Abstract
Single-molecule microscopy techniques have emerged as useful tools to image individual molecules and analyze their dynamics inside cells, but their application has mostly been restricted to cell cultures. Here, a light-sheet fluorescence microscopy setup is presented for imaging individual proteins inside living zebrafish embryos. The optical configuration makes this design accessible to many laboratories and a dedicated sample-mounting system ensures sample viability and mounting flexibility. Using this setup, we have analyzed the dynamics of individual glucocorticoid receptors, which demonstrates that this approach creates multiple possibilities for the analysis of intracellular protein dynamics in intact living organisms.
Highlights
A quantitative imaging-based analysis of the biochemical processes and protein interactions inside cells requires microscopy techniques that go beyond ensemble averaging and a static molecular view [1]
Imaging of individual proteins in living cells is enabled by fluorescent labeling of these proteins, using either genetically encoded fusion to autofluorescent proteins such as green fluorescent protein (GFP) and photoactivatable or switchable variants of these proteins, or linking of nanoparticles or organic dyes to peptide or protein tags, that have been fused to the protein of interest [2,3,4,5]
Our Light Sheet Fluorescence Single-Molecule Microscopy (LSFSMM) implementation is built on a Nikon Eclipse body, on top of which two perpendicular objectives are mounted in iSPIM configuration [25]
Summary
A quantitative imaging-based analysis of the biochemical processes and protein interactions inside cells requires microscopy techniques that go beyond ensemble averaging and a static molecular view [1]. In WM, while technically simple, the entire sample is illuminated by the excitation beam, which creates a high background over the weak signal from the single molecules. At the same time, TIRF limits the detection of molecules to the basal membrane of the cells mounted on the coverslip. In HILO microscopy an oblique sheet of light is used to illuminate a section of the specimen, enabling excitation of fluorophores deeper inside cells [7]. Since the illumination is oblique with respect to the detection focal plane, the size of the lateral Field-of-View (FoV) of the produced images is limited. All these approaches are optimized for, and limited to, coverslip-based sample mounting
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