Dimerization of the Alzheimer's disease pathogenic receptor SORLA regulates its association with retromer.

SORL1, the gene encoding the large multidomain SORLA protein, has emerged as only the fourth gene that when mutated can by itself cause Alzheimer's disease (AD), and as a gene reliably linked to both the early- and late-onset forms of the disease. SORLA is known to interact with the endosomal trafficking regulatory complex called retromer in regulating the recycling of endosomal cargo, including the amyloid precursor protein (APP) and the glutamate receptor GluA1. Nevertheless, SORLA's precise structural-functional relationship in endosomal recycling tubules remains unknown. Here, we address these outstanding questions by relying on crystallographic and artificial-intelligence evidence to generate a structural model for how SORLA folds and fits into retromer-positive endosomal tubules, where it is found to dimerize via both SORLA's fibronectin-type-III (3Fn)- and VPS10p-domains. Moreover, we identify a SORLA fragment comprising the 3Fn-, transmembrane, and cytoplasmic domains that has the capacity to form a dimer, and to enhance retromer-dependent recycling of APP by decreasing its amyloidogenic processing. Collectively, these observations generate a model for how SORLA dimer (and possibly polymer) formation can function in stabilizing and enhancing retromer function at endosome tubules. These findings can inform investigation of the many AD-associated SORL1 variants for evidence of pathogenicity and can guide discovery of novel drugs for the disease.

Characterization of Immune Cell Populations of Cutaneous Neurofibromas in Neurofibromatosis 1.

Cutaneous neurofibromas (cNFs) are characteristic of neurofibromatosis 1 (NF1), yet their immune microenvironment is incompletely known. A total of 61 cNFs from 10 patients with NF1 were immunolabeled for different types of T cells and macrophages, and the cell densities were correlated with clinical characteristics. Eight cNFs and their overlying skin were analyzed for T cell receptor CDR domain sequences, and mass spectrometry of 15 cNFs and the overlying skin was performed to study immune-related processes. Intratumoral T cells were detected in all cNFs. Tumors from individuals younger than the median age of the study participants (33 years), growing tumors, and tumors smaller than the data set median showed increased T cell density. Most samples displayed intratumoral or peritumoral aggregations of CD3-positive cells. T cell receptor sequencing demonstrated that the skin and cNFs host distinct T cell populations, whereas no dominant cNF-specific T cell clones were detected. Unique T cell clones were fewer in cNFs than in skin, and mass spectrometry suggested lower expression of proteins related to T cell-mediated immunity in cNFs than in skin. CD163-positive cells, suggestive of M2 macrophages, were abundant in cNFs. Human cNFs have substantial T cell and macrophage populations that may be tumor-specific.

A familial missense variant in the Alzheimer's disease gene SORL1 impairs its maturation and endosomal sorting.

The SORL1 gene has recently emerged as a strong Alzheimer's Disease (AD) risk gene. Over 500 different variants have been identified in the gene and the contribution of individual variants to AD development and progression is still largely unknown. Here, we describe a family consisting of 2 parents and 5 offspring. Both parents were affected with dementia and one had confirmed AD pathology with an age of onset > 75 years. All offspring were affected with AD with ages at onset ranging from 53 years to 74 years. DNA was available from the parent with confirmed AD and 5 offspring. We identified a coding variant, p.(Arg953Cys), in SORL1 in 5 of 6 individuals affected by AD. Notably, variant carriers had severe AD pathology, and the SORL1 variant segregated with TDP-43 pathology (LATE-NC). We further characterized this variant and show that this Arginine substitution occurs at a critical position in the YWTD-domain of the SORL1 translation product, SORL1. Functional studies further show that the p.R953C variant leads to retention of the SORL1 protein in the endoplasmic reticulum which leads to decreased maturation and shedding of the receptor and prevents its normal endosomal trafficking. Together, our analysis suggests that p.R953C is a pathogenic variant of SORL1 and sheds light on mechanisms of how missense SORL1 variants may lead to AD.

Recognising the importance and impact of Imaging Scientists: Global guidelines for establishing career paths within core facilities.

In the dynamic landscape of scientific research, imaging core facilities are vital hubs propelling collaboration and innovation at the technology development and dissemination frontier. Here, we present a collaborative effort led by Global BioImaging (GBI), introducing international recommendations geared towards elevating the careers of Imaging Scientists in core facilities. Despite the critical role of Imaging Scientists in modern research ecosystems, challenges persist in recognising their value, aligning performance metrics and providing avenues for career progression and job security. The challenges encompass a mismatch between classic academic career paths and service-oriented roles, resulting in a lack of understanding regarding the value and impact of Imaging Scientists and core facilities and how to evaluate them properly. They further include challenges around sustainability, dedicated training opportunities and the recruitment and retention of talent. Structured across these interrelated sections, the recommendations within this publication aim to propose globally applicable solutions to navigate these challenges. These recommendations apply equally to colleagues working in other core facilities and research institutions through which access to technologies is facilitated and supported. This publication emphasises the pivotal role of Imaging Scientists in advancing research programs and presents a blueprint for fostering their career progression within institutions all around the world.

