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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.
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Processamento de Imagem Assistida por Computador , Reprodutibilidade dos Testes , Microscopia de Fluorescência/métodosRESUMO
Cellular senescence is a well-established driver of aging and age-related diseases. There are many challenges to mapping senescent cells in tissues such as the absence of specific markers and their relatively low abundance and vast heterogeneity. Single-cell technologies have allowed unprecedented characterization of senescence; however, many methodologies fail to provide spatial insights. The spatial component is essential, as senescent cells communicate with neighboring cells, impacting their function and the composition of extracellular space. The Cellular Senescence Network (SenNet), a National Institutes of Health (NIH) Common Fund initiative, aims to map senescent cells across the lifespan of humans and mice. Here, we provide a comprehensive review of the existing and emerging methodologies for spatial imaging and their application toward mapping senescent cells. Moreover, we discuss the limitations and challenges inherent to each technology. We argue that the development of spatially resolved methods is essential toward the goal of attaining an atlas of senescent cells.
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Envelhecimento , Senescência Celular , Estados Unidos , Humanos , Animais , Camundongos , LongevidadeRESUMO
Technological advancements in biology and microscopy have empowered a transition from bioimaging as an observational method to a quantitative one. However, as biologists are adopting quantitative bioimaging and these experiments become more complex, researchers need additional expertise to carry out this work in a rigorous and reproducible manner. This Essay provides a navigational guide for experimental biologists to aid understanding of quantitative bioimaging from sample preparation through to image acquisition, image analysis, and data interpretation. We discuss the interconnectedness of these steps, and for each, we provide general recommendations, key questions to consider, and links to high-quality open-access resources for further learning. This synthesis of information will empower biologists to plan and execute rigorous quantitative bioimaging experiments efficiently.
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Processamento de Imagem Assistida por Computador , MicroscopiaRESUMO
BACKGROUND: Tunneling nanotubes (TNTs) are cellular structures connecting cell membranes and mediating intercellular communication. TNTs are manually identified and counted by a trained investigator; however, this process is time-intensive. We therefore sought to develop an automated approach for quantitative analysis of TNTs. METHODS: We used a convolutional neural network (U-Net) deep learning model to segment phase contrast microscopy images of both cancer and non-cancer cells. Our method was composed of preprocessing and model development. We developed a new preprocessing method to label TNTs on a pixel-wise basis. Two sequential models were employed to detect TNTs. First, we identified the regions of images with TNTs by implementing a classification algorithm. Second, we fed parts of the image classified as TNT-containing into a modified U-Net model to estimate TNTs on a pixel-wise basis. RESULTS: The algorithm detected 49.9% of human expert-identified TNTs, counted TNTs, and calculated the number of TNTs per cell, or TNT-to-cell ratio (TCR); it detected TNTs that were not originally detected by the experts. The model had 0.41 precision, 0.26 recall, and 0.32 f-1 score on a test dataset. The predicted and true TCRs were not significantly different across the training and test datasets (p = 0.78). CONCLUSIONS: Our automated approach labeled and detected TNTs and cells imaged in culture, resulting in comparable TCRs to those determined by human experts. Future studies will aim to improve on the accuracy, precision, and recall of the algorithm.
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Magnetic resonance imaging (MRI) has transformed our understanding of the human brain through well-replicated mapping of abilities to specific structures (for example, lesion studies) and functions1-3 (for example, task functional MRI (fMRI)). Mental health research and care have yet to realize similar advances from MRI. A primary challenge has been replicating associations between inter-individual differences in brain structure or function and complex cognitive or mental health phenotypes (brain-wide association studies (BWAS)). Such BWAS have typically relied on sample sizes appropriate for classical brain mapping4 (the median neuroimaging study sample size is about 25), but potentially too small for capturing reproducible brain-behavioural phenotype associations5,6. Here we used three of the largest neuroimaging datasets currently available-with a total sample size of around 50,000 individuals-to quantify BWAS effect sizes and reproducibility as a function of sample size. BWAS associations were smaller than previously thought, resulting in statistically underpowered studies, inflated effect sizes and replication failures at typical sample sizes. As sample sizes grew into the thousands, replication rates began to improve and effect size inflation decreased. More robust BWAS effects were detected for functional MRI (versus structural), cognitive tests (versus mental health questionnaires) and multivariate methods (versus univariate). Smaller than expected brain-phenotype associations and variability across population subsamples can explain widespread BWAS replication failures. In contrast to non-BWAS approaches with larger effects (for example, lesions, interventions and within-person), BWAS reproducibility requires samples with thousands of individuals.
