Guided-deconvolution for correlative light and electron microscopy.

Correlative light and electron microscopy is a powerful tool to study the internal structure of cells. It combines the mutual benefit of correlating light (LM) and electron (EM) microscopy information. The EM images only contain contrast information. Therefore, some of the detailed structures cannot be specified from these images alone, especially when different cell organelle are contacted. However, the classical approach of overlaying LM onto EM images to assign functional to structural information is hampered by the large discrepancy in structural detail visible in the LM images. This paper aims at investigating an optimized approach which we call EM-guided deconvolution. This applies to living cells structures before fixation as well as previously fixed sample. It attempts to automatically assign fluorescence-labeled structures to structural details visible in the EM image to bridge the gaps in both resolution and specificity between the two imaging modes. We tested our approach on simulations, correlative data of multi-color beads and previously published data of biological samples.

Correlative super-resolution fluorescence and electron cryo-microscopy based on cryo-SOFI.

The combination of super-resolution fluorescence microscopy and electron microscopy at ambient temperatures has become an established technique and a broad variety of modalities are now available to the cell biology community. In contrast, correlative cryogenic super-resolution fluorescence and electron microscopy (super-resolution cryo-CLEM) is just emerging. Aside from technical challenges, one of the major issues is the risk of devitrification of the specimen caused by the laser intensities required for super-resolution imaging. Cryo-SOFI (cryogenic super-resolution optical fluctuation imaging) allows the reconstruction of super-resolution images at particularly low laser intensities. It is fully compatible with the standard sample preparation for cryogenic electron microscopy (cryo-EM) and fairly easy to implement in any standard cryogenic fluorescence microscope.

Structural Basis of Teneurin-Latrophilin Interaction in Repulsive Guidance of Migrating Neurons.

Teneurins are ancient metazoan cell adhesion receptors that control brain development and neuronal wiring in higher animals. The extracellular C terminus binds the adhesion GPCR Latrophilin, forming a trans-cellular complex with synaptogenic functions. However, Teneurins, Latrophilins, and FLRT proteins are also expressed during murine cortical cell migration at earlier developmental stages. Here, we present crystal structures of Teneurin-Latrophilin complexes that reveal how the lectin and olfactomedin domains of Latrophilin bind across a spiraling beta-barrel domain of Teneurin, the YD shell. We couple structure-based protein engineering to biophysical analysis, cell migration assays, and in utero electroporation experiments to probe the importance of the interaction in cortical neuron migration. We show that binding of Latrophilins to Teneurins and FLRTs directs the migration of neurons using a contact repulsion-dependent mechanism. The effect is observed with cell bodies and small neurites rather than their processes. The results exemplify how a structure-encoded synaptogenic protein complex is also used for repulsive cell guidance.

Cryo-SOFI enabling low-dose super-resolution correlative light and electron cryo-microscopy.

Correlative light and electron cryo-microscopy (cryo-CLEM) combines information from the specific labeling of fluorescence cryo-microscopy (cryo-FM) with the high resolution in environmental context of electron cryo-microscopy (cryo-EM). Exploiting super-resolution methods for cryo-FM is advantageous, as it enables the identification of rare events within the environmental background of cryo-EM at a sensitivity and resolution beyond that of conventional methods. However, due to the need for relatively high laser intensities, current super-resolution cryo-CLEM methods require cryo-protectants or support films which can severely reduce image quality in cryo-EM and are not compatible with many samples, such as mammalian cells. Here, we introduce cryogenic super-resolution optical fluctuation imaging (cryo-SOFI), a low-dose super-resolution imaging scheme based on the SOFI principle. As cryo-SOFI does not require special sample preparation, it is fully compatible with conventional cryo-EM specimens, and importantly, it does not affect the quality of cryo-EM imaging. By applying cryo-SOFI to a variety of biological application examples, we demonstrate resolutions up to ∼135 nm, an improvement of up to three times compared with conventional cryo-FM, while maintaining the specimen in a vitrified state for subsequent cryo-EM. Cryo-SOFI presents a general solution to the problem of specimen devitrification in super-resolution cryo-CLEM. It does not require a complex optical setup and can easily be implemented in any existing cryo-FM system.

