Tissue-Like 3D Standard and Protocols for Microscope Quality Management.

This article outlines a global study conducted by the Association of Biomedical Resource Facilities (ABRF) Light Microscopy Research Group (LMRG). The results present a novel 3D tissue-like biologically relevant standard sample that is affordable and straightforward to prepare. Detailed sample preparation, instrument-specific image acquisition protocols and image analysis methods are presented and made available to the community. The standard consists of sub-resolution and large well characterized relative intensity fluorescence microspheres embedded in a 120 µm thick 3D gel with a refractive index of 1.365. The standard allows the evaluation of several properties as a function of depth. These include the following: 1) microscope resolution with automated analysis of the point-spread function (PSF), 2) automated signal-to-noise ratio analysis, 3) calibration and correction of fluorescence intensity loss, and 4) quantitative relative intensity. Results demonstrate expected refractive index mismatch dependent losses in intensity and resolution with depth, but the relative intensities of different objects at similar depths are maintained. This is a robust standard showing reproducible results across laboratories, microscope manufacturers and objective lens types (e.g., magnification, immersion medium). Thus, these tools will be valuable for the global community to benchmark fluorescence microscopes and will contribute to improved scientific rigor and reproducibility.

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.

Best practices and tools for reporting reproducible fluorescence microscopy methods.

Although fluorescence microscopy is ubiquitous in biomedical research, microscopy methods reporting is inconsistent and perhaps undervalued. We emphasize the importance of appropriate microscopy methods reporting and seek to educate researchers about how microscopy metadata impact data interpretation. We provide comprehensive guidelines and resources to enable accurate reporting for the most common fluorescence light microscopy modalities. We aim to improve microscopy reporting, thus improving the quality, rigor and reproducibility of image-based science.

Combined iDISCO and CUBIC tissue clearing and lightsheet microscopy for in toto analysis of the adult mouse ovary†.

At any given time, the ovary contains a number of follicles in distinct growth stages, each with a set of identifying characteristics. Although follicle counting and staging using histological stains on paraffin-embedded ovary sections has been the gold standard in assessing ovarian health in fertility studies, the final counts rely on extrapolation factors that diverge greatly among studies. These methods also limit our ability to investigate spatial aspects of ovary organization. Recent advances in optical tissue clearing and lightsheet microscopy have permitted comprehensive analysis of intact tissues. In this study, we set out to determine the best clearing and imaging methods to generate 3D images of the complete adult mouse ovary that could be used for accurate assessments of ovarian follicles. We found that a combination of iDISCO and CUBIC was the best method to clear the immunostained ovary. Using lightsheet microscopy, we generated 3D images of the intact ovary and performed qualitative assessments of follicles at all stages of development. This study is an important step toward developing quantitative computational models that allow rapid and accurate assessments of growing and quiescent primordial follicles, and to investigate the integrity of extrinsic ovarian components including vascular and neuronal networks.

Intermittent gastric feeds lower insulin requirements without worsening dysglycemia: A pilot randomized crossover trial.

INTRODUCTION: We hypothesized that critically ill medical patients would require less insulin when fed intermittently. METHODS: First, 26 patients were randomized to receive intermittent or continuous gastric feeds. Once at goal nutrition, data were collected for the first 4-hr data collection period. Next, the enteral feed type was switched, goal nutrition was repeated, and a second 4-h data collection period was completed. The primary endpoint was the total amount of insulin infused; secondary endpoints were glucose concentration mean, maximum, minimum, and standard deviation, as well as episodes of hypoglycemia. RESULTS: Sixteen of the 26 patients successfully completed the protocol. One patient experienced a large, rapid, and sustained decline in insulin requirement from liver failure, creating a bias of lesser insulin in the intermittent arm; this patient was removed from the analysis. For the remaining 15 patients, the average total amount of insulin infused was 1.4 U/patient/h less following intermittent feeds: P =0.027, 95% confidence interval (0.02, 11.17), and effect size 0.6. Secondary endpoints were statistically similar. CONCLUSIONS: Critically ill medical patients who require an insulin infusion have a reduced insulin requirement when fed intermittently, whereas dysglycemia metrics are not adversely affected. A larger clinical study is required to confirm these findings.

