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In vivo labeling method using a genetic construct for nanoscale resolution microscopy.

ABSTRACT: We demonstrate beam scanning-stimulated emission depletion microscopy with in vivo labeled cells. A red emitting fluorescent dye is introduced into membrane protein fused to a multifunctional reporter protein (HaloTag, Promega, Madison, WI) in live cells. This approach allows superresolution stimulated emission depletion imaging without the limitations of immunofluorescence-based staining.

SUBMITTER: Schroder J

PROVIDER: S-EPMC2710050 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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In vivo labeling method using a genetic construct for nanoscale resolution microscopy.

Schröder Jan J Benink Hélène H Dyba Marcus M Los Georgyi V GV

Biophysical journal 20090101 1

We demonstrate beam scanning-stimulated emission depletion microscopy with in vivo labeled cells. A red emitting fluorescent dye is introduced into membrane protein fused to a multifunctional reporter protein (HaloTag, Promega, Madison, WI) in live cells. This approach allows superresolution stimulated emission depletion imaging without the limitations of immunofluorescence-based staining. ...[more]

PMID: 19134467

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Project description:Super-resolution (SR) microscopy has been used to observe structural details beyond the diffraction limit of ∼250 nm in a variety of biological and materials systems. By combining this imaging technique with both computer-vision algorithms and topological methods, we reveal and quantify the nanoscale morphology of the primary cilium, a tiny tubular cellular structure (∼2-6 μm long and 200-300 nm in diameter). The cilium in mammalian cells protrudes out of the plasma membrane and is important in many signaling processes related to cellular differentiation and disease. After tagging individual ciliary transmembrane proteins, specifically Smoothened, with single fluorescent labels in fixed cells, we use three-dimensional (3D) single-molecule SR microscopy to determine their positions with a precision of 10-25 nm. We gain a dense, pointillistic reconstruction of the surfaces of many cilia, revealing large heterogeneity in membrane shape. A Poisson surface reconstruction algorithm generates a fine surface mesh, allowing us to characterize the presence of deformations by quantifying the surface curvature. Upon impairment of intracellular cargo transport machinery by genetic knockout or small-molecule treatment of cells, our quantitative curvature analysis shows significant morphological differences not visible by conventional fluorescence microscopy techniques. Furthermore, using a complementary SR technique, two-color, two-dimensional stimulated emission depletion microscopy, we find that the cytoskeleton in the cilium, the axoneme, also exhibits abnormal morphology in the mutant cells, similar to our 3D results on the Smoothened-measured ciliary surface. Our work combines 3D SR microscopy and computational tools to quantitatively characterize morphological changes of the primary cilium under different treatments and uses stimulated emission depletion to discover correlated changes in the underlying structure. This approach can be useful for studying other biological or nanoscale structures of interest.

Dataset Information

In vivo labeling method using a genetic construct for nanoscale resolution microscopy.

Publications

In vivo labeling method using a genetic construct for nanoscale resolution microscopy.

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