Mast Cells in Human Cutaneous Neurofibromas: Density, Subtypes, and Association with Clinical Features in Neurofibromatosis 1.

BACKGROUND: Cutaneous neurofibromas (cNFs) are hallmarks of neurofibromatosis 1 (NF1) and cause the main disease burden in adults with NF1. Mast cells are a known component of cNFs. However, no comprehensive characterization of mast cells in cNFs is available, and their contributions to cNF growth and symptoms such as itch are not known. METHODS: We collected 60 cNFs from ten individuals with NF1, studied their mast cell proteinase content, and compared the mast cell numbers to selected clinical features of the tumors and patients. The tumors were immunolabeled for the mast cell markers CD117, tryptase, and chymase, and the percentage of immunopositive cells was determined using computer-assisted methods. RESULTS: The median proportions of positive cells were 5.5% (range 0.1-14.4) for CD117, 4.0% (1.2-7.0) for tryptase, and 5.0% (1.1-15.9) for chymase. The median densities of cells immunopositive for CD117, tryptase, and chymase were 280, 243, and 250 cells/mm2, respectively. Small tumors, growing tumors, and tumors from patients below the median age of 33 years displayed a high proportion of mast cells. Cells expressing both tryptase and chymase were the predominant mast cell type in cNFs, followed by cells expressing chymase only. CONCLUSION: The results highlight the abundance of mast cells in cNFs and that their number and subtypes clearly differ from those previously reported in unaffected skin.

Novel long noncoding RNAs upregulation may have synergistic effects on the CYP24A1 and PFDN4 biomarker role in human colorectal cancer.

Long noncoding RNAs (lncRNAs) are emerging as the master regulators of tumor initiation, proliferation, and metastasis; however, their diagnostic value as potential biomarkers should be clarified. Vitamin D influences the expression of several genes in various pathways, including the CYP24A1 gene in the vitamin D metabolism pathway. In the present research, we surveyed the expression levels and clinical significance of novel lncRNAs related to CYP24A1 and PFDN4 genes in colorectal cancer (CRC) using real-time polymerase chain reaction. Furthermore, we assessed the expression of these genes after vitamin D treatment in HCT-116 and HT-29 colon cancer cell lines. Our results indicated that the transcription of CYP24A1, PFDN4, and nearby lncRNAs was affected by vitamin D treatment in HCT-116 and HT-29 cell lines. Moreover, CYP24A1, PFDN4, lnc-CYP24A1-3:1, and lnc-TSHZ2-19:1 were upregulated and had the potential to distinguish colorectal cancer tissues from the adjacent tissues by the large area under the receiver operating characteristic curve (0.94, 0.66, 0.70, and 0.60, respectively). lnc-TSHZ2-19:1 expression level significantly correlated with gender (p = .03). In conclusion, CYP24A1, PFDN4, lnc-CYP24A1-3:1, and lnc-TSHZ2-19:1 can be used as potential diagnostic biomarkers in the specific and sensitive assessment of CRC. Besides this, vitamin D treatment may modulate the expression of these genes in a cell-specific manner.

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.

CYP24A1 expression analysis in uterine leiomyoma regarding MED12 mutation profile.

INTRODUCTION: Uterine leiomyoma (ULM) is the most common gynecological tumor. Recent studies have revealed the role of hypovitaminosis D as a major risk factor in the disease development. CYP24A, a mitochondrial enzyme that catalyzes the degradation of 1,25(OH)2D3, is reported to be over-expressed in several human cancers. In this study, we aimed to investigate the expression level of CYP24A1 in leiomyoma samples compared with the adjacent tissues regarding the MED12 mutation profile. MATERIALS AND METHODS: In the present study, 61 ULMs and adjacent tissue samples were collected from 51 women undergoing hysterectomy and myomectomy. The samples were Sanger sequenced for MED12 mutation, and the expression level of CYP24A1 was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: The results demonstrated that CYP24A1 gene was ectopically expressed in 18% of uterine leiomyoma tissues, although this expression was independent of the MED12 mutation profile. CONCLUSION: The findings of the present study support current evidence that dysregulation of vitamin D signaling and metabolic pathways may be involved in at least some subtypes of ULMs.