The Impact of Sex Differences on Odor Identification and Facial Affect Recognition in Patients with Schizophrenia Spectrum Disorders.

BACKGROUND: Social interactive functions such as facial emotion recognition and smell identification have been shown to differ between women and men. However, little is known about how these differences are mirrored in patients with schizophrenia and how these abilities interact with each other and with other clinical variables in patients vs. healthy controls. METHODS: Standardized instruments were used to assess facial emotion recognition [Facially Expressed Emotion Labelling (FEEL)] and smell identification [University of Pennsylvania Smell Identification Test (UPSIT)] in 51 patients with schizophrenia spectrum disorders and 79 healthy controls; furthermore, working memory functions and clinical variables were assessed. RESULTS: In both the univariate and the multivariate results, illness showed a significant influence on UPSIT and FEEL. The inclusion of age and working memory in the MANOVA resulted in a differential effect with sex and working memory as remaining significant factors. Duration of illness was correlated with both emotion recognition and smell identification in men only, whereas immediate general psychopathology and negative symptoms were associated with emotion recognition only in women. CONCLUSION: Being affected by schizophrenia spectrum disorder impacts one's ability to correctly recognize facial affects and identify odors. Converging evidence suggests a link between the investigated basic and social cognitive abilities in patients with schizophrenia spectrum disorders with a strong contribution of working memory and differential effects of modulators in women vs. men.

A combined 3D-SIM/SMLM approach allows centriole proteins to be localized with a precision of ∼4-5 nm.

Centrioles are small barrel-shaped structures that form centrosomes and cilia [1]. Centrioles assemble around a central cartwheel comprising the Sas-6 and Ana2/STIL proteins. The amino termini of nine Sas-6 dimers form a central hub of ∼12 nm radius from which nine dimer spokes radiate, placing the Sas-6 carboxyl termini at the outer edge of the ∼60 nm radius cartwheel [2]. Several centriole proteins are distributed in a toroid around the cartwheel, and super-resolution light microscopy studies have measured the average radii of these ∼100-200 nm radius toroids with a 'precision' - or standard deviation (s.d. or 1σ) - of ±âˆ¼10-40 nm. The organization of Ana2/STIL within the cartwheel, however, has not been resolvable. Here, we develop methods to calculate the average toroidal radius of centriolar proteins in the ∼20-60 nm range with a s.d. of just ±âˆ¼4-5 nm, revealing that the amino and carboxyl termini of Ana2 are located in the outer cartwheel region.

Correlative In-Resin Super-Resolution Fluorescence and Electron Microscopy of Cultured Cells.

Correlative super-resolution light and electron microscopy (super-resolution CLEM) is a powerful and emerging tool in biological research. The practical realization of these two very different microscopy techniques with their individual requirements remains a challenging task. There is a broad range of approaches to choose from, each with their own advantages and limitations. Here, we present a detailed protocol for in-resin super-resolution CLEM of high-pressure frozen and freeze substituted cultured cells. The protocol makes use of a strategy to preserve the fluorescence and photo-switching capabilities of standard fluorescent proteins, such as GFP and YFP, to enable single-molecule localization microscopy (SMLM) in-resin sections followed by transmission electron microscopy (TEM) imaging. This results in a fivefold improvement in resolution in the fluorescence image and a more precise correlation of the distribution of fluorescently labeled molecules with EM ultrastructure compared with conventional CLEM.

Preserving the photoswitching ability of standard fluorescent proteins for correlative in-resin super-resolution and electron microscopy.

