Bridging structural and cell biology with cryo-electron microscopy.

Most life scientists would agree that understanding how cellular processes work requires structural knowledge about the macromolecules involved. For example, deciphering the double-helical nature of DNA revealed essential aspects of how genetic information is stored, copied and repaired. Yet, being reductionist in nature, structural biology requires the purification of large amounts of macromolecules, often trimmed off larger functional units. The advent of cryogenic electron microscopy (cryo-EM) greatly facilitated the study of large, functional complexes and generally of samples that are hard to express, purify and/or crystallize. Nevertheless, cryo-EM still requires purification and thus visualization outside of the natural context in which macromolecules operate and coexist. Conversely, cell biologists have been imaging cells using a number of fast-evolving techniques that keep expanding their spatial and temporal reach, but always far from the resolution at which chemistry can be understood. Thus, structural and cell biology provide complementary, yet unconnected visions of the inner workings of cells. Here we discuss how the interplay between cryo-EM and cryo-electron tomography, as a connecting bridge to visualize macromolecules in situ, holds great promise to create comprehensive structural depictions of macromolecules as they interact in complex mixtures or, ultimately, inside the cell itself.

A Compound Loss Function With Shape Aware Weight Map for Microscopy Cell Segmentation.

Microscopy cell segmentation is a crucial step in biological image analysis and a challenging task. In recent years, deep learning has been widely used to tackle this task, with promising results. A critical aspect of training complex neural networks for this purpose is the selection of the loss function, as it affects the learning process. In the field of cell segmentation, most of the recent research in improving the loss function focuses on addressing the problem of inter-class imbalance. Despite promising achievements, more work is needed, as the challenge of cell segmentation is not only the inter-class imbalance but also the intra-class imbalance (the cost imbalance between the false positives and false negatives of the inference model), the segmentation of cell minutiae, and the missing annotations. To deal with these challenges, in this paper, we propose a new compound loss function employing a shape aware weight map. The proposed loss function is inspired by Youden's J index to handle the problem of inter-class imbalance and uses a focal cross-entropy term to penalize the intra-class imbalance and weight easy/hard samples. The proposed shape aware weight map can handle the problem of missing annotations and facilitate valid segmentation of cell minutiae. Results of evaluations on all ten 2D+time datasets from the public cell tracking challenge demonstrate 1) the superiority of the proposed loss function with the shape aware weight map, and 2) that the performance of recent deep learning-based cell segmentation methods can be improved by using the proposed compound loss function.

A Flexible Chamber for Time-Lapse Live-Cell Imaging with Stimulated Raman Scattering Microscopy.

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many biological and biomedical applications. However, long-term time-lapse SRS imaging of live cells has not been widely adopted. SRS microscopy often uses a high numerical aperture (NA) water-immersion objective and a high NA oil-immersion condenser to achieve high-resolution imaging. In this case, the gap between the objective and the condenser is only a few millimeters. Therefore, most commercial stage-top environmental chambers cannot be used for SRS imaging because of their large thickness with a rigid glass cover. This paper describes the design and fabrication of a flexible chamber that can be used for time-lapse live-cell imaging with transmitted SRS signal detection on an upright microscope frame. The flexibility of the chamber is achieved by using a soft material - a thin natural rubber film. The new enclosure and chamber design can be easily added to an existing SRS imaging setup. The testing and preliminary results demonstrate that the flexible chamber system enables stable, long-term, time-lapse SRS imaging of live cells, which can be used for various bioimaging applications in the future.

Correlative all-optical quantification of mass density and mechanics of subcellular compartments with fluorescence specificity.

Quantitative measurements of physical parameters become increasingly important for understanding biological processes. Brillouin microscopy (BM) has recently emerged as one technique providing the 3D distribution of viscoelastic properties inside biological samples - so far relying on the implicit assumption that refractive index (RI) and density can be neglected. Here, we present a novel method (FOB microscopy) combining BM with optical diffraction tomography and epifluorescence imaging for explicitly measuring the Brillouin shift, RI, and absolute density with specificity to fluorescently labeled structures. We show that neglecting the RI and density might lead to erroneous conclusions. Investigating the nucleoplasm of wild-type HeLa cells, we find that it has lower density but higher longitudinal modulus than the cytoplasm. Thus, the longitudinal modulus is not merely sensitive to the water content of the sample - a postulate vividly discussed in the field. We demonstrate the further utility of FOB on various biological systems including adipocytes and intracellular membraneless compartments. FOB microscopy can provide unexpected scientific discoveries and shed quantitative light on processes such as phase separation and transition inside living cells.