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Mapeamento Encefálico , Encéfalo , Imageamento por Ressonância Magnética , Mapeamento Encefálico/métodos , Cognição , Conjuntos de Dados como Assunto , Humanos , Imageamento por Ressonância Magnética/métodos , Neuroimagem , Fenótipo , Reprodutibilidade dos TestesRESUMO
Numerous interventions have been explored in animal models using cells differentiated from human induced pluripotent stem cells (iPSCs) in the context of neural injury with some success. Our work seeks to transplant cells that are generated from hiPSCs into regionally specific spinal neural progenitor cells (sNPCs) utilizing a novel accelerated differentiation protocol designed for clinical translation. We chose a xenotransplantation model because our laboratory is focused on the behaviour of human cells in order to bring this potential therapy to translation. Cells were transplanted into adult immunodeficient rats after moderate contusion spinal cord injury (SCI). Twelve weeks later, cells derived from the transplanted sNPCs survived and differentiated into neurons and glia that filled the lesion cavity and produced a thoracic spinal cord transcriptional program in vivo. Furthermore, neurogenesis and ionic channel expression were promoted within the adjacent host spinal cord tissue. Transplanted cells displayed robust integration properties including synapse formation and myelination by host oligodendrocytes. Axons from transplanted hiPSC sNPC-derived cells extended both rostrally and caudally from the SCI transplant site, rostrally approximately 6 cm into supraspinal structures. Thus, iPSC-derived sNPCs may provide a patient-specific cell source for patients with SCI that could provide a relay system across the site of injury.
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Células-Tronco Pluripotentes Induzidas , Células-Tronco Neurais , Traumatismos da Medula Espinal , Animais , Axônios/patologia , Diferenciação Celular/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neurais/metabolismo , Ratos , Recuperação de Função Fisiológica , Medula Espinal/patologia , Traumatismos da Medula Espinal/patologia , Sinapses/patologiaRESUMO
Effective treatment of glioblastoma remains a daunting challenge. One of the major hurdles in the development of therapeutics is their inability to cross the blood-brain tumor barrier (BBTB). Local delivery is an alternative approach that can still suffer from toxicity in the absence of target selectivity. Here, we show that nanotubes formed from self-assembly of ssDNA-amphiphiles are stable in serum and nucleases. After bilateral brain injections, nanotubes show preferential retention by tumors compared to normal brain and are taken up by glioblastoma cells through scavenger receptor binding and macropinocytosis. After intravenous injection, they cross the BBTB and internalize in glioblastoma cells. In a minimal residual disease model, local delivery of doxorubicin showed signs of toxicity in the spleen and liver. In contrast, delivery of doxorubicin by the nanotubes resulted in no systemic toxicity and enhanced mouse survival. Our results demonstrate that ssDNA nanotubes are a promising drug delivery vehicle to glioblastoma.
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Núcleo Celular/ultraestrutura , Cromatina/ultraestrutura , Processamento de Imagem Assistida por Computador/normas , Informática Médica/estatística & dados numéricos , Metadados/estatística & dados numéricos , Microscopia/métodos , Software , Humanos , Processamento de Imagem Assistida por Computador/métodosRESUMO
Recurrence of metastatic breast cancer stemming from acquired endocrine and chemotherapy resistance remains a health burden for women with luminal (ER+) breast cancer. Disseminated ER+ tumor cells can remain viable but quiescent for years to decades. Contributing factors to metastatic spread include the maintenance and expansion of breast cancer stem cells (CSCs). Breast CSCs frequently exist as a minority population in therapy resistant tumors. In this study, we show that cytoplasmic complexes composed of steroid receptor (SR) co-activators, PELP1 and SRC-3, modulate breast CSC expansion through upregulation of the HIF-activated metabolic target genes PFKFB3 and PFKFB4. Seahorse metabolic assays demonstrated that cytoplasmic PELP1 influences cellular metabolism by increasing both glycolysis and mitochondrial respiration. PELP1 interacts with PFKFB3 and PFKFB4 proteins, and inhibition of PFKFB3 and PFKFB4 kinase activity blocks PELP1-induced tumorspheres and protein-protein interactions with SRC-3. PFKFB4 knockdown inhibited in vivo emergence of circulating tumor cell (CTC) populations in mammary intraductal (MIND) models. Application of PFKFB inhibitors in combination with ER targeted therapies blocked tumorsphere formation in multiple models of advanced breast cancer including tamoxifen (TamR) and paclitaxel (TaxR) resistant models, murine tumor cells, and ER+ patient-derived organoids (PDxO). Together, our data suggest that PELP1, SRC-3, and PFKFBs cooperate to drive ER+ tumor cell populations that include CSCs and CTCs. Identifying non-ER pharmacological targets offers a useful approach to blocking metastatic escape from standard of care ER/estrogen (E2)-targeted strategies to overcome endocrine and chemotherapy resistance.