Ectopic expression of CYP24A1 circular RNA hsa_circ_0060927 in uterine leiomyomas.

BACKGROUND: As a novel class of non-coding RNAs, the role of circular RNAs (circRNAs) in tumor biogenesis and progression has been proved in a number of human tumors; however, up to now, the relation between circRNAs and uterine leiomyomas (ULM) remains unclear. METHODS: In this study, we have estimated the expression level of CYP24A1 hsa_circ_0060927 in uterine leiomyoma and adjacent tissues considering the mediator complex subunit 12 gene (MED12) mutation profile by quantitative real-time polymerase chain reaction (qRT-PCRs). RESULTS: Using Sanger sequencing method, somatic mutations in the MED12 exon 2 were detected in 14 (35.90%) ULM samples, including 10 (71.43%) missense mutations and 4 (28.57%) in-frame deletions. Our results revealed that hsa_circ_0060927 was ectopically expressed in 33.33% of ULM tissues; although, this expression was independent of the MED12 mutation profile in the ULM samples. CONCLUSIONS: Present results provide primary evidence for the role of circular RNAs in the leiomyoma development; however, further studies are essential to confirm the importance of these molecules as potential biomarkers for diagnosis and/or prognosis in ULM.

Sphingolipids inhibit vimentin-dependent cell migration.

The sphingolipids, sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC), can induce or inhibit cellular migration. The intermediate filament protein vimentin is an inducer of migration and a marker for epithelial-mesenchymal transition. Given that keratin intermediate filaments are regulated by SPC, with consequences for cell motility, we wanted to determine whether vimentin is also regulated by sphingolipid signalling and whether it is a determinant for sphingolipid-mediated functions. In cancer cells where S1P and SPC inhibited migration, we observed that S1P and SPC induced phosphorylation of vimentin on S71, leading to a corresponding reorganization of vimentin filaments. These effects were sphingolipid-signalling-dependent, because inhibition of either the S1P2 receptor (also known as S1PR2) or its downstream effector Rho-associated kinase (ROCK, for which there are two isoforms ROCK1 and ROCK2) nullified the sphingolipid-induced effects on vimentin organization and S71 phosphorylation. Furthermore, the anti-migratory effect of S1P and SPC could be prevented by expressing S71-phosphorylation-deficient vimentin. In addition, we demonstrated, by using wild-type and vimentin-knockout mouse embryonic fibroblasts, that the sphingolipid-mediated inhibition of migration is dependent on vimentin. These results imply that this newly discovered sphingolipid-vimentin signalling axis exerts brake-and-throttle functions in the regulation of cell migration.

Ratiometric Sensing and Imaging of Intracellular pH Using Polyethylenimine-Coated Photon Upconversion Nanoprobes.

Measurement of changes of pH at various intracellular compartments has potential to solve questions concerning the processing of endocytosed material, regulation of the acidification process, and also acidification of vesicles destined for exocytosis. To monitor these events, the nanosized optical pH probes need to provide ratiometric signals in the optically transparent biological window, target to all relevant intracellular compartments, and to facilitate imaging at subcellular resolution without interference from the biological matrix. To meet these criteria we sensitize the surface conjugated pH sensitive indicator via an upconversion process utilizing an energy transfer from the nanoparticle to the indicator. Live cells were imaged with a scanning confocal microscope equipped with a low-energy 980 nm laser excitation, which facilitated high resolution and penetration depth into the specimen, and low phototoxicity needed for long-term imaging. Our upconversion nanoparticle resonance energy transfer based sensor with polyethylenimine-coating provides high colloidal stability, enhanced cellular uptake, and distribution across cellular compartments. This distribution was modulated with membrane integrity perturbing treatment that resulted into total loss of lysosomal compartments and a dramatic pH shift of endosomal compartments. These nanoprobes are well suited for detection of pH changes in in vitro models with high biological background fluorescence and in in vivo applications, e.g., for the bioimaging of small animal models.

Diversity of actin architecture in human osteoclasts: network of curved and branched actin supporting cell shape and intercellular micrometer-level tubes.