Effect of topography parameters on cellular morphology during guided cell migration on a graded micropillar surface.

PURPOSE: Guided cell migration refers to the engineering of local cell environment to specifically direct cell migration and has important applications such as utilization in cell sorting and wound healing assays. Graded micropillar surfaces have been utilized for achieving guided cell migration. Topographic parameters such as micropillar diameter and spacing gradient may have effects on the morphology of attached cells. It is critical to understand this interaction between the cells and the underlying microscale structures. METHODS: In this study, a graded micropillar substrate has been fabricated to investigate the effects of the microtopography on the cell morphology in terms of the cell aspect ratio and cell circularity. RESULTS: It is found that 1) with the increase of the micropillar diameter, the cell aspect ratio has no significance change. At the small spacing gradients, the aspect ratio is smaller than that at the large spacing gradients; 2) statistical analysis shows both the micropillar diameter and spacing gradient have no significant effect on the cell aspect ratio compared to the flat surface; 3) the cell circularity at the small micropillar diameters is higher than that at the large micropillar diameters. The cell circularity at the micropillar gradient of 0.1 µm is higher than those at the other micropillar gradients; 4) three microtopographic conditions are considered to have statistically significant effect on the cell circularity compared to the flat surface, including the micropillar diameters of 5 µm and 10 µm and the spacing gradient of 0.1 µm.

Deciphering cell signaling networks with massively multiplexed biosensor barcoding.

Genetically encoded fluorescent biosensors are powerful tools for monitoring biochemical activities in live cells, but their multiplexing capacity is limited by the available spectral space. We overcome this problem by developing a set of barcoding proteins that can generate over 100 barcodes and are spectrally separable from commonly used biosensors. Mixtures of barcoded cells expressing different biosensors are simultaneously imaged and analyzed by deep learning models to achieve massively multiplexed tracking of signaling events. Importantly, different biosensors in cell mixtures show highly coordinated activities, thus facilitating the delineation of their temporal relationship. Simultaneous tracking of multiple biosensors in the receptor tyrosine kinase signaling network reveals distinct mechanisms of effector adaptation, cell autonomous and non-autonomous effects of KRAS mutations, as well as complex interactions in the network. Biosensor barcoding presents a scalable method to expand multiplexing capabilities for deciphering the complexity of signaling networks and their interactions between cells.

Repeat and single dose administration of gadodiamide to rats to investigate concentration and location of gadolinium and the cell ultrastructure.

Gadolinium based contrast agents (GBCA) are used to image patients using magnetic resonance (MR) imaging. In recent years, there has been controversy around gadolinium retention after GBCA administration. We sought to evaluate the potential toxicity of gadolinium in the rat brain up to 1-year after repeated gadodiamide dosing and tissue retention kinetics after a single administration. Histopathological and ultrastructural transmission electron microscopy (TEM) analysis revealed no findings in rats administered a cumulative dose of 12 mmol/kg. TEM-energy dispersive X-ray spectroscopy (TEM-EDS) localization of gadolinium in the deep cerebellar nuclei showed ~ 100 nm electron-dense foci in the basal lamina of the vasculature. Laser ablation-ICP-MS (LA-ICP-MS) showed diffuse gadolinium throughout the brain but concentrated in perivascular foci of the DCN and globus pallidus with no observable tissue injury or ultrastructural changes. A single dose of gadodiamide (0.6 mmol/kg) resulted in rapid cerebrospinal fluid (CSF) and blood clearance. Twenty-weeks post administration gadolinium concentrations in brain regions was reduced by 16-72-fold and in the kidney (210-fold), testes (194-fold) skin (44-fold), liver (42-fold), femur (6-fold) and lung (64-fold). Our findings suggest that gadolinium does not lead to histopathological or ultrastructural changes in the brain and demonstrate in detail the kinetics of a human equivalent dose over time in a pre-clinical model.

Cryo-Structured Illumination Microscopic Data Collection from Cryogenically Preserved Cells.