Photoreceptor disc membranes are formed through an Arp2/3-dependent lamellipodium-like mechanism.

The light-sensitive outer segment of the vertebrate photoreceptor is a highly modified primary cilium filled with disc-shaped membranes that provide a vast surface for efficient photon capture. The formation of each disc is initiated by a ciliary membrane evagination driven by an unknown molecular mechanism reportedly requiring actin polymerization. Since a distinct F-actin network resides precisely at the site of disc morphogenesis, we employed a unique proteomic approach to identify components of this network potentially driving disc morphogenesis. The only identified actin nucleator was the Arp2/3 complex, which induces the polymerization of branched actin networks. To investigate the potential involvement of Arp2/3 in the formation of new discs, we generated a conditional knockout mouse lacking its essential ArpC3 subunit in rod photoreceptors. This knockout resulted in the complete loss of the F-actin network specifically at the site of disc morphogenesis, with the time course of ArpC3 depletion correlating with the time course of F-actin loss. Without the actin network at this site, the initiation of new disc formation is completely halted, forcing all newly synthesized membrane material to be delivered to the several nascent discs whose morphogenesis had already been in progress. As a result, these discs undergo uncontrolled expansion instead of normal enclosure, which leads to formation of unusual, large membrane whorls. These data suggest a model of photoreceptor disc morphogenesis in which Arp2/3 initiates disc formation in a "lamellipodium-like" mechanism.

Vitamin D receptor regulates autophagy in the normal mammary gland and in luminal breast cancer cells.

Women in North America have a one in eight lifetime risk of developing breast cancer (BC), and a significant proportion of these individuals will develop recurrent BC and will eventually succumb to the disease. Metastatic, therapy-resistant BC cells are refractory to cell death induced by multiple stresses. Here, we document that the vitamin D receptor (VDR) acts as a master transcriptional regulator of autophagy. Activation of the VDR by vitamin D induces autophagy and an autophagic transcriptional signature in BC cells that correlates with increased survival in patients; strikingly, this signature is present in the normal mammary gland and is progressively lost in patients with metastatic BC. A number of epidemiological studies have shown that sufficient vitamin D serum levels might be protective against BC. We observed that dietary vitamin D supplementation in mice increases basal levels of autophagy in the normal mammary gland, highlighting the potential of vitamin D as a cancer-preventive agent. These findings point to a role of vitamin D and the VDR in modulating autophagy and cell death in both the normal mammary gland and BC cells.

Two familial ALS proteins function in prevention/repair of transcription-associated DNA damage.

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron dysfunction disease that leads to paralysis and death. There is currently no established molecular pathogenesis pathway. Multiple proteins involved in RNA processing are linked to ALS, including FUS and TDP43, and we propose a disease mechanism in which loss of function of at least one of these proteins leads to an accumulation of transcription-associated DNA damage contributing to motor neuron cell death and progressive neurological symptoms. In support of this hypothesis, we find that FUS or TDP43 depletion leads to increased sensitivity to a transcription-arresting agent due to increased DNA damage. Thus, these proteins normally contribute to the prevention or repair of transcription-associated DNA damage. In addition, both FUS and TDP43 colocalize with active RNA polymerase II at sites of DNA damage along with the DNA damage repair protein, BRCA1, and FUS and TDP43 participate in the prevention or repair of R loop-associated DNA damage, a manifestation of aberrant transcription and/or RNA processing. Gaining a better understanding of the role(s) that FUS and TDP43 play in transcription-associated DNA damage could shed light on the mechanisms underlying ALS pathogenesis.

The coupling between sister kinetochore directional instability and oscillations in centromere stretch in metaphase PtK1 cells.

