Rainer Heintzmann

A guide to super-resolution fluorescence microscopy

For centuries, cell biology has been based on light microscopy and at the same time been limited by its optical resolution. However, several new technologies have been developed recently that bypass this limit. These new super-resolution technologies are either based on tailored illumination, nonlinear fluorophore responses, or the precise localization of single molecules. Overall, these new approaches have created unprecedented new possibilities to investigate the structure and function of cells.

Quantum dot ligands provide new insights into erbB/HER receptor–mediated signal transduction

The erbB/HER family of transmembrane receptor tyrosine kinases (RTKs) mediate cellular responses to epidermal growth factor (EGF) and related ligands. We have imaged the early stages of RTK-dependent signaling in living cells using: (i) stable expression of erbB1/2/3 fused with visible fluorescent proteins (VFPs), (ii) fluorescent quantum dots (QDs) bearing epidermal growth factor (EGF-QD) and (iii) continuous confocal laser scanning microscopy and flow cytometry. Here we demonstrate that EGF-QDs are highly specific and potent in the binding and activation of the EGF receptor (erbB1), being rapidly internalized into endosomes that exhibit active trafficking and extensive fusion. EGF-QDs bound to erbB1 expressed on filopodia revealed a previously unreported mechanism of retrograde transport to the cell body. When erbB2-monomeric yellow fluorescent protein (mYFP) or erbB3-monomeric Citrine (mCitrine) were coexpressed with erbB1, the rates and extent of endocytosis of EGF-QD and the RTK-VFP demonstrated that erbB2 but not erbB3 heterodimerizes with erbB1 after EGF stimulation, thereby modulating EGF-induced signaling. QD-ligands will find widespread use in basic research and biotechnological developments.

Saturated patterned excitation microscopy—a concept for optical resolution improvement

The resolution of optical microscopy is limited by the numerical aperture and the wavelength of light. Many strategies for improving resolution such as 4Pi and I5M have focused on an increase of the numerical aperture. Other approaches have based resolution improvement in fluorescence microscopy on the establishment of a nonlinear relationship between local excitation light intensity in the sample and in the emitted light. However, despite their innovative character, current techniques such as stimulated emission depletion (STED) and ground-state depletion (GSD) microscopy require complex optical configurations and instrumentation to narrow the point-spread function. We develop the theory of nonlinear patterned excitation microscopy for achieving a substantial improvement in resolution by deliberate saturation of the fluorophore excited state. The postacquisition manipulation of the acquired data is computationally more complex than in STED or GSD, but the experimental requirements are simple. Simulations comparing saturated patterned excitation microscopy with linear patterned excitation microscopy (also referred to in the literature as structured illumination or harmonic excitation light microscopy) and ordinary widefield microscopy are presented and discussed. The effects of photon noise are included in the simulations.

Bayesian localization microscopy reveals nanoscale podosome dynamics

We describe a localization microscopy analysis method that is able to extract results in live cells using standard fluorescent proteins and xenon arc lamp illumination. Our Bayesian analysis of the blinking and bleaching (3B analysis) method models the entire dataset simultaneously as being generated by a number of fluorophores that may or may not be emitting light at any given time. The resulting technique allows many overlapping fluorophores in each frame and unifies the analysis of the localization from blinking and bleaching events. By modeling the entire dataset, we were able to use each reappearance of a fluorophore to improve the localization accuracy. The high performance of this technique allowed us to reveal the nanoscale dynamics of podosome formation and dissociation throughout an entire cell with a resolution of 50 nm on a 4-s timescale.

Silencing of EphA3 through a cis interaction with ephrinA5

EphAs and ephrinAs are expressed in multiple areas of the developing brain in overlapping countergradients, notably in the retina and tectum. Here they are involved in targeting retinal axons to their correct topographic position in the tectum. We have used truncated versions of EphA3, single–amino acid point mutants of ephrinA5 and fluorescence resonance energy transfer technology to uncover a cis interaction between EphA3 and ephrinA5 that is independent of the established ligand-binding domain of EphA3. This cis interaction abolishes the induction of tyrosine phosphorylation of EphA3 and results in a loss of sensitivity of retinal axons to ephrinAs in trans. Our data suggest that formation of this complex transforms the uniform expression of EphAs in the nasal part of the retina into a gradient of functional EphAs and has a key role in controlling retinotectal mapping.

