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Resolving the internal morphology of core-shell microgels with super-resolution fluorescence microscopy.

ABSTRACT: We investigate the internal morphology of smart core-shell microgels by super-resolution fluorescence microscopy exploiting a combination of 3D single molecule localization and structured illumination microscopy utilizing freely diffusing fluorescent dyes. This approach does not require any direct chemical labeling and does not perturb the network structure of these colloidal gels. Hence, it allows us to study the morphology of the particles with very high precision. We found that the structure of the core-forming seed particles is drastically changed by the second synthesis step necessary for making the shell, resulting in a core region with highly increased dye localization density. The present work shows that super-resolution microscopy has great potential with respect to the study of soft colloidal systems.

SUBMITTER: Otto P

PROVIDER: S-EPMC9416983 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Resolving the internal morphology of core-shell microgels with super-resolution fluorescence microscopy.

Otto Pia P Bergmann Stephan S Sandmeyer Alice A Dirksen Maxim M Wrede Oliver O Hellweg Thomas T Huser Thomas T

Nanoscale advances 20191128 1

We investigate the internal morphology of smart core-shell microgels by super-resolution fluorescence microscopy exploiting a combination of 3D single molecule localization and structured illumination microscopy utilizing freely diffusing fluorescent dyes. This approach does not require any direct chemical labeling and does not perturb the network structure of these colloidal gels. Hence, it allows us to study the morphology of the particles with very high precision. We found that the structure ...[more]

PMID: 36134006

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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

Resolving the internal morphology of core-shell microgels with super-resolution fluorescence microscopy.

Publications

Resolving the internal morphology of core-shell microgels with super-resolution fluorescence microscopy.

Similar Datasets

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