Kinetochores bound to kinetochore microtubules (kMTs) exhibit directional instability in mammalian and other mitotic vertebrate cells, oscillating between poleward (P) and away-from-the-pole (AP) movements. These oscillations are coupled to changes in length of kMTs in a way that maintains a net stretch of the centromere. To understand how sister kinetochore directional instability and kMT plus-end dynamic instability are coupled to oscillations in centromere stretch, we tracked at high resolution the positions of fluorescent kinetochores and their poles for oscillating chromosomes within spindles of metaphase PtK1 cells. We found that the kinetics of P and AP movement are nonlinear and different. By subtracting contributions from the poleward flux of kMTs, we found that maximum centromere stretch occurred when the leading kinetochore switched from depolymerization to polymerization, whereas minimum centromere stretch occurred on average 7 s after the initially trailing kinetochore switched from polymerization to depolymerization. These differences produce oscillations in centromere stretch at about twice the frequency of kinetochore directional instability and at about twice the frequency of centromere oscillations back and forth across the spindle equator.

Proinvasion metastasis drivers in early-stage melanoma are oncogenes.

Clinical and genomic evidence suggests that the metastatic potential of a primary tumor may be dictated by prometastatic events that have additional oncogenic capability. To test this "deterministic" hypothesis, we adopted a comparative oncogenomics-guided function-based strategy involving: (1) comparison of global transcriptomes of two genetically engineered mouse models with contrasting metastatic potential, (2) genomic and transcriptomic profiles of human melanoma, (3) functional genetic screen for enhancers of cell invasion, and (4) evidence of expression selection in human melanoma tissues. This integrated effort identified six genes that are potently proinvasive and oncogenic. Furthermore, we show that one such gene, ACP5, confers spontaneous metastasis in vivo, engages a key pathway governing metastasis, and is prognostic in human primary melanomas.

Fluorescent speckle microscopy.

INTRODUCTION: Fluorescent speckle microscopy (FSM) is a live imaging and quantitative measurement technique used for analyzing motion and turnover of macromolecular assemblies in vivo and in vitro. It differs from related imaging techniques such as photobleaching and photoactivation in its use of substantially lower concentrations of fluorescently labeled assembly subunits. When small numbers of labeled subunits and large numbers of unlabeled subunits become randomly incorporated together into a macromolecular structure, the random distribution of fluorophores generates nonuniform fluorescence intensity patterns that appear as distinct puncta against low background fluorescence. These puncta, called speckles, serve as fiduciary markers so that motion and turnover of the structure are visualized. Computational analysis of speckle image data transforms FSM into a powerful tool for high-resolution quantitative analysis of macromolecular assembly dynamics. Successful application of FSM depends on the ability to reliably generate and image speckles, which are characterized by their weak emission signals, and to effectively extract quantitative information through computational analysis of speckle image data, which are characterized by their stochastic fluctuations, low signal-to-noise ratios, and high spatiotemporal complexity. This article aims to provide a practical introduction to basic principles, experimental implementation, and computational data analysis of FSM. Examples are used to show the application of FSM in analyzing the dynamic organization and assembly/disassembly of cytoskeletal filament networks, an area in which FSM analysis has found great success.

Synchronizing chromosome segregation by flux-dependent force equalization at kinetochores.

The synchronous movement of chromosomes during anaphase ensures their correct inheritance in every cell division. This reflects the uniformity of spindle forces acting on chromosomes and their simultaneous entry into anaphase. Although anaphase onset is controlled by the spindle assembly checkpoint, it remains unknown how spindle forces are uniformly distributed among different chromosomes. In this paper, we show that tension uniformity at metaphase kinetochores and subsequent anaphase synchrony in Drosophila S2 cells are promoted by spindle microtubule flux. These results can be explained by a mechanical model of the spindle where microtubule poleward translocation events associated with flux reflect relaxation of the kinetochore-microtubule interface, which accounts for the redistribution and convergence of kinetochore tensions in a timescale comparable to typical metaphase duration. As predicted by the model, experimental acceleration of mitosis precludes tension equalization and anaphase synchrony. We propose that flux-dependent equalization of kinetochore tensions ensures a timely and uniform maturation of kinetochore-microtubule interfaces necessary for error-free and coordinated segregation of chromosomes in anaphase.

Regional variation of microtubule flux reveals microtubule organization in the metaphase meiotic spindle.

