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3D single-molecule super-resolution microscopy with a tilted light sheet.

Tilted light sheet microscopy with 3D point spread functions (TILT3D) combines a novel, tilted light sheet illumination strategy with long axial range point spread functions (PSFs) for low-background, 3D super-localization of single molecules as well as 3D super-resolution imaging in thick cells. Because the axial positions of the single emitters are encoded in the shape of each single-molecule image rather than in the position or thickness of the light sheet, the light sheet need not be extremely thin. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The result is simple and flexible 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validate TILT3D for 3D super-resolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed tetrapod PSFs for fiducial bead tracking and live axial drift correction.

Tilted Light Sheet Microscopy with 3D Point Spread Functions for Single-Molecule Super-Resolution Imaging in Mammalian Cells.

To obtain a complete picture of subcellular nanostructures, cells must be imaged with high resolution in all three dimensions (3D). Here, we present tilted light sheet microscopy with 3D point spread functions (TILT3D), an imaging platform that combines a novel, tilted light sheet illumination strategy with engineered long axial range point spread functions (PSFs) for low-background, 3D super localization of single molecules as well as 3D super-resolution imaging in thick cells. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The axial positions of the single molecules are encoded in the shape of the PSF rather than in the position or thickness of the light sheet, and the light sheet can therefore be formed using simple optics. The result is flexible and user-friendly 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validated TILT3D for 3D super-resolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed Tetrapod PSF for fiducial bead tracking and live axial drift correction. We envision TILT3D to become an important tool not only for 3D super-resolution imaging, but also for live whole-cell single-particle and single-molecule tracking.

Observation of live chromatin dynamics in cells via 3D localization microscopy using Tetrapod point spread functions.

We report the observation of chromatin dynamics in living budding yeast (Saccharomyces cerevisiae) cells, in three-dimensions (3D). Using dual color localization microscopy and employing a Tetrapod point spread function, we analyze the spatio-temporal dynamics of two fluorescently labeled DNA loci surrounding the GAL locus. From the measured trajectories, we obtain different dynamical characteristics in terms of inter-loci distance and temporal variance; when the GAL locus is activated, the 3D inter-loci distance and temporal variance increase compared to the inactive state. These changes are visible in spite of the large thermally- and biologically-driven heterogeneity in the relative motion of the two loci. Our observations are consistent with current euchromatin vs. heterochromatin models.

Enhanced DNA imaging using super-resolution microscopy and simultaneous single-molecule orientation measurements.

Single-molecule orientation measurements provide unparalleled insight into a multitude of biological and polymeric systems. We report a simple, high-throughput technique for measuring the azimuthal orientation and rotational dynamics of single fluorescent molecules, which is compatible with localization microscopy. Our method involves modulating the polarization of an excitation laser, and analyzing the corresponding intensities emitted by single dye molecules and their modulation amplitudes. To demonstrate our approach, we use intercalating and groove-binding dyes to obtain super-resolved images of stretched DNA strands through binding-induced turn-on of fluorescence. By combining our image data with thousands of dye molecule orientation measurements, we develop a means of probing the structure of individual DNA strands, while also characterizing dye-DNA interactions. This approach may hold promise as a method for monitoring DNA conformation changes resulting from DNA-binding proteins.

Multicolour localization microscopy by point-spread-function engineering.

Super-resolution microscopy has revolutionized cellular imaging in recent years1-4. Methods relying on sequential localization of single point emitters enable spatial tracking at ~10-40 nm resolution. Moreover, tracking and imaging in three dimensions is made possible by various techniques, including point-spread-function (PSF) engineering5-9 -namely, encoding the axial (z) position of a point source in the shape that it creates in the image plane. However, a remaining challenge for localization-microscopy is efficient multicolour imaging - a task of the utmost importance for contextualizing biological data. Normally, multicolour imaging requires sequential imaging10, 11, multiple cameras12, or segmented dedicated fields of view13, 14. Here, we demonstrate an alternate strategy, the encoding of spectral information (colour), in addition to 3D position, directly in the image. By exploiting chromatic dispersion, we design a new class of optical phase masks that simultaneously yield controllably different PSFs for different wavelengths, enabling simultaneous multicolour tracking or super-resolution imaging in a single optical path.

Correcting field-dependent aberrations with nanoscale accuracy in three-dimensional single-molecule localization microscopy.

The localization of single fluorescent molecules enables the imaging of molecular structure and dynamics with subdiffraction precision and can be extended to three dimensions using point spread function (PSF) engineering. However, the nanoscale accuracy of localization throughout a 3D single-molecule microscope's field of view has not yet been rigorously examined. By using regularly spaced subdiffraction apertures filled with fluorescent dyes, we reveal field-dependent aberrations as large as 50-100 nm and show that they can be corrected to less than 25 nm over an extended 3D focal volume. We demonstrate the applicability of this technique for two engineered PSFs, the double-helix PSF and the astigmatic PSF. We expect these results to be broadly applicable to 3D single-molecule tracking and superresolution methods demanding high accuracy.