Osteoclasts are multinucleated bone-resorbing cells with a dynamic actin cytoskeleton. Osteoclasts are derived from circulating mononuclear precursors. Confocal and stimulated emission depletion (STED) super-resolution microscopy was used to investigate peripheral blood-derived human osteoclasts cultured on glass surfaces. STED and confocal microscopy demonstrated that the actin was curved and branched, for instance, in the vicinity of membrane ruffles. The overall architecture of the curved actin network extended from the podosomes to the top of the cell. The other novel finding was that a micrometer-level tube containing actin bridged the osteoclasts well above the level of the culture glass. The actin filaments of the tubes originated from the network of curved actin often surrounding a group of nuclei. Furthermore, nuclei were occasionally located inside the tubes. Our findings demonstrated the accumulation of c-Src, cortactin, cofilin, and actin around nuclei suggesting their role in nuclear processes such as the locomotion of nuclei. ARP2/3 labeling was abundant at the substratum level of osteoclasts and in the branched actin network, where it localized to the branching points. We speculate that the actin-containing tubes of osteoclasts may provide a means of transportation of nuclei, e.g., during the fusion of osteoclasts. These novel findings can pave the way for future studies aiming at the elucidation of the differentiation of multinucleated osteoclasts.

Advanced Cellulose Fibers for Efficient Immobilization of Enzymes.

Biocatalytic pulp fibers were prepared using surface functionalization of bleached kraft pulp with amino groups (F) and further immobilization of a cross-linked glucose oxidase (G*) from Aspergillus niger. The cross-linked enzymes (G*) were characterized using X-ray spectroscopy, Fourier transform infrared spectroscopy, dynamic scanning calorimetry, and dynamic light scattering. According to standard assays, the G* content on the resulting fibers (FG*) was of 11 mg/g of fiber, and enzyme activity was of 215 U/g. The results from confocal- and stimulated emission depletion microscopy techniques demonstrated that glucose oxidase do not penetrate the interlayers of fibers. The benefit of pulp fiber functionalization was evident in the present case, as the introduction of amino groups allowed the immobilization of larger amount of enzymes and rendered more efficient systems. Using the approach described on this paper, several advanced materials from wood pulp fibers and new bioprocesses might be developed by selecting the correct enzyme for the target applications.

Functional characterization of SORL1 variants in cell-based assays to investigate variant pathogenicity.

SORLA, the protein encoded by the SORL1 gene, has an important role in recycling cargo proteins to the cell surface. While SORLA loss-of-function variants occur almost exclusively in Alzheimer's disease cases, the majority of SORL1 variants are missense variants that are individually rare and can have individual mechanisms how they impair SORLA function as well as have individual effect size on disease risk. However, since carriers mostly come from small pedigrees, it is challenging to determine variant penetrance, leaving clinical significance associated with most missense variants unclear. In this article, we present functional approaches to evaluate the pathogenicity of a SORL1 variant, p.D1105H. First, we generated our mutant receptor by inserting the D1105H variant into the full-length SORLA-WT receptor. Then using western blot analysis we quantified the effect of the mutation on maturation and shedding of the receptor for transfected cells, and finally applied a flow cytometry approach to quantify SORLA expression at the cell surface. The results showed decreased maturation, decreased shedding, and decreased cell surface expression of D1105H compared with wild-type SORLA. We propose how these approaches can be used to functionally assess the pathogenicity of SORL1 variants in the future. This article is part of a discussion meeting issue 'Understanding the endo-lysosomal network in neurodegeneration'.

A familial missense variant in the Alzheimer's Disease gene SORL1 impairs its maturation and endosomal sorting.

The SORL1 gene has recently emerged as a strong Alzheimer's Disease (AD) risk gene. Over 500 different variants have been identified in the gene and the contribution of individual variants to AD development and progression is still largely unknown. Here, we describe a family consisting of 2 parents and 5 offspring. Both parents were affected with dementia and one had confirmed AD pathology with an age of onset >75 years. All offspring were affected with AD with ages at onset ranging from 53yrs-74yrs. DNA was available from the parent with confirmed AD and 5 offspring. We identified a coding variant, p.(Arg953Cys), in SORL1 in 5 of 6 individuals affected by AD. Notably, variant carriers had severe AD pathology, and the SORL1 variant segregated with TDP-43 pathology (LATE-NC). We further characterized this variant and show that this Arginine substitution occurs at a critical position in the YWTD-domain of the SORL1 translation product, SORL1. Functional studies further show that the p.R953C variant leads to retention of the SORL1 protein in the endoplasmic reticulum which leads to decreased maturation and shedding of the receptor and prevents its normal endosomal trafficking. Together, our analysis suggests that p.R953C is a pathogenic variant of SORL1 and sheds light on mechanisms of how missense SORL1 variants may lead to AD.

A Hitchhiker's guide through the bio-image analysis software universe.