There are many different correlative light and electron microscopy (CLEM) techniques available. The use of super-resolution microscopy in CLEM is an emerging application and while offering the obvious advantages of improved resolution in the fluorescence image, and therefore more precise correlation to electron microscopy (EM) ultrastructure, it also presents new challenges. Choice of fluorophore, method of fixation, and timing of the fluorescence imaging are critical to the success of super-resolution CLEM and the relative importance, and technical difficulty, of each of these factors depends on the type of super-resolution microscopy being employed. This chapter details the method we developed for in-resin super-resolution CLEM using single molecule localization microscopy (SMLM) with standard fluorescent proteins (e.g., GFP and mVenus). The key to this approach is being able to preserve not only the fluorescence, but also, and more importantly, the photoswitching ability of the fluorescent proteins throughout the EM sample preparation procedure. Cells are cryofixed using high pressure freezing for optimal structural preservation and then freeze substituted in tannic acid, which preserves the photoswitching ability of the fluorescent proteins and is essential for high-quality SMLM imaging. Resin sections are then imaged using SMLM, achieving a structural resolution of 40-50nm and a localization precision of ∼17nm, followed by transmission electron microscopy. This produces high quality correlative images without the use of specialized fluorescent proteins or antibodies.

Structural Basis for Plexin Activation and Regulation.

Class A plexins (PlxnAs) act as semaphorin receptors and control diverse aspects of nervous system development and plasticity, ranging from axon guidance and neuron migration to synaptic organization. PlxnA signaling requires cytoplasmic domain dimerization, but extracellular regulation and activation mechanisms remain unclear. Here we present crystal structures of PlxnA (PlxnA1, PlxnA2, and PlxnA4) full ectodomains. Domains 1-9 form a ring-like conformation from which the C-terminal domain 10 points away. All our PlxnA ectodomain structures show autoinhibitory, intermolecular "head-to-stalk" (domain 1 to domain 4-5) interactions, which are confirmed by biophysical assays, live cell fluorescence microscopy, and cell-based and neuronal growth cone collapse assays. This work reveals a 2-fold role of the PlxnA ectodomains: imposing a pre-signaling autoinhibitory separation for the cytoplasmic domains via intermolecular head-to-stalk interactions and supporting dimerization-based PlxnA activation upon ligand binding. More generally, our data identify a novel molecular mechanism for preventing premature activation of axon guidance receptors.

Imaging cellular structures in super-resolution with SIM, STED and Localisation Microscopy: A practical comparison.

Many biological questions require fluorescence microscopy with a resolution beyond the diffraction limit of light. Super-resolution methods such as Structured Illumination Microscopy (SIM), STimulated Emission Depletion (STED) microscopy and Single Molecule Localisation Microscopy (SMLM) enable an increase in image resolution beyond the classical diffraction-limit. Here, we compare the individual strengths and weaknesses of each technique by imaging a variety of different subcellular structures in fixed cells. We chose examples ranging from well separated vesicles to densely packed three dimensional filaments. We used quantitative and correlative analyses to assess the performance of SIM, STED and SMLM with the aim of establishing a rough guideline regarding the suitability for typical applications and to highlight pitfalls associated with the different techniques.

Towards correlative super-resolution fluorescence and electron cryo-microscopy.

Correlative light and electron microscopy (CLEM) has become a powerful tool in life sciences. Particularly cryo-CLEM, the combination of fluorescence cryo-microscopy (cryo-FM) permitting for non-invasive specific multi-colour labelling, with electron cryo-microscopy (cryo-EM) providing the undisturbed structural context at a resolution down to the Ångstrom range, has enabled a broad range of new biological applications. Imaging rare structures or events in crowded environments, such as inside a cell, requires specific fluorescence-based information for guiding cryo-EM data acquisition and/or to verify the identity of the structure of interest. Furthermore, cryo-CLEM can provide information about the arrangement of specific proteins in the wider structural context of their native nano-environment. However, a major obstacle of cryo-CLEM currently hindering many biological applications is the large resolution gap between cryo-FM (typically in the range of ∼400 nm) and cryo-EM (single nanometre to the Ångstrom range). Very recently, first proof of concept experiments demonstrated the feasibility of super-resolution cryo-FM imaging and the correlation with cryo-EM. This opened the door towards super-resolution cryo-CLEM, and thus towards direct correlation of structural details from both imaging modalities.

