Organic (opto)electronic materials have received considerable attention due to their applications in thin-film-transistors, light-emitting diodes, solar cells, sensors, photorefractive devices, and many others. The technological promises include low cost of these materials and the possibility of their room-temperature deposition from solution on large-area and/or flexible substrates. The article reviews the current understanding of the physical mechanisms that determine the (opto)electronic properties of high-performance organic materials. The focus of the review is on photoinduced processes and on electronic properties important for optoelectronic applications relying on charge carrier photogeneration. Additionally, it highlights the capabilities of various experimental techniques for characterization of these materials, summarizes top-of-the-line device performance, and outlines recent trends in the further development of the field. The properties of materials based both on small molecules and on conjug...

Organic Optoelectronic Materials: Mechanisms and Applications

Fluorescence nanoscopy uniquely combines minimally invasive optical access to the internal nanoscale structure and dynamics of cells and tissues with molecular detection specificity. While the basic physical principles of 'super-resolution' imaging were discovered in the 1990s, with initial experimental demonstrations following in 2000, the broad application of super-resolution imaging to address cell-biological questions has only more recently emerged. Nanoscopy approaches have begun to facilitate discoveries in cell biology and to add new knowledge. One current direction for method improvement is the ambition to quantitatively account for each molecule under investigation and assess true molecular colocalization patterns via multi-colour analyses. In pursuing this goal, the labelling of individual molecules to enable their visualization has emerged as a central challenge. Extending nanoscale imaging into (sliced) tissue and whole-animal contexts is a further goal. In this Review we describe the successes to date and discuss current obstacles and possibilities for further development.

Fluorescence nanoscopy in cell biology

Super-resolution microscopy (SRM) bypasses the diffraction limit, a physical barrier that restricts the optical resolution to roughly 250 nm and was previously thought to be impenetrable. SRM techniques allow the visualization of subcellular organization with unprecedented detail, but also confront biologists with the challenge of selecting the best-suited approach for their particular research question. Here, we provide guidance on how to use SRM techniques advantageously for investigating cellular structures and dynamics to promote new discoveries.

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Super-resolution microscopy demystified

This Perspective introduces the development and use of adaptive optics in correcting aberrations in deep optical imaging applications.

Adaptive optical fluorescence microscopy

Single-molecule super-resolution fluorescence microscopy and single-particle tracking are two imaging modalities that illuminate the properties of cells and materials on spatial scales down to tens of nanometers or with dynamical information about nanoscale particle motion in the millisecond range, respectively. These methods generally use wide-field microscopes and two-dimensional camera detectors to localize molecules to much higher precision than the diffraction limit. Given the limited total photons available from each single-molecule label, both modalities require careful mathematical analysis and image processing. Much more information can be obtained about the system under study by extending to three-dimensional (3D) single-molecule localization: without this capability, visualization of structures or motions extending in the axial direction can easily be missed or confused, compromising scientific understanding. A variety of methods for obtaining both 3D super-resolution images and 3D tracking inf...

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Three-Dimensional Localization of Single Molecules for Super-Resolution Imaging and Single-Particle Tracking

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.

Multicolour localization microscopy by point-spread-function engineering

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.

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

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 orientations 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.

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

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.

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

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.

Maurice Y. Lee

Papers

Multicolour localization microscopy by point-spread-function engineering

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

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

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

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