Community-developed checklists for publishing images and image analyses.

Images document scientific discoveries and are prevalent in modern biomedical research. Microscopy imaging in particular is currently undergoing rapid technological advancements. However, for scientists wishing to publish obtained images and image-analysis results, there are currently no unified guidelines for best practices. Consequently, microscopy images and image data in publications may be unclear or difficult to interpret. Here, we present community-developed checklists for preparing light microscopy images and describing image analyses for publications. These checklists offer authors, readers and publishers key recommendations for image formatting and annotation, color selection, data availability and reporting image-analysis workflows. The goal of our guidelines is to increase the clarity and reproducibility of image figures and thereby to heighten the quality and explanatory power of microscopy data.

Multisite assessment of reproducibility in high-content cell migration imaging data.

High-content image-based cell phenotyping provides fundamental insights into a broad variety of life science disciplines. Striving for accurate conclusions and meaningful impact demands high reproducibility standards, with particular relevance for high-quality open-access data sharing and meta-analysis. However, the sources and degree of biological and technical variability, and thus the reproducibility and usefulness of meta-analysis of results from live-cell microscopy, have not been systematically investigated. Here, using high-content data describing features of cell migration and morphology, we determine the sources of variability across different scales, including between laboratories, persons, experiments, technical repeats, cells, and time points. Significant technical variability occurred between laboratories and, to lesser extent, between persons, providing low value to direct meta-analysis on the data from different laboratories. However, batch effect removal markedly improved the possibility to combine image-based datasets of perturbation experiments. Thus, reproducible quantitative high-content cell image analysis of perturbation effects and meta-analysis depend on standardized procedures combined with batch correction.

Actomyosin contractility requirements and reciprocal cell-tissue mechanics for cancer cell invasion through collagen-based channels.

The interstitial tumor microenvironment is composed of heterogeneously organized collagen-rich porous networks as well as channel-like structures and interfaces which provide both barriers and guidance for invading cells. Tumor cells invading 3D random porous collagen networks depend upon actomyosin contractility to deform and translocate the nucleus, whereas Rho/Rho-associated kinase-dependent contractility is largely dispensable for migration in stiff capillary-like confining microtracks. To investigate whether this dichotomy of actomyosin contractility dependence also applies to physiological, deformable linear collagen environments, we developed nearly barrier-free collagen-scaffold microtracks of varying cross section using two-photon laser ablation. Both very narrow and wide tracks supported single-cell migration by either outward pushing of collagen up to four times when tracks were narrow, or cell pulling on collagen walls down to 50% of the original diameter by traction forces of up to 40 nN when tracks were wide, resulting in track widths optimized to single-cell diameter. Targeting actomyosin contractility by synthetic inhibitors increased cell elongation and nuclear shape change in narrow tracks and abolished cell-mediated deformation of both wide and narrow tracks. Accordingly, migration speeds in all channel widths reduced, with migration rates of around 45-65% of the original speed persisting. Together, the data suggest that cells engage actomyosin contraction to reciprocally adjust both own morphology and linear track width to optimal size for effective cellular locomotion.

QUAREP-LiMi: A community-driven initiative to establish guidelines for quality assessment and reproducibility for instruments and images in light microscopy.

