Claudia Geisler

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.

Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores.

We demonstrate three-dimensional (3D) super-resolution imaging of stochastically switched fluorophores distributed across whole cells. By evaluating the higher moments of the diffraction spot provided by a 4Pi detection scheme, single markers can be simultaneously localized with <10 nm precision in three dimensions in a layer of 650 nm thickness at an arbitrarily selected depth in the sample. By splitting the fluorescence light into orthogonal polarization states, our 4Pi setup also facilitates the 3D nanoscopy of multiple fluorophores. Offering a combination of multicolor recording, nanoscale resolution and extended axial depth, our method substantially advances the noninvasive 3D imaging of cells and of other transparent materials.

Aptamers as potential tools for super-resolution microscopy

(a) The TfnR aptamer c2, the EGFR aptamer E07 and their respective control aptamers (random sequences) were incubated at 37°C with human A431 cells as described in Supplementary Methods. Similarly, HeLa cells stably transfected with a human PSMA construct were incubated with the PSMA A9 aptamer or its random control. The pairs of images (control and aptamer) are equally scaled to allow a direct visual comparison. The insets in the control images correspond to the same images, scaled to a level where autofluorescence can be visualized. Scale bar, 10 μm. (b) Colocalization of the different aptamers with endosomal labels. We co-incubated the cells (same as above) with aptamers against TfnR (c2) or PSMA (A9) and Alexa488-transferrin (Invitrogen), since the latter constitutes an ideal marker for early endosomes.

Isotropic 3D Nanoscopy based on single emitter switching

We propose and analyze a method for isotropic resolution in far-field fluorescence nanoscopy based on switching and mathematically localizing individual emitters. Under typical imaging conditions, the coherent detection of fluorescence light through two opposing high angle lenses strongly improves the 3D-resolution down to 5-10nm in all directions. Furthermore, we give a detailed analysis of the resolution of this and other single molecule switching based approaches using the Fisher information matrix.We verify the results by Monte-Carlo simulations of the imaging process and by applying a simple maximum-likelihood estimator for position determination.

Drift estimation for single marker switching based imaging schemes.

In recent years, the diffraction barrier in fluorescence imaging has been broken and optical nanoscopes now routinely image with resolutions of down to 20 nm, an improvement of more than 10 fold. Because this allows imaging much smaller features and because all super-resolution approaches trade off speed for spatial resolution, mechanical instabilities of the microscopes become a limiting factor. Here, we propose a fully data-driven statistical registration method for drift detection and drift correction for single marker switching (SMS) imaging schemes, including a guideline for parameter choice and quality checks of the drift analysis. The necessary assumptions about the drift are minimal, allowing a model-free approach, but more specific models can easily be integrated. We determine the resulting performance on standard SMS measurements and show that the drift determination can be routinely brought to the range of precision achievable by fiducial marker-tracking methods.

Nanoscale separation of molecular species based on their rotational mobility

We combine far-field fluorescence nanoscopy through serialized recording of switchable emitters with polarization-sensitive fluorescence detection. In addition to imaging with nanoscale spatial resolution, this technique allows determination of the fluorescence anisotropy of each detected dipole emitter and thus an estimate of its rotational mobility. Sub-populations of fluorescent markers can thus be separated based on their interaction with the sample. We applied this new functional nanoscopy to imaging of living mammalian cells.

Dominant negative SNARE peptides stabilize the fusion pore in a narrow, release-unproductive state

Key support for vesicle-based release of gliotransmitters comes from studies of transgenic mice with astrocyte-specific expression of a dominant-negative domain of synaptobrevin 2 protein (dnSNARE). To determine how this peptide affects exocytosis, we used super-resolution stimulated emission depletion microscopy and structured illumination microscopy to study the anatomy of single vesicles in astrocytes. Smaller vesicles contained amino acid and peptidergic transmitters and larger vesicles contained ATP. Discrete increases in membrane capacitance, indicating single-vesicle fusion, revealed that astrocyte stimulation increases the frequency of predominantly transient fusion events in smaller vesicles, whereas larger vesicles transitioned to full fusion. To determine whether this reflects a lower density of SNARE proteins in larger vesicles, we treated astrocytes with botulinum neurotoxins D and E, which reduced exocytotic events of both vesicle types. dnSNARE peptide stabilized the fusion-pore diameter to narrow, release-unproductive diameters in both vesicle types, regardless of vesicle diameter.