Continuous poleward movement of tubulin is a hallmark of metaphase spindle dynamics in higher eukaryotic cells and is essential for stable spindle architecture and reliable chromosome segregation. We use quantitative fluorescent speckle microscopy to map with high resolution the spatial organization of microtubule flux in Xenopus laevis egg extract meiotic spindles. We find that the flux velocity decreases near spindle poles by approximately 20%. The regional variation is independent of functional kinetochores and centrosomes and is suppressed by inhibition of dynein/dynactin, kinesin-5, or both. Statistical analysis reveals that tubulin flows in two distinct velocity modes. We propose an association of these modes with two architecturally distinct yet spatially overlapping and dynamically cross-linked arrays of microtubules: focused polar microtubule arrays of a uniform polarity and slower flux velocities are interconnected by a dense barrel-like microtubule array of antiparallel polarities and faster flux velocities.

Kinesin 5-independent poleward flux of kinetochore microtubules in PtK1 cells.

Forces in the spindle that align and segregate chromosomes produce a steady poleward flux of kinetochore microtubules (MTs [kMTs]) in higher eukaryotes. In several nonmammalian systems, flux is driven by the tetrameric kinesin Eg5 (kinesin 5), which slides antiparallel MTs toward their minus ends. However, we find that the inhibition of kinesin 5 in mammalian cultured cells (PtK1) results in only minor reduction in the rate of kMT flux from approximately 0.7 to approximately 0.5 microm/min, the same rate measured in monopolar spindles that lack antiparallel MTs. These data reveal that the majority of poleward flux of kMTs in these cells is not driven by Eg5. Instead, we favor a polar "pulling-in" mechanism in which a depolymerase localized at kinetochore fiber minus ends makes a major contribution to poleward flux. One candidate, Kif2a (kinesin 13), was detected at minus ends of fluxing kinetochore fibers. Kif2a remains associated with the ends of K fibers upon disruption of the spindle by dynein/dynactin inhibition, and these K fibers flux.

Anaphase spindle mechanics prevent mis-segregation of merotelically oriented chromosomes.

Merotelic kinetochore orientation is a kinetochore misattachment in which a single kinetochore is attached to microtubules from both spindle poles instead of just one. It can be favored in specific circumstances, is not detected by the mitotic checkpoint, and induces lagging chromosomes in anaphase. In mammalian cells, it occurs at high frequency in early mitosis, but few anaphase cells show lagging chromosomes. We developed live-cell imaging methods to determine whether and how the mitotic spindle prevents merotelic kinetochores from producing lagging chromosomes. We found that merotelic kinetochores entering anaphase never lost attachment to the spindle poles; they remained attached to both microtubule bundles, but this did not prevent them from segregating correctly. The two microtubule bundles usually showed different fluorescence intensities, the brighter bundle connecting the merotelic kinetochore to the correct pole. During anaphase, the dimmer bundle lengthened much more than the brighter bundle as spindle elongation occurred. This resulted in correct segregation of the merotelically oriented chromosome. We propose a model based on the ratios of microtubules to the correct versus incorrect pole for how anaphase spindle dynamics and microtubule polymerization at kinetochores prevent potential segregation errors deriving from merotelic kinetochore orientation.

XRHAMM functions in ran-dependent microtubule nucleation and pole formation during anastral spindle assembly.

BACKGROUND: The regulated assembly of microtubules is essential for bipolar spindle formation. Depending on cell type, microtubules nucleate through two different pathways: centrosome-driven or chromatin-driven. The chromatin-driven pathway dominates in cells lacking centrosomes. RESULTS: Human RHAMM (receptor for hyaluronic-acid-mediated motility) was originally implicated in hyaluronic-acid-induced motility but has since been shown to associate with centrosomes and play a role in astral spindle pole integrity in mitotic systems. We have identified the Xenopus ortholog of human RHAMM as a microtubule-associated protein that plays a role in focusing spindle poles and is essential for efficient microtubule nucleation during spindle assembly without centrosomes. XRHAMM associates both with gamma-TuRC, a complex required for microtubule nucleation and with TPX2, a protein required for microtubule nucleation and spindle pole organization. CONCLUSIONS: XRHAMM facilitates Ran-dependent, chromatin-driven nucleation in a process that may require coordinate activation of TPX2 and gamma-TuRC.