Rebecca Medda

Direct observation of the nanoscale dynamics of membrane lipids in a living cell

Cholesterol-mediated lipid interactions are thought to have a functional role in many membrane-associated processes such as signalling events. Although several experiments indicate their existence, lipid nanodomains (‘rafts’) remain controversial owing to the lack of suitable detection techniques in living cells. The controversy is reflected in their putative size of 5–200 nm, spanning the range between the extent of a protein complex and the resolution limit of optical microscopy. Here we demonstrate the ability of stimulated emission depletion (STED) far-field fluorescence nanoscopy to detect single diffusing (lipid) molecules in nanosized areas in the plasma membrane of living cells. Tuning of the probed area to spot sizes ∼70-fold below the diffraction barrier reveals that unlike phosphoglycerolipids, sphingolipids and glycosylphosphatidylinositol-anchored proteins are transiently (∼10–20 ms) trapped in cholesterol-mediated molecular complexes dwelling within <20-nm diameter areas. The non-invasive optical recording of molecular time traces and fluctuation data in tunable nanoscale domains is a powerful new approach to study the dynamics of biomolecules in living cells.

Fluorescence nanoscopy by ground-state depletion and single-molecule return.

We introduce far-field fluorescence nanoscopy with ordinary fluorophores based on switching the majority of them to a metastable dark state, such as the triplet, and calculating the position of those left or those that spontaneously returned to the ground state. Continuous widefield illumination by a single laser and a continuously operating camera yielded dual-color images of rhodamine- and fluorescent protein–labeled (living) samples, proving a simple yet powerful super-resolution approach.

STED microscopy with continuous wave beams

We report stimulated emission depletion (STED) fluorescence microscopy with continuous wave (CW) laser beams. Lateral fluorescence confinement from the scanning focal spot delivered a resolution of 29–60 nm in the focal plane, corresponding to a 5–8-fold improvement over the diffraction barrier. Axial spot confinement increased the axial resolution by 3.5-fold. We observed three-dimensional (3D) subdiffraction resolution in 3D image stacks. Viable for fluorophores with low triplet yield, the use of CW light sources greatly simplifies the implementation of this concept of far-field fluorescence nanoscopy.

Nanoscale resolution in GFP-based microscopy

We report attainment of subdiffraction resolution using stimulated emission depletion (STED) microscopy with GFP-labeled samples. The ∼70 nm lateral resolution attained in this study is demonstrated by imaging GFP-labeled viruses and the endoplasmic reticulum (ER) of a mammalian cell. Our results mark the advent of nanoscale biological microscopy with genetically encoded markers.

Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters

We demonstrate nanoscale resolution in far-field fluorescence microscopy using reversible photoswitching and localization of individual fluorophores at comparatively fast recording speeds and from the interior of intact cells. These advancements have become possible by asynchronously recording the photon bursts of individual molecular switching cycles. We present images from the microtubular network of an intact mammalian cell with a resolution of 40 nm.

A compact STED microscope providing 3D nanoscale resolution

The advent of supercontinuum laser sources has enabled the implementation of compact and tunable stimulated emission depletion fluorescence microscopes for imaging far below the diffraction barrier. Here we report on an enhanced version of this approach displaying an all‐physics based resolution down to (19 ± 3) nm in the focal plane. Alternatively, this single objective lens system can be configured for 3D imaging with resolution down to 45 × 45 × 108 nm in a cell. The obtained results can be further improved by mathematical restoration algorithms. The far‐field optical nanoscale resolution is attained in a variety of biological samples featuring strong variations in the local density of features.

Multicolor Far-Field Fluorescence Nanoscopy through Isolated Detection of Distinct Molecular Species

By combining the photoswitching and localization of individual fluorophores with spectroscopy on the single molecule level, we demonstrate simultaneous multicolor imaging with low crosstalk and down to 15 nm spatial resolution using only two detection color channels. The applicability of the method to biological specimens is demonstrated on mammalian cells. The combination of far-field fluorescence nanoscopy with the recording of a single switchable molecular species at a time opens up a new class of functional imaging techniques.

Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength.

Current far-field fluorescence nanoscopes provide subdiffraction resolution by exploiting a mechanism of fluorescence inhibition. This mechanism is implemented such that features closer than the diffraction limit emit separately when simultaneously exposed to excitation light. A basic mechanism for such transient fluorescence inhibition is the depletion of the fluorophore ground state by transferring it (via a triplet) in a dark state, a mechanism which is workable in most standard dyes. Here we show that microscopy based on ground state depletion followed by individual molecule return (GSDIM) can effectively provide multicolor diffraction-unlimited resolution imaging of immunolabeled fixed and SNAP-tag labeled living cells. Implemented with standard labeling techniques, GSDIM is demonstrated to separate up to four different conventional fluorophores using just two detection channels and a single laser line. The method can be expanded to even more colors by choosing optimized dichroic mirrors and selecting marker molecules with negligible inhomogeneous emission broadening.

Dual‐Color STED Microscopy at 30‐nm Focal‐Plane Resolution

Owing to its sensitivity and noninvasiveness, far-field fluo-rescence microscopy would be almost ideal for biologicalimaging if the resolution of its established variants were notlimitedbydiffractiontoDr l=ð2n sinaÞ,withldenotingthewavelength of light, n the index of refraction, and a theaperture angle of the objective lens.

Fast STED microscopy with continuous wave fiber lasers.

We report on fast beam-scanning stimulated-emission-depletion (STED) microscopy in the visible range using for resolution enhancement compact, low cost and turn-key continuous wave (CW) fiber lasers emitting at 592 nm. Spatial resolutions of 35 to 65 nm in the focal plane are shown for various samples including fluorescent nanoparticles, immuno-stained cells with a non-exhaustive selection of 5 commonly used organic fluorescent markers, and living cells expressing the yellow fluorescent protein Citrine. The potential of the straightforward combination of CW-STED and fast beam scanning is illustrated in a movie of the endoplasmic reticulum (ER) of a living cell, composed of 100 frames (6 microm x 12 microm), each of them acquired in a time shorter than 0.2 s.