A modern day light microscope has evolved from a tool devoted to making primarily empirical observations to what is now a sophisticated , quantitative device that is an integral part of both physical and life science research. Nowadays, microscopes are found in nearly every experimental laboratory. However, despite their prevalent use in capturing and quantifying scientific phenomena, neither a thorough understanding of the principles underlying quantitative imaging techniques nor appropriate knowledge of how to calibrate, operate and maintain microscopes can be taken for granted. This is clearly demonstrated by the well-documented and widespread difficulties that are routinely encountered in evaluating acquired data and reproducing scientific experiments. Indeed, studies have shown that more than 70% of researchers have tried and failed to repeat another scientist's experiments, while more than half have even failed to reproduce their own experiments. One factor behind the reproducibility crisis of experiments published in scientific journals is the frequent underreporting of imaging methods caused by a lack of awareness and/or a lack of knowledge of the applied technique. Whereas quality control procedures for some methods used in biomedical research, such as genomics (e.g. DNA sequencing, RNA-seq) or cytometry, have been introduced (e.g. ENCODE), this issue has not been tackled for optical microscopy instrumentation and images. Although many calibration standards and protocols have been published, there is a lack of awareness and agreement on common standards and guidelines for quality assessment and reproducibility. In April 2020, the QUality Assessment and REProducibility for instruments and images in Light Microscopy (QUAREP-LiMi) initiative was formed. This initiative comprises imaging scientists from academia and industry who share a common interest in achieving a better understanding of the performance and limitations of microscopes and improved quality control (QC) in light microscopy. The ultimate goal of the QUAREP-LiMi initiative is to establish a set of common QC standards, guidelines, metadata models and tools, including detailed protocols, with the ultimate aim of improving reproducible advances in scientific research. This White Paper (1) summarizes the major obstacles identified in the field that motivated the launch of the QUAREP-LiMi initiative; (2) identifies the urgent need to address these obstacles in a grassroots manner, through a community of stakeholders including, researchers, imaging scientists, bioimage analysts, bioimage informatics developers, corporate partners, funding agencies, standards organizations, scientific publishers and observers of such; (3) outlines the current actions of the QUAREP-LiMi initiative and (4) proposes future steps that can be taken to improve the dissemination and acceptance of the proposed guidelines to manage QC. To summarize, the principal goal of the QUAREP-LiMi initiative is to improve the overall quality and reproducibility of light microscope image data by introducing broadly accepted standard practices and accurately captured image data metrics.

Third harmonic generation microscopy of cells and tissue organization.

The interaction of cells within their microenvironmental niche is fundamental to cell migration, positioning, growth and differentiation in order to form and maintain complex tissue organization and function. Third harmonic generation (THG) microscopy is a label-free scatter process that is elicited by water-lipid and water-protein interfaces, including intra- and extracellular membranes, and extracellular matrix structures. In applied life sciences, THG delivers a versatile contrast modality to complement multi-parameter fluorescence, second harmonic generation and fluorescence lifetime microscopy, which allows detection of cellular and molecular cell functions in three-dimensional tissue culture and small animals. In this Commentary, we review the physical and technical basis of THG, and provide considerations for optimal excitation, detection and interpretation of THG signals. We further provide an overview on how THG has versatile applications in cell and tissue research, with a particular focus on analyzing tissue morphogenesis and homeostasis, immune cell function and cancer research, as well as the emerging applicability of THG in clinical practice.

Single cell-based automated quantification of therapy responses of invasive cancer spheroids in organotypic 3D culture.

Organotypic in vitro culture of 3D spheroids in an extracellular matrix represent a promising cancer therapy prediction model for personalized medicine screens due to their controlled experimental conditions and physiological similarities to in vivo conditions. As in tumors in vivo, 3D invasion cultures identify intratumor heterogeneity of growth, invasion and apoptosis induction by cytotoxic therapy. We here combine in vitro 3D spheroid invasion culture with irradiation and automated nucleus-based segmentation for single cell analysis to quantify growth, survival, apoptosis and invasion response during experimental radiation therapy. As output, multi-parameter histogram-based representations deliver an integrated insight into therapy response and resistance. This workflow may be suited for high-throughput screening and identification of invasive and therapy-resistant tumor sub-populations.

Lateral mobility of individual integrin nanoclusters orchestrates the onset for leukocyte adhesion.

Integrins are cell membrane adhesion receptors involved in morphogenesis, immunity, tissue healing, and metastasis. A central, yet unresolved question regarding the function of integrins is how these receptors regulate both their conformation and dynamic nanoscale organization on the membrane to generate adhesion-competent microclusters upon ligand binding. Here we exploit the high spatial (nanometer) accuracy and temporal resolution of single-dye tracking to dissect the relationship between conformational state, lateral mobility, and microclustering of the integrin receptor lymphocyte function-associated antigen 1 (LFA-1) expressed on immune cells. We recently showed that in quiescent monocytes, LFA-1 preorganizes in nanoclusters proximal to nanoscale raft components. We now show that these nanoclusters are primarily mobile on the cell surface with a small (ca. 5%) subset of conformational-active LFA-1 nanoclusters preanchored to the cytoskeleton. Lateral mobility resulted crucial for the formation of microclusters upon ligand binding and for stable adhesion under shear flow. Activation of high-affinity LFA-1 by extracellular Ca(2+) resulted in an eightfold increase on the percentage of immobile nanoclusters and cytoskeleton anchorage. Although having the ability to bind to their ligands, these active nanoclusters failed to support firm adhesion in static and low shear-flow conditions because mobility and clustering capacity were highly compromised. Altogether, our work demonstrates an intricate coupling between conformation and lateral diffusion of LFA-1 and further underscores the crucial role of mobility for the onset of LFA-1 mediated leukocyte adhesion.