RNAi knockdown of hPrp31 leads to an accumulation of U4/U6 di-snRNPs in Cajal bodies

Cajal bodies (CBs) are subnuclear organelles of animal and plant cells. A role of CBs in the assembly and maturation of small nuclear ribonucleoproteins (snRNP) has been proposed but is poorly understood. Here we have addressed the question where U4/U6.U5 tri‐snRNP assembly occurs in the nucleus. The U4/U6.U5 tri‐snRNP is a central unit of the spliceosome and must be re‐formed from its components after each round of splicing. By combining RNAi and biochemical methods, we demonstrate that, after knockdown of the U4/U6‐specific hPrp31 (61 K) or the U5‐specific hPrp6 (102 K) protein in HeLa cells, tri‐snRNP formation is inhibited and stable U5 mono‐snRNPs and U4/U6 di‐snRNPs containing U4/U6 proteins and the U4/U6 recycling factor p110 accumulate. Thus, hPrp31 and hPrp6 form an essential connection between the U4/U6 and U5 snRNPs in vivo. Using fluorescence microscopy, we show that, in the absence of either hPrp31 or hPrp6, U4/U6 di‐snRNPs as well as p110 accumulate in Cajal bodies. In contrast, U5 snRNPs largely remain in nucleoplasmic speckles. Our data support the idea that CBs may play a role in tri‐snRNP recycling.

Polycomb group protein complexes exchange rapidly in living Drosophila

Fluorescence recovery after photobleaching (FRAP) microscopy was used to determine the kinetic properties of Polycomb group (PcG) proteins in whole living Drosophila organisms (embryos) and tissues (wing imaginal discs and salivary glands). PcG genes are essential genes in higher eukaryotes responsible for the maintenance of the spatially distinct repression of developmentally important regulators such as the homeotic genes. Their absence, as well as overexpression, causes transformations in the axial organization of the body. Although protein complexes have been isolated in vitro, little is known about their stability or exact mechanism of repression in vivo. We determined the translational diffusion constants of PcG proteins, dissociation constants and residence times for complexes in vivo at different developmental stages. In polytene nuclei, the rate constants suggest heterogeneity of the complexes. Computer simulations with new models for spatially distributed protein complexes were performed in systems showing both diffusion and binding equilibria, and the results compared with our experimental data. We were able to determine forward and reverse rate constants for complex formation. Complexes exchanged within a period of 1-10 minutes, more than an order of magnitude faster than the cell cycle time, ruling out models of repression in which access of transcription activators to the chromatin is limited and demonstrating that long-term repression primarily reflects mass-action chemical equilibria.

The 2015 super-resolution microscopy roadmap.

Far-field optical microscopy using focused light is an important tool in a number of scientific disciplines including chemical, (bio) physical and biomedical research, particularly with respect to ...

Superresolution by image scanning microscopy using pixel reassignment

The effect of detector array size on resolution and signal collection efficiency of image scanning microscopy based on pixel reassignment is studied. It is shown how the method can also be employed if there is a Stokes shift in fluorescence emission wavelength. With no Stokes shift, the width of the point spread function can be sharpened by a factor of 1.53, and its peak intensity increased by a factor of 1.84.

A dual path programmable array microscope (PAM): simultaneous acquisition of conjugate and non-conjugate images

A programmable array microscope (PAM) incorporates a spatial light modulator (SLM) placed in the primary image plane of a widefield microscope, where it is used to define patterns of illumination and/or detection. We describe the characteristics of a special type of PAM collecting two images simultaneously. The conjugate image (Ic) is formed by light originating from the object plane and returning along the optical path of the illumination light. The non‐conjugate image (Inc) receives light from only those regions of the SLM that are not used for illuminating the sample. The dual‐signal PAM provides much more time‐efficient excitation than the confocal laser scanning microscope (CLSM) and greater utilization of the available emission light. It has superior noise characteristics in comparison to single‐sided instruments. The axial responses of the system under a variety of conditions were measured and the behaviour of the novel Inc image characterized. As in systems in which only Ic images are collected (Nipkow‐disc microscopes, and previously characterized PAMs), the axial response to thin fluorescent films showed a sharpening of the axial response as the unit cell of the repetitive patterns decreased in size.