SIMcheck: a Toolbox for Successful Super-resolution Structured Illumination Microscopy.

Three-dimensional structured illumination microscopy (3D-SIM) is a versatile and accessible method for super-resolution fluorescence imaging, but generating high-quality data is challenging, particularly for non-specialist users. We present SIMcheck, a suite of ImageJ plugins enabling users to identify and avoid common problems with 3D-SIM data, and assess resolution and data quality through objective control parameters. Additionally, SIMcheck provides advanced calibration tools and utilities for common image processing tasks. This open-source software is applicable to all commercial and custom platforms, and will promote routine application of super-resolution SIM imaging in cell biology.

Radiation induced chromatin conformation changes analysed by fluorescent localization microscopy, statistical physics, and graph theory.

It has been well established that the architecture of chromatin in cell nuclei is not random but functionally correlated. Chromatin damage caused by ionizing radiation raises complex repair machineries. This is accompanied by local chromatin rearrangements and structural changes which may for instance improve the accessibility of damaged sites for repair protein complexes. Using stably transfected HeLa cells expressing either green fluorescent protein (GFP) labelled histone H2B or yellow fluorescent protein (YFP) labelled histone H2A, we investigated the positioning of individual histone proteins in cell nuclei by means of high resolution localization microscopy (Spectral Position Determination Microscopy = SPDM). The cells were exposed to ionizing radiation of different doses and aliquots were fixed after different repair times for SPDM imaging. In addition to the repair dependent histone protein pattern, the positioning of antibodies specific for heterochromatin and euchromatin was separately recorded by SPDM. The present paper aims to provide a quantitative description of structural changes of chromatin after irradiation and during repair. It introduces a novel approach to analyse SPDM images by means of statistical physics and graph theory. The method is based on the calculation of the radial distribution functions as well as edge length distributions for graphs defined by a triangulation of the marker positions. The obtained results show that through the cell nucleus the different chromatin re-arrangements as detected by the fluorescent nucleosomal pattern average themselves. In contrast heterochromatic regions alone indicate a relaxation after radiation exposure and re-condensation during repair whereas euchromatin seemed to be unaffected or behave contrarily. SPDM in combination with the analysis techniques applied allows the systematic elucidation of chromatin re-arrangements after irradiation and during repair, if selected sub-regions of nuclei are investigated.

Correlative in-resin super-resolution and electron microscopy using standard fluorescent proteins.

We introduce a method for correlative in-resin super-resolution fluorescence and electron microscopy (EM) of biological structures in mammalian culture cells. Cryo-fixed resin embedded samples offer superior structural preservation, performing in-resin super-resolution, however, remains a challenge. We identified key aspects of the sample preparation procedure of high pressure freezing, freeze substitution and resin embedding that are critical for preserving fluorescence and photo-switching of standard fluorescent proteins, such as mGFP, mVenus and mRuby2. This enabled us to combine single molecule localization microscopy with transmission electron microscopy imaging of standard fluorescent proteins in cryo-fixed resin embedded cells. We achieved a structural resolution of 40-50 nm (~17 nm average single molecule localization accuracy) in the fluorescence images without the use of chemical fixation or special fluorophores. Using this approach enabled the correlation of fluorescently labeled structures to the ultrastructure in the same cell at the nanometer level and superior structural preservation.

Super-resolution microscopy using standard fluorescent proteins in intact cells under cryo-conditions.