Approaches to Determine Nuclear Shape in Cells During Migration Through Collagen Matrices.

Fibrillar collagen is an abundant extracellular matrix (ECM) component of interstitial tissues which supports the structure of many organs, including the skin and breast. Many different physiological processes, but also pathological processes such as metastatic cancer invasion, involve interstitial cell migration. Often, cell movement takes place through small ECM gaps and pores and depends upon the ability of the cell and its stiff nucleus to deform. Such nuclear deformation during cell migration may impact nuclear integrity, such as of chromatin or the nuclear envelope, and therefore the morphometric analysis of nuclear shapes can provide valuable insight into a broad variety of biological processes. Here, we describe a protocol on how to generate a cell-collagen model in vitro and how to use confocal microscopy for the static and dynamic visualization of labeled nuclei in single migratory cells. We developed, and here provide, two scripts that (Fidler, Nat Rev Cancer 3(6):453-458, 2003) enable the semi-automated and fast quantification of static single nuclear shape descriptors, such as aspect ratio or circularity, and the nuclear irregularity index that forms a combination of four distinct shape descriptors, as well as (Frantz et al., J Cell Sci 123 (Pt 24):4195-4200, 2010) a quantification of their changes over time. Finally, we provide quantitative measurements on nuclear shapes from cells that migrated through collagen either in the presence or the absence of an inhibitor of collagen degradation, showing the distinctive power of this approach. This pipeline can also be applied to cell migration studied in different assays, ranging from 3D microfluidics to migration in the living organism.

Community-developed checklists for publishing images and image analyses.

Images document scientific discoveries and are prevalent in modern biomedical research. Microscopy imaging in particular is currently undergoing rapid technological advancements. However for scientists wishing to publish the obtained images and image analyses results, there are to date no unified guidelines. Consequently, microscopy images and image data in publications may be unclear or difficult to interpret. Here we present community-developed checklists for preparing light microscopy images and image analysis for publications. These checklists offer authors, readers, and publishers key recommendations for image formatting and annotation, color selection, data availability, and for reporting image analysis workflows. The goal of our guidelines is to increase the clarity and reproducibility of image figures and thereby heighten the quality and explanatory power of microscopy data is in publications.

Intravital deep-tumor single-beam 3-photon, 4-photon, and harmonic microscopy.

Three-photon excitation has recently been demonstrated as an effective method to perform intravital microscopy in deep, previously inaccessible regions of the mouse brain. The applicability of 3-photon excitation for deep imaging of other, more heterogeneous tissue types has been much less explored. In this work, we analyze the benefit of high-pulse-energy 1 MHz pulse-repetition-rate infrared excitation near 1300 and 1700 nm for in-depth imaging of tumorous and bone tissue. We show that this excitation regime provides a more than 2-fold increased imaging depth in tumor and bone tissue compared to the illumination conditions commonly used in 2-photon excitation, due to improved excitation confinement and reduced scattering. We also show that simultaneous 3- and 4-photon processes can be effectively induced with a single laser line, enabling the combined detection of blue to far-red fluorescence together with second and third harmonic generation without chromatic aberration, at excitation intensities compatible with live tissue imaging. Finally, we analyze photoperturbation thresholds in this excitation regime and derive setpoints for safe cell imaging. Together, these results indicate that infrared high-pulse-energy low-repetition-rate excitation opens novel perspectives for intravital deep-tissue microscopy of multiple parameters in strongly scattering tissues and organs.

Adenosine A2a Receptor Antagonism Restores Additive Cytotoxicity by Cytotoxic T Cells in Metabolically Perturbed Tumors.

Cytotoxic T lymphocytes (CTL) are antigen-specific effector cells with the ability to eradicate cancer cells in a contact-dependent manner. Metabolic perturbation compromises the CTL effector response in tumor subregions, resulting in failed cancer cell elimination despite the infiltration of tumor-specific CTLs. Restoring the functionality of these tumor-infiltrating CTLs is key to improve immunotherapy. Extracellular adenosine is an immunosuppressive metabolite produced within the tumor microenvironment. Here, by applying single-cell reporter strategies in 3D collagen cocultures in vitro and progressing tumors in vivo, we show that adenosine weakens one-to-one pairing of activated effector CTLs with target cells, thereby dampening serial cytotoxic hit delivery and cumulative death induction. Adenosine also severely compromised CTL effector restimulation and expansion. Antagonization of adenosine A2a receptor (ADORA2a) signaling stabilized and prolonged CTL-target cell conjugation and accelerated lethal hit delivery by both individual contacts and CTL swarms. Because adenosine signaling is a near-constitutive confounding parameter in metabolically perturbed tumors, ADORA2a targeting represents an orthogonal adjuvant strategy to enhance immunotherapy efficacy.

