3D d STORM Imaging of Fixed Brain Tissue.

Central nervous system tissue contains a high density of synapses each composed of an intricate molecular machinery mediating precise transmission of information. Deciphering the molecular nanostructure of pre- and postsynaptic specializations within such a complex tissue architecture poses a particular challenge for light microscopy. Here, we describe two approaches suitable to examine the molecular nanostructure of synapses at 20-30 nm lateral and 50-70 nm axial resolution within an area of 500 μm × 500 μm and a depth of 0.

Kar1 binding to Sfi1 C-terminal regions anchors the SPB bridge to the nuclear envelope.

The yeast spindle pole body (SPB) is the functional equivalent of the mammalian centrosome. The half bridge is a SPB substructure on the nuclear envelope (NE), playing a key role in SPB duplication. Its cytoplasmic components are the membrane-anchored Kar1, the yeast centrin Cdc31, and the Cdc31-binding protein Sfi1.

Integrated and correlative high-throughput and super-resolution microscopy.

We have developed a method to perform microscopic temporal and spacial multi-scale experiments by imaging cellular phenotypes of interest on complementary fluorescence microscopy systems. In a low-resolution fast data acquisition screen for phenotypic cellular responses induced by small interfering RNA (siRNA), cells in spots of siRNA cell arrays showing characteristic alterations have been selected automatically by feature space analysis. These objects were imaged on a second super-resolution dSTORM microscope (direct stochastic optical reconstruction microscopy).

Single-molecule super-resolution imaging by tryptophan-quenching-induced photoswitching of phalloidin-fluorophore conjugates.

Photophysical properties of any fluorophore are governed by the chemical nanoenvironment. In the context of imaging biological samples, this translates to different photophysical properties for different labels and probes. Here, we demonstrate that the nanoenvironment of fluorophores within a probe can be advantageously used to induce particular properties such as light-induced photoswitching.

Correlative light microscopy for high-content screening.

High-throughput microscopy is an effective tool for rapidly collecting data on a large scale. However, high throughput comes at the cost of low spatial resolution. Here we introduce correlative light microscopy by combining fast automated widefield imaging, confocal microscopy and super-resolution microscopy.

Photoswitchable fluorophores for single-molecule localization microscopy.

Over the past decade, fluorescence microscopy has been revolutionized by the development of novel techniques that allow near-molecular resolution. Many such methods-collectively referred to as "single-molecule localization microscopy" (SMLM)-are based upon the repeated imaging of sparse stochastic subsets of the fluorophores in a sample. Active fluorophores are localized by finding the centers of their point spread functions, and a super-resolution image is constructed.

Hybridization and reaction-based fluorogenic nucleic acid probes.

We developed fluorogenic probes, which are photoactivated in the presence of specific nucleic acid templates with the release of fluorescent dyes. This templated reaction can be used to target specific nucleic acids in complex mixtures. We further demonstrate that this reaction can be monitored by single-molecule fluorescence imaging.

Three-dimensional, tomographic super-resolution fluorescence imaging of serially sectioned thick samples.

Three-dimensional fluorescence imaging of thick tissue samples with near-molecular resolution remains a fundamental challenge in the life sciences. To tackle this, we developed tomoSTORM, an approach combining single-molecule localization-based super-resolution microscopy with array tomography of structurally intact brain tissue. Consecutive sections organized in a ribbon were serially imaged with a lateral resolution of 28 nm and an axial resolution of 40 nm in tissue volumes of up to 50 µm×50 µm×2.

Chemically induced photoswitching of fluorescent probes--a general concept for super-resolution microscopy.

We review fluorescent probes that can be photoswitched or photoactivated and are suited for single-molecule localization based super-resolution microscopy. We exploit the underlying photochemical mechanisms that allow photoswitching of many synthetic organic fluorophores in the presence of reducing agents, and study the impact of these on the photoswitching properties of various photoactivatable or photoconvertible fluorescent proteins. We have identified mEos2 as a fluorescent protein that exhibits reversible photoswitching under various imaging buffer conditions and present strategies to characterize reversible photoswitching.