Three-dimensional (3D) structured illumination microscopy (SIM) allows imaging of fluorescently labelled cellular structures at higher resolution than conventional fluorescence microscopy. This super-resolution (SR) technique enables visualization of molecular processes in whole cells and has the potential to be used in conjunction with electron microscopy and X-ray tomography to correlate structural and functional information. A SIM microscope for cryogenically preserved samples (cryoSIM) has recently been commissioned at the correlative cryo-imaging beamline B24 at the UK synchrotron. It was designed specifically for 3D imaging of biological samples at cryogenic temperatures in a manner compatible with subsequent imaging of the same samples by X-ray microscopy methods such as cryo-soft X-ray tomography. This video article provides detailed methods and protocols for successful imaging using the cryoSIM. In addition to instructions on the operation of the cryoSIM microscope, recommendations have been included regarding the choice of samples, fluorophores, and parameter settings. The protocol is demonstrated in U2OS cell samples whose mitochondria and tubulin have been fluorescently labelled.

Cryo-Focused Ion Beam Lamella Preparation Protocol for in Situ Structural Biology.

The advances in electron cryo-microscopy have enabled high-resolution structural studies of vitrified macromolecular complexes in situ by cryo-electron tomography (cryo-ET). Since utilization of cryo-ET is generally limited to the specimens with thickness < 500 nm, a complex sample preparation protocol to study larger samples such as single eukaryotic cells by cryo-ET was developed and optimized over the last decade. The workflow is based on the preparation of a thin cellular lamella by cryo-focused ion beam milling (cryo-FIBM) from the vitrified cells. The sample preparation protocol is a multi-step process which includes utilization of several high-end instruments and comprises sample manipulation prone to sample deterioration. Here, we present a workflow for preparation of three different model specimens that was optimized to provide high-quality lamellae for cryo-ET or electron diffraction tomography with high reproducibility. Preparation of lamellae from large adherent mammalian cells, small suspension eukaryotic cell line, and protein crystals of intermediate size is described which represents examples of the most frequently studied samples used for cryo-FIBM in life sciences.

Correlative fluorescence microscopy, transmission electron microscopy and secondary ion mass spectrometry (CLEM-SIMS) for cellular imaging.

Electron microscopy (EM) has been employed for decades to analyze cell structure. To also analyze the positions and functions of specific proteins, one typically relies on immuno-EM or on a correlation with fluorescence microscopy, in the form of correlated light and electron microscopy (CLEM). Nevertheless, neither of these procedures is able to also address the isotopic composition of cells. To solve this, a correlation with secondary ion mass spectrometry (SIMS) would be necessary. SIMS has been correlated in the past to EM or to fluorescence microscopy in biological samples, but not to CLEM. We achieved this here, using a protocol based on transmission EM, conventional epifluorescence microscopy and nanoSIMS. The protocol is easily applied, and enables the use of all three technologies at high performance parameters. We suggest that CLEM-SIMS will provide substantial information that is currently beyond the scope of conventional correlative approaches.

[Cryo-electron microcopy for a new vision of the cell and its components]. / La cryo-microscopie électronique révèle une nouvelle vision de la cellule et de ses composants.

Cryo-electron microscopy (cryo-EM) is a technique for imaging biological samples that plays a central role in structural biology, with high impact on research fields such as cell and developmental biology, bioinformatics, cell physics and applied mathematics. It allows the determination of structures of purified proteins within cells. This review describes the main recent advances in cryo-EM, illustrated by examples of proteins of biomedical interest, and the avenues for future development.

TITLE: La cryo-microscopie électronique révèle une nouvelle vision de la cellule et de ses composants. ABSTRACT: La cryo-microscopie électronique (cryo-EM) est une technique d'imagerie du vivant qui prend désormais une place prépondérante en biologie structurale, avec des retombées en biologie cellulaire et du développement, en bioinformatique, en biomédecine ou en physique de la cellule. Elle permet de déterminer des structures de protéines purifiées in vitro ou au sein des cellules. Cette revue décrit les principales avancées récentes de la cryo-EM, illustrées par des exemples d'élucidation de structures de protéines d'intérêt en biomédecine, et les pistes de développements futurs.

Towards high-throughput in situ structural biology using electron cryotomography.

Electron cryotomography is a rapidly evolving method for imaging macromolecules directly within the native environment of cells and tissues. Combined with sub-tomogram averaging, it allows structural and cell biologists to obtain sub-nanometre resolution structures in situ. However, low throughput in cryo-ET sample preparation and data acquisition, as well as difficulties in target localisation and sub-tomogram averaging image processing, limit its widespread usability. In this review, we discuss new advances in the field that address these throughput and technical problems. We focus on recent efforts made to resolve issues in sample thinning, improvement in data collection speed at the microscope, strategies for localisation of macromolecules using correlated light and electron microscopy and advancements made to improve resolution in sub-tomogram averaging. These advances will considerably decrease the amount of time and effort required for cryo-ET and sub-tomogram averaging, ushering in a new era of structural biology where in situ macromolecular structure determination will be routine.