Two-photon laser-generated microtracks in 3D collagen lattices: principles of MMP-dependent and -independent collective cancer cell invasion.

Cancer invasion into an extracellular matrix (ECM) results from a biophysical reciprocal interplay between the expanding cancer lesion and tissue barriers imposed by the adjacent microenvironment. In vivo, connective tissue provides both densely packed ECM barriers adjacent to channel/track-like spaces and loosely organized zones, both of which may impact cancer invasion mode and efficiency; however little is known about how three-dimensional (3D) spaces and aligned tracks present in interstitial tissue guide cell invasion. We here describe a two-photon laser ablation procedure to generate 3D microtracks in dense 3D collagen matrices that support and guide collective cancer cell invasion. Whereas collective invasion of mammary tumor (MMT) breast cancer cells into randomly organized collagen networks required matrix metalloproteinase (MMP) activity for cell-derived collagen breakdown, re-alignment and track generation, preformed tracks supported MMP-independent collective invasion down to a track caliber of 3 µm. Besides contact guidance along the track of least resistance and initial cell deformation (squeezing), MMP-independent collective cell strands led to secondary track expansion by a pushing mechanism. Thus, two-photon laser ablation is useful to generate barrier-free microtracks in a 3D ECM which guide collective invasion independently of pericellular proteolysis.

Cell-cell adhesion and 3D matrix confinement determine jamming transitions in breast cancer invasion.

Plasticity of cancer invasion and metastasis depends on the ability of cancer cells to switch between collective and single-cell dissemination, controlled by cadherin-mediated cell-cell junctions. In clinical samples, E-cadherin-expressing and -deficient tumours both invade collectively and metastasize equally, implicating additional mechanisms controlling cell-cell cooperation and individualization. Here, using spatially defined organotypic culture, intravital microscopy of mammary tumours in mice and in silico modelling, we identify cell density regulation by three-dimensional tissue boundaries to physically control collective movement irrespective of the composition and stability of cell-cell junctions. Deregulation of adherens junctions by downregulation of E-cadherin and p120-catenin resulted in a transition from coordinated to uncoordinated collective movement along extracellular boundaries, whereas single-cell escape depended on locally free tissue space. These results indicate that cadherins and extracellular matrix confinement cooperate to determine unjamming transitions and stepwise epithelial fluidization towards, ultimately, cell individualization.

Community standards for open cell migration data.

Cell migration research has become a high-content field. However, the quantitative information encapsulated in these complex and high-dimensional datasets is not fully exploited owing to the diversity of experimental protocols and non-standardized output formats. In addition, typically the datasets are not open for reuse. Making the data open and Findable, Accessible, Interoperable, and Reusable (FAIR) will enable meta-analysis, data integration, and data mining. Standardized data formats and controlled vocabularies are essential for building a suitable infrastructure for that purpose but are not available in the cell migration domain. We here present standardization efforts by the Cell Migration Standardisation Organisation (CMSO), an open community-driven organization to facilitate the development of standards for cell migration data. This work will foster the development of improved algorithms and tools and enable secondary analysis of public datasets, ultimately unlocking new knowledge of the complex biological process of cell migration.

Changes in membrane sphingolipid composition modulate dynamics and adhesion of integrin nanoclusters.

Sphingolipids are essential constituents of the plasma membrane (PM) and play an important role in signal transduction by modulating clustering and dynamics of membrane receptors. Changes in lipid composition are therefore likely to influence receptor organisation and function, but how this precisely occurs is difficult to address given the intricacy of the PM lipid-network. Here, we combined biochemical assays and single molecule dynamic approaches to demonstrate that the local lipid environment regulates adhesion of integrin receptors by impacting on their lateral mobility. Induction of sphingomyelinase (SMase) activity reduced sphingomyelin (SM) levels by conversion to ceramide (Cer), resulting in impaired integrin adhesion and reduced integrin mobility. Dual-colour imaging of cortical actin in combination with single molecule tracking of integrins showed that this reduced mobility results from increased coupling to the actin cytoskeleton brought about by Cer formation. As such, our data emphasizes a critical role for the PM local lipid composition in regulating the lateral mobility of integrins and their ability to dynamically increase receptor density for efficient ligand binding in the process of cell adhesion.