Modern research in the life sciences is unthinkable without computational methods for extracting, quantifying and visualising information derived from microscopy imaging data of biological samples. In the past decade, we observed a dramatic increase in available software packages for these purposes. As it is increasingly difficult to keep track of the number of available image analysis platforms, tool collections, components and emerging technologies, we provide a conservative overview of software that we use in daily routine and give insights into emerging new tools. We give guidance on which aspects to consider when choosing the platform that best suits the user's needs, including aspects such as image data type, skills of the team, infrastructure and community at the institute and availability of time and budget.

A candidate intronic CYP24A1 gene variant affects the risk of colorectal cancer.

Aim: rs2585428 and rs4809960 polymorphisms were significantly associated with overall cancer risk, but there is no evidence regarding the overall colorectal cancer (CRC) risk. Materials & methods: A total of 505 subjects, including 246 patients with CRC and 259 noncancer controls participated in the study. The genotyping was performed using tetra-primer amplification refractory mutation systems PCR. Results: Analysis of genotypes revealed that CYP24A1 rs4809960 CC genotype decreased the risk of CRC (p = 0.009). In addition, the genotype frequencies showed a significant difference under the dominant and recessive inheritance models (p = 0.019 and p = 0.02, respectively). Conclusion: Our findings indicate that the CYP24A1 rs4809960 polymorphism decreased the risk of CRC in the studied population.

Automated cell tracking using StarDist and TrackMate.

The ability of cells to migrate is a fundamental physiological process involved in embryonic development, tissue homeostasis, immune surveillance, and wound healing. Therefore, the mechanisms governing cellular locomotion have been under intense scrutiny over the last 50 years. One of the main tools of this scrutiny is live-cell quantitative imaging, where researchers image cells over time to study their migration and quantitatively analyze their dynamics by tracking them using the recorded images. Despite the availability of computational tools, manual tracking remains widely used among researchers due to the difficulty setting up robust automated cell tracking and large-scale analysis. Here we provide a detailed analysis pipeline illustrating how the deep learning network StarDist can be combined with the popular tracking software TrackMate to perform 2D automated cell tracking and provide fully quantitative readouts. Our proposed protocol is compatible with both fluorescent and widefield images. It only requires freely available and open-source software (ZeroCostDL4Mic and Fiji), and does not require any coding knowledge from the users, making it a versatile and powerful tool for the field. We demonstrate this pipeline's usability by automatically tracking cancer cells and T cells using fluorescent and brightfield images. Importantly, we provide, as supplementary information, a detailed step-by-step protocol to allow researchers to implement it with their images.

Delivery of genome editing tools: A promising strategy for HPV-related cervical malignancy therapy.

INTRODUCTION: Persistent high-risk human papillomavirus infection is the main cause of various types of cancer especially cervical cancer. The E6 and E7 oncoproteins of HPV play critical roles in promoting carcinogenesis and cancer cell growth. As a result, E6 and E7 oncogenes are considered as promising therapeutic targets for cervical cancer. Recently, the development of genome-editing technologies including transcription activator-like effector nucleases (TALEN), meganucleases (MNs), zinc finger nucleases (ZFN), and more importantly clustered regularly interspaced short palindromic repeat-CRISPR-associated protein (CRISPR-Cas) has sparked a revolution in the cervical cancer-targeted therapy. However, due to immunogenicity, off-target effect, renal clearance, guide RNA (gRNA) nuclease degradation, and difficult direct transportation into the cytoplasm and nucleus, the safe and effective delivery is considered as the Achilles' heel of this robust strategy. AREAS COVERED: In this review, we discuss cutting-edge available strategies for in vivo delivery of genome-editing technologies for HPV-induced cervical cancer therapy. Moreover, the combination of genome-editing tools and other therapies has been fully discussed. EXPERT OPINION: The combination of nanoparticle-based delivery systems and genome-editing tools is a promising powerful strategy for cervical cancer therapy. The most significant limitations of this strategy that need to be focused on are low efficiency and off-target events.

Image Quality Ranking Method for Microscopy.

Automated analysis of microscope images is necessitated by the increased need for high-resolution follow up of events in time. Manually finding the right images to be analyzed, or eliminated from data analysis are common day-to-day problems in microscopy research today, and the constantly growing size of image datasets does not help the matter. We propose a simple method and a software tool for sorting images within a dataset, according to their relative quality. We demonstrate the applicability of our method in finding good quality images in a STED microscope sample preparation optimization image dataset. The results are validated by comparisons to subjective opinion scores, as well as five state-of-the-art blind image quality assessment methods. We also show how our method can be applied to eliminate useless out-of-focus images in a High-Content-Screening experiment. We further evaluate the ability of our image quality ranking method to detect out-of-focus images, by extensive simulations, and by comparing its performance against previously published, well-established microscopy autofocus metrics.