Epifluorescence-based three-dimensional traction force microscopy.

We introduce a novel method to compute three-dimensional (3D) displacements and both in-plane and out-of-plane tractions on nominally planar transparent materials using standard epifluorescence microscopy. Despite the importance of out-of-plane components to fully understanding cell behavior, epifluorescence images are generally not used for 3D traction force microscopy (TFM) experiments due to limitations in spatial resolution and measuring out-of-plane motion. To extend an epifluorescence-based technique to 3D, we employ a topology-based single particle tracking algorithm to reconstruct high spatial-frequency 3D motion fields from densely seeded single-particle layer images. Using an open-source finite element (FE) based solver, we then compute the 3D full-field stress and strain and surface traction fields. We demonstrate this technique by measuring tractions generated by both single human neutrophils and multicellular monolayers of Madin-Darby canine kidney cells, highlighting its acuity in reconstructing both individual and collective cellular tractions. In summary, this represents a new, easily accessible method for calculating fully three-dimensional displacement and 3D surface tractions at high spatial frequency from epifluorescence images. We released and support the complete technique as a free and open-source code package.

A novel epitope tagging system to visualize and monitor antigens in live cells with chromobodies.

Epitope tagging is a versatile approach to study different proteins using a well-defined and established methodology. To date, most epitope tags such as myc, HA, V5 and FLAG tags are recognized by antibodies, which limits their use to fixed cells, tissues or protein samples. Here we introduce a broadly applicable tagging strategy utilizing a short peptide tag (PepTag) which is specifically recognized by a nanobody (PepNB). We demonstrated that the PepNB can be easily functionalized for immunoprecipitation or direct immunofluorescence staining of Pep-tagged proteins in vitro. For in cellulo studies we converted the PepNB into a fluorescently labeled Pep-chromobody (PepCB) which is functionally expressed in living cells. The addition of the small PepTag does not interfere with the examined structures in different cellular compartments and its detection with the PepCB enables optical antigen tracing in real time. By employing the phenomenon of antigen-mediated chromobody stabilization (AMCBS) using a turnover-accelerated PepCB we demonstrated that the system is suitable to visualize and quantify changes in Pep-tagged antigen concentration by quantitative live-cell imaging. We expect that this novel tagging strategy offers new opportunities to study the dynamic regulation of proteins, e.g. during cellular signaling, cell differentiation, or upon drug action.

Dielectrophoretic Manipulation of Cancer Cells and Their Electrical Characterization.

Electromanipulation and electrical characterization of cancerous cells is becoming a topic of high interest as the results reported to date demonstrate a good differentiation among various types of cells from an electrical viewpoint. Dielectrophoresis and broadband dielectric spectroscopy are complementary tools for sorting, identification, and characterization of malignant cells and were successfully used on both primary tumor cells and culture cells as well. However, the literature is presenting a plethora of studies with respect to electrical evaluation of these type of cells, and this review is reporting a collection of information regarding the functioning principles of different types of dielectrophoresis setups, theory of cancer cell polarization, and electrical investigation (including here the polarization mechanisms). The interpretation of electrical characteristics against frequency is discussed with respect to interfacial/Maxwell-Wagner polarization and the parasitic influence of electrode polarization. Moreover, the electrical equivalent circuits specific to biological cells polarizations are discussed for a good understanding of the cells' morphology influence. The review also focuses on advantages of specific low-conductivity buffers employed currently for improving the efficiency of dielectrophoresis and provides a set of synthesized data from the literature highlighting clear differentiation between the crossover frequencies of different cancerous cells.

Apparent diffusion coefficient measured by diffusion MRI of moving and deforming domains.

The modeling of the diffusion MRI signal from moving and deforming organs such as the heart is challenging due to significant motion and deformation of the imaged medium during the signal acquisition. Recently, a mathematical formulation of the Bloch-Torrey equation, describing the complex transverse magnetization due to diffusion-encoding magnetic field gradients, was developed to account for the motion and deformation. In that work, the motivation was to cancel the effect of the motion and deformation in the MRI image and the space scale of interest spans multiple voxels. In the present work, we adapt the mathematical equation to study the diffusion MRI signal at the much smaller scale of biological cells. We start with the Bloch-Torrey equation defined on a cell that is moving and deforming and linearize the equation around the magnitude of the diffusion-encoding gradient. The result is a second order signal model in which the linear term gives the imaginary part of the diffusion MRI signal and the quadratic term gives the apparent diffusion coefficient (ADC) attributable to the biological cell. We numerically validate this model for a variety of motions and deformations.

