness of LFC in assaying, analyzing, and enumerating intricate subcellular morphology, function, and heterogeneity using various phantoms and biological specimens. The advancement offered by the LFC system presents a promising methodological pathway for broad cell biological and translational discoveries, with the potential for widespread adoption in biomedical research.

Portable light-sheet optofluidic microscopy for 3D fluorescence imaging flow cytometry.

Imaging flow cytometry (IFC) combines conventional flow cytometry with optical microscopy, allowing for high-throughput, multi-parameter screening of single-cell specimens with morphological and spatial information. However, current 3D IFC systems are limited by instrumental complexity and incompatibility with available microfluidic devices or operations. Here, we report portable light-sheet optofluidic microscopy (PLSOM) for 3D fluorescence cytometric imaging. PLSOM exploits a compact, open-top light-sheet configuration compatible with commonly adopted microfluidic chips. The system offers a subcellular resolution (2-4 µm) in all three dimensions, high throughput (∼1000 cells per s), and portability (30 cm (l) × 10 cm (w) × 26 cm (h)). We demonstrated PLSOM for 3D IFC using various phantom and cell systems. The low-cost and custom-built architecture of PLSOM permits easy adaptability and dissemination for broad 3D flow cytometric investigations.

In vivo mRNA delivery to virus-specific T cells by light-induced ligand exchange of MHC class I antigen-presenting nanoparticles.

Simultaneous delivery of mRNA to multiple populations of antigen (Ag)-specific CD8+ T cells is challenging given the diversity of peptide epitopes and polymorphism of class I major histocompatibility complexes (MHCI). We developed Ag-presenting nanoparticles (APNs) for mRNA delivery using pMHCI molecules that were refolded with photocleavable peptides to allow rapid ligand exchange by UV light and site-specifically conjugated with a lipid tail for postinsertion into preformed mRNA lipid nanoparticles. Across different TCR transgenic mouse models (P14, OT-1, and Pmel), UV-exchanged APNs bound and transfected their cognate Ag-specific CD8+ T cells equivalent to APNs produced using conventionally refolded pMHCI molecules. In mice infected with PR8 influenza, multiplexed delivery of UV-exchanged APNs against three immunodominant epitopes led to ~50% transfection of a VHH mRNA reporter in cognate Ag-specific CD8+ T cells. Our data show that UV-mediated peptide exchange can be used to rapidly produce APNs for mRNA delivery to multiple populations of Ag-specific T cells in vivo.