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13 Janelia Publications
Showing 11-13 of 13 resultsExtending three-dimensional (3D) single-molecule localization microscopy away from the coverslip and into thicker specimens will greatly broaden its biological utility. However, because of the limitations of both conventional imaging modalities and conventional labeling techniques, it is a challenge to localize molecules in three dimensions with high precision in such samples while simultaneously achieving the labeling densities required for high resolution of densely crowded structures. Here we combined lattice light-sheet microscopy with newly developed, freely diffusing, cell-permeable chemical probes with targeted affinity for DNA, intracellular membranes or the plasma membrane. We used this combination to perform high-localization precision, ultrahigh-labeling density, multicolor localization microscopy in samples up to 20 μm thick, including dividing cells and the neuromast organ of a zebrafish embryo. We also demonstrate super-resolution correlative imaging with protein-specific photoactivable fluorophores, providing a mutually compatible, single-platform alternative to correlative light-electron microscopy over large volumes.
Fluorogenic molecules are important tools for biological and biochemical research. The majority of fluorogenic compounds have a simple input-output relationship, where a single chemical input yields a fluorescent output. Development of new systems where multiple inputs converge to yield an optical signal could refine and extend fluorogenic compounds by allowing greater spatiotemporal control over the fluorescent signal. Here, we introduce a new red-shifted fluorescein derivative, Virginia Orange, as an exceptional scaffold for single- and dual-input fluorogenic molecules. Unlike fluorescein, installation of a single masking group on Virginia Orange is sufficient to fully suppress fluorescence, allowing preparation of fluorogenic enzyme substrates with rapid, single-hit kinetics. Virginia Orange can also be masked with two independent moieties; both of these masking groups must be removed to induce fluorescence. This allows facile construction of multi-input fluorogenic probes for sophisticated sensing regimes and genetic targeting of latent fluorophores to specific cellular populations.
The structure of axonal arbors controls how signals from individual neurons are routed within the mammalian brain. However, the arbors of very few long-range projection neurons have been reconstructed in their entirety, as axons with diameters as small as 100 nm arborize in target regions dispersed over many millimeters of tissue. We introduce a platform for high-resolution, three-dimensional fluorescence imaging of complete tissue volumes that enables the visualization and reconstruction of long-range axonal arbors. This platform relies on a high-speed two-photon microscope integrated with a tissue vibratome and a suite of computational tools for large-scale image data. We demonstrate the power of this approach by reconstructing the axonal arbors of multiple neurons in the motor cortex across a single mouse brain.