Liquid-phase electron microscopy imaging of cellular and biomolecular systems.

The ongoing development of liquid-phase electron microscopy methods-in which specimens are kept fully solvated in the microscope by encapsulation in transparent, vacuum-tight chambers-is making it possible to investigate a wide variety of nanoscopic dynamic phenomena at the single-particle level, and with nanometer to atomic resolution. As such, there has been growing motivation to make liquid-phase electron microscopy tools applicable not only to inorganic materials, like metals, semiconductors, and ceramics, but also to "soft" materials such as biomolecules and cells, whose nanoscale dynamics and organization are intricately tied to their functionality. Here we review efforts toward making this an experimental reality, summarizing recent liquid-phase electron microscopy studies of whole cells, assembling peptides, and even individual proteins. Successes and challenges are discussed, as well as strategies to maximize the amount of accessible information and minimize the impact of the electron beam. We conclude with an outlook on the potential of liquid-phase electron microscopy to provide new insight into the rich and functional dynamics occurring in biological systems at the microscopic to molecular level.

The long dark teatime of the cell.

Florian Maderspacher introduces the special issue 'the cell in evolution'.

Colorimetric and fluorescence sensing of Zn2+ ion and its bio-imaging applications based on macrocyclic "tet a" derivative.

We report on the synthesis and characterization of trans N, N'-di-substituted macrocyclic "tet a" probe (L) for metal ion sensing. Both the colorimetric and fluorescent titration studies are performed with different metal ions. The results have suggested that the probe L is very selective and sensitive towards Zn2+ ions with significant changes in color. The pendant armed macrocyclic "tet a" probe has exhibited 1.28× 105 M-1 binding constant and virtuous selectivity for Zn2+ ion than other common metal ions. The detection limit of the probe towards Zn2+ ion is 0.027 nM. The selective sensing of Zn2+ ion is efficiently reversible with EDTA, which is demonstrated for five cycles without losing sensitivity. The time-resolved single-photon counting (TCSPC) studies have determined the average lifetime value for the probe L and L+ Zn2+ ion of 1.29 and 2.96 ns, respectively. The theoretical DFT studies have well supported the experimental outcomes. The practical application of the probe in visualizing intracellular Zn2+ ion distribution in live Artemia salina has proved the low cytotoxicity and cell membrane permeability of probe, which makes it capable of sensing Zn2+ ion in HeLa cells. Thus, the probe L can act as a selective recognition of Zn2+ ion in living cell applications.

The Quantitative-Phase Dynamics of Apoptosis and Lytic Cell Death.

Cell viability and cytotoxicity assays are highly important for drug screening and cytotoxicity tests of antineoplastic or other therapeutic drugs. Even though biochemical-based tests are very helpful to obtain preliminary preview, their results should be confirmed by methods based on direct cell death assessment. In this study, time-dependent changes in quantitative phase-based parameters during cell death were determined and methodology useable for rapid and label-free assessment of direct cell death was introduced. The goal of our study was distinction between apoptosis and primary lytic cell death based on morphologic features. We have distinguished the lytic and non-lytic type of cell death according to their end-point features (Dance of Death typical for apoptosis versus swelling and membrane rupture typical for all kinds of necrosis common for necroptosis, pyroptosis, ferroptosis and accidental cell death). Our method utilizes Quantitative Phase Imaging (QPI) which enables the time-lapse observation of subtle changes in cell mass distribution. According to our results, morphological and dynamical features extracted from QPI micrographs are suitable for cell death detection (76% accuracy in comparison with manual annotation). Furthermore, based on QPI data alone and machine learning, we were able to classify typical dynamical changes of cell morphology during both caspase 3,7-dependent and -independent cell death subroutines. The main parameters used for label-free detection of these cell death modalities were cell density (pg/pixel) and average intensity change of cell pixels further designated as Cell Dynamic Score (CDS). To the best of our knowledge, this is the first study introducing CDS and cell density as a parameter typical for individual cell death subroutines with prediction accuracy 75.4% for caspase 3,7-dependent and -independent cell death.