Directing collagen fibers using counter-rotating cone extrusion.

The bio-inspired engineering of tissue equivalents should take into account anisotropic morphology and the mechanical properties of the extracellular matrix. This especially applies to collagen fibrils, which have various, but highly defined, orientations throughout tissues and organs. There are several methods available to control the alignment of soluble collagen monomers, but the options to direct native insoluble collagen fibers are limited. Here we apply a controlled counter-rotating cone extrusion technology to engineer tubular collagen constructs with defined anisotropy. Driven by diverging inner and outer cone rotation speeds, collagen fibrils from bovine skin were extruded and precipitated onto mandrels as tubes with oriented fibers and bundles, as examined by second harmonic generation microscopy and quantitative image analysis. A clear correlation was found whereby the direction and extent of collagen fiber alignment during extrusion were a function of the shear forces caused by a combination of the cone rotation and flow direction. A gradual change in the fiber direction, spanning +50 to -40°, was observed throughout the sections of the sample, with an average decrease ranging from 2.3 to 2.6° every 10µm. By varying the cone speeds, the collagen constructs showed differences in elasticity and toughness, spanning 900-2000kPa and 19-35mJ, respectively. Rotational extrusion presents an enabling technology to create and control the (an)isotropic architecture of collagen constructs for application in tissue engineering and regenerative medicine.

Intravital third harmonic generation microscopy of collective melanoma cell invasion: Principles of interface guidance and microvesicle dynamics.

Cancer cell invasion is an adaptive process based on cell-intrinsic properties to migrate individually or collectively, and their adaptation to encountered tissue structure acting as barrier or providing guidance. Whereas molecular and physical mechanisms of cancer invasion are well-studied in 3D in vitro models, their topographic relevance, classification and validation toward interstitial tissue organization in vivo remain incomplete. Using combined intravital third and second harmonic generation (THG, SHG), and three-channel fluorescence microscopy in live tumors, we here map B16F10 melanoma invasion into the dermis with up to 600 µm penetration depth and reconstruct both invasion mode and tissue tracks to establish invasion routes and outcome. B16F10 cells preferentially develop adaptive invasion patterns along preformed tracks of complex, multi-interface topography, combining single-cell and collective migration modes, without immediate anatomic tissue remodeling or destruction. The data suggest that the dimensionality (1D, 2D, 3D) of tissue interfaces determines the microanatomy exploited by invading tumor cells, emphasizing non-destructive migration along microchannels coupled to contact guidance as key invasion mechanisms. THG imaging further detected the presence and interstitial dynamics of tumor-associated microparticles with submicron resolution, revealing tumor-imposed conditioning of the microenvironment. These topographic findings establish combined THG, SHG and fluorescence microscopy in intravital tumor biology and provide a template for rational in vitro model development and context-dependent molecular classification of invasion modes and routes.

Fluorescence lifetime microscopy of tumor cell invasion, drug delivery, and cytotoxicity.

Fluorescence lifetime imaging microscopy (FLIM) enables detection of complex molecular assemblies within a single voxel for studies of cell function and communication with subcellular resolution in optically transparent tissue. We describe a fast FLIM technique consisting of a novel time-correlated single-photon counting (TCSPC) detector that features 80 MHz average count rate and the phasor analysis for efficient data acquisition and evaluation. This method in combination with multiphoton microscopy enables acquisition of a lifetime image every 1-2 s in 3D live organotypic tissue culture. 3D time-lapse fluorescence lifetime data were acquired over up to 20 h and analyzed by using exponential fitting and phasor analysis. By correlating specific areas in the phasor plot to the actual image, we obtained direct insight into cancer-cell invasion into a 3D collagen matrix, the differential uptake of doxorubicin by cells, and the consequences on cell invasion and apoptosis induction. Based on the fast acquisition and simplified image postprocessing and quantification, time-lapse 3D FLIM is a versatile approach for monitoring the 3D topography, kinetics, and biological output of structurally and spectrally complex cell and tissue models.