We introduce a super-resolution technique for fluorescence cryo-microscopy based on photoswitching of standard genetically encoded fluorescent marker proteins in intact mammalian cells at low temperature (81 K). Given the limit imposed by the lack of cryo-immersion objectives, current applications of fluorescence cryo-microscopy to biological specimens achieve resolutions between 400-500 nm only. We demonstrate that the single molecule characteristics of reversible photobleaching of mEGFP and mVenus at liquid nitrogen temperature are suitable for the basic concept of single molecule localization microscopy. This enabled us to perform super-resolution imaging of vitrified biological samples and to visualize structures in unperturbed fast frozen cells for the first time with a structural resolution of ∼125 nm (average single molecule localization accuracy ∼40 nm), corresponding to a 3-5 fold resolution improvement.

Fluorescence cryo-microscopy: current challenges and prospects.

Studying biological structures with fine details does not only require a microscope with high resolution, but also a sample preparation process that preserves the structures in a near-native state. Live-cell imaging is restricted mostly to the field of light microscopy. For studies requiring much higher resolution, fast freezing techniques (vitrification) are successfully used to immobilize the sample in a near-native state for imaging with electron and X-ray cryo-microscopy. Fluorescence cryo-microscopy combines imaging of vitrified samples with the advantages of fluorescence labeling of biological structures. Technical considerations as well as the behavior of fluorophores at low temperatures have to be taken into account for developing or adapting super-resolution methods under cryo conditions to exploit the full potential of this technique.

Spatial distribution and structural arrangement of a murine cytomegalovirus glycoprotein detected by SPDM localization microscopy.

Novel approaches of localization microscopy have opened new insights into the molecular nano-cosmos of cells. We applied a special embodiment called spectral position determination microscopy (SPDM) that has the advantage to run with standard fluorescent dyes or proteins under standard preparation conditions. Pointillist images with a resolution in the order of 10 nm can be obtained by SPDM. Therefore, vector pEYFP-m164, encoding the murine cytomegalovirus glycoprotein gp36.5/m164 fused to enhanced yellow fluorescent protein, was transiently transfected into COS-7 cells. This protein shows exceptional intracellular trafficking dynamics, moving within the endoplasmic reticulum (ER) and outer nuclear membrane. The molecular positions of gp36.5/m164 were visualized and determined by SPDM imaging. From the position point patterns of the protein molecules, their arrangements were quantified by next neighbour distance analyses. Three different structural arrangements were discriminated: (a) a linear distribution along the membrane, (b) a highly structured distribution in the ER, and (c) a homogenous distribution in the cellular cytoplasm. The results indicate that the analysis of next neighbour distances on the nano-scale allows the identification and discrimination of different structural arrangements of molecules within their natural cellular environment.

Quantitative analysis of individual hepatocyte growth factor receptor clusters in influenza A virus infected human epithelial cells using localization microscopy.

In this report, we applied a special localization microscopy technique (Spectral Precision Distance/Spatial Position Determination Microscopy/SPDM) to quantitatively analyze the effect of influenza A virus (IAV) infection on the spatial distribution of individual HGFR (Hepatocyte Growth Factor Receptor) proteins on the membrane of human epithelial cells at the single molecule resolution level. We applied this SPDM method to Alexa 488 labeled HGFR proteins with two different ligands. The ligands were either HGF (Hepatocyte Growth Factor), or IAV. In addition, the HGFR distribution in a control group of mock-incubated cells without any ligands was investigated. The spatial distribution of 1×10(6) individual HGFR proteins localized in large regions of interest on membranes of 240 cells was quantitatively analyzed and found to be highly non-random. Between 21% and 24% of the HGFR molecules were located in 44,304 small clusters with an average diameter of 54nm. The mean density of HGFR molecule signals per individual cluster was very similar in control cells, in cells with ligand only, and in IAV infected cells, independent of the incubation time. From the density of HGFR molecule signals in the clusters and the diameter of the clusters, the number of HGFR molecule signals per cluster was estimated to be in the range between 4 and 11 (means 5-6). This suggests that the membrane bound HGFR clusters form small molecular complexes with a maximum diameter of few tens of nm, composed of a relatively low number of HGFR molecules. This article is part of a Special Issue entitled: Viral Membrane Proteins - Channels for Cellular Networking.