Complement triggers relocation of Mortalin/GRP75 from mitochondria to the plasma membrane

Mortalin/GRP75 is a ubiquitously expressed mitochondrial chaperon that is overexpressed in cancer. Mortalin protects cells from complement-dependent cytotoxicity (CDC) and facilitates elimination of the complement C5b-9 complexes from the cell surface. We performed a nanoscopical study aimed at imaging the distribution of the C5b-9 complexes in the plasma membrane and the postulated relocation of mortalin from the mitochondria to the plasma membrane. To gain a resolution of 35nm, the locations of the C5b-9 complex and mortalin were imaged with a STED (Stimulated Emission Depletion) microscope at sub-diffraction resolution. Early changes in the spatial distribution of the C5b-9 on the cell surface are described. Juxtaposition of the labeled mortalin and C5b-9 at the plasma membrane region within minutes after complement attack is evident. Microscopical analysis of the distribution of mortalin in the vicinity of the mitochondria of complement-treated cells shows a more diffused pattern relative to control cells, proposing exit of mortalin from the mitochondria in response to complement-induced stress. In support, analysis of cytoplasmic mortalin by immunoblotting shows enhanced level of mortalin in the cytoplasm in complement-treated cells. Our data demonstrates that cells can sense complement activation at the plasma membrane and in response, swiftly send mortalin to this region in order to deactivate it.

Chromatin swelling drives neutrophil extracellular trap release

Neutrophilic granulocytes are able to release their own DNA as neutrophil extracellular traps (NETs) to capture and eliminate pathogens. DNA expulsion (NETosis) has also been documented for other cells and organisms, thus highlighting the evolutionary conservation of this process. Moreover, dysregulated NETosis has been implicated in many diseases, including cancer and inflammatory disorders. During NETosis, neutrophils undergo dynamic and dramatic alterations of their cellular as well as sub-cellular morphology whose biophysical basis is poorly understood. Here we investigate NETosis in real-time on the single-cell level using fluorescence and atomic force microscopy. Our results show that NETosis is highly organized into three distinct phases with a clear point of no return defined by chromatin status. Entropic chromatin swelling is the major physical driving force that causes cell morphology changes and the rupture of both nuclear envelope and plasma membrane. Through its material properties, chromatin thus directly orchestrates this complex biological process.Neutrophilic granulocytes release their own DNA (NETosis) as neutrophil extracellular traps to capture pathogens. Here, the authors use time-resolved fluorescence and atomic force microscopy and reveal that NETosis is highly organized into three distinct phases with a clear point of no return defined by chromatin status.

Modeling superresolution reflection microscopy via absorbance modulation (Conference Presentation)

Absorbance modulation enables lateral superresolution in optical lithography and transmission microscopy by generating a dynamic aperture within a photochromic absorbance-modulation layer (AML) coated on a substrate or a specimen. The absorbance-modulation is the property of photochromic molecules modulated between two states. The process is therefore solely controlled by far-field radiation at different wavelengths. The applicability of this concept to reflection microscopy has not been addressed so far, although reflection imaging exhibits the important ability to image a wide range of samples, transparent or opaque, dielectric or metallic. We will present a simulation model for absorbance-modulation imaging (AMI) in confocal reflection microscopy and it is shown that imaging well beyond the diffraction limit is feasible. Our model includes the imaging properties of confocal microscopy, reflections at the boundaries, the photochromic process and diffraction due to propagation through a subwavelength aperture. We derive an analytical design equation which estimates the dependence of the achievable resolution on relevant parameters, such as the AML properties and the applied light powers. This equation is very similar to the corresponding equation for STED (Stimulated emission depletion) microscopy and it is helpful for a fast design of the arrangement of optical setup and AML. As rapid scanning is relevant for a short imaging duration, we further derived an estimation for the pixel dwell time. We prove the validity of these equations by comparing the estimations with the complex numerical simulations. In addition, we show that a resolution enhancement down to 1/5 of the diffraction limit is possible.