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Neoplasias da Mama/genética , Proteínas Correpressoras/genética , Resistencia a Medicamentos Antineoplásicos/genética , Coativador 3 de Receptor Nuclear/genética , Fosfofrutoquinase-2/genética , Receptores de Estrogênio/genética , Fatores de Transcrição/genética , Animais , Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Estrogênios/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Células MCF-7 , Camundongos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Paclitaxel/farmacologia , Fosforilação/genética , Tamoxifeno/farmacologia , Regulação para Cima/genéticaRESUMO
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.
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Pesquisa Biomédica/métodos , Pesquisa Biomédica/normas , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Microscopia de Fluorescência/normas , Convallaria , Escherichia coli/metabolismo , Corantes Fluorescentes , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imageamento Tridimensional , Microscopia Confocal/métodos , Reprodutibilidade dos Testes , Projetos de Pesquisa , Razão Sinal-Ruído , SoftwareRESUMO
Tumor-infiltrating lymphocytes in colorectal cancer (CRC) predict better survival. However, associations between T-lymphocyte count in histologically normal tissues from patients with CRC and survival remain uncertain. We examined associations of CD3+ T-cells in colorectal tumor and histologically normal tissues with CRC-specific and all-cause mortality in the prospective Iowa Women's Health Study. Tissue microarrays were constructed using paraffin-embedded colorectal tissue samples from 464 women with tumor tissues and 314 women with histologically normal tissues (55-69 years at baseline) diagnosed with incident CRC from 1986 to 2002 and followed through 2014 (median follow-up 20.5 years). Three tumor and two histologically normal tissue cores for each patient were immunostained using CD3+ antibody and quantified, and the counts were averaged across the cores in each tissue. Cox proportional hazards regression estimated hazard ratios (HR) and 95% confidence interval (CI) for CRC-specific and all-cause mortality. After adjustment for age at diagnosis, body mass index, smoking status, tumor grade, and stage, HRs (95% CI) for the highest versus lowest tertile of tumor CD3+ score were 0.59 (0.38-0.89) for CRC-specific mortality and 0.82 (0.63-1.05) for all-cause mortality; for histologically normal CD3+ score, the corresponding HRs (95% CI) were 0.47 (0.19-1.17) and 0.50 (0.27-0.90), respectively. The CD3+ score combining the tumor and histologically normal scores was inversely associated with CRC-specific and all-cause mortality. Although the association between tumor CD3+ score and all-cause mortality was not significant, both higher CD3+ T-lymphocyte counts in tumor and histologically normal scores tended to be associated with lower CRC-specific and all-cause mortality.
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Complexo CD3/análise , Neoplasias Colorretais/patologia , Linfócitos T/patologia , Idoso , Colo/patologia , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/epidemiologia , Feminino , Humanos , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Reto/patologia , Análise de SobrevidaRESUMO
A variety of microscopy techniques are used by researchers in the life and biomedical sciences. As these techniques become more powerful and more complex, it is vital that scientific articles containing images obtained with advanced microscopes include full details about how each image was obtained. To explore the reporting of such details we examined 240 original research articles published in eight journals. We found that the quality of reporting was poor, with some articles containing no information about how images were obtained, and many articles lacking important basic details. Efforts by researchers, funding agencies, journals, equipment manufacturers and staff at shared imaging facilities are required to improve the reporting of experiments that rely on microscopy techniques.
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Pesquisa Biomédica/estatística & dados numéricos , Microscopia/métodos , Editoração/estatística & dados numéricos , Microscopia/estatística & dados numéricosRESUMO
The proto-oncogene MET, the hepatocyte growth factor (HGF) receptor, is a transmembrane receptor tyrosine kinase (RTK) with a prominent role in tumor metastasis and resistance to anti-cancer therapies. Melanoma demonstrates relatively frequent MET aberrations, including MET gene amplification. Concurrently, programmed death-ligand 1 (PD-L1), with its ability to evade anti-tumor immune responses, has emerged as a prominent therapeutic target in melanoma and other malignancies and its expression is used as a predictive biomarker of response to immunotherapy. We performed immunohistochemistry analysis of MET and PD-L1 in 18 human melanoma cell lines derived from both primary and metastatic lesions, and in a human melanoma tissue microarray containing one hundreds melanocytic lesions, including primary cutaneous melanomas, primary mucosal melanomas, metastatic melanomas and benign melanocytic nevi as controls. After color deconvolution, each core was segmented to isolate staining and calculate the percentage of positive cells. Overall, MET expression was higher in tumors with increased PD-L1 expression. Moreover, a robust correlation between MET and PD-L1 expression was found in samples from metastatic melanoma and not in primary cutaneous or mucosal melanoma. These data suggest that relative expression levels of these proteins in combination is a marker of advanced disease and testing for expression of these markers should be considered in patients with melanoma.
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BACKGROUND: Synaptic dysfunction prevalent in Alzheimer's disease (AD) brain is closely associated with increased accumulation of amyloid-ß (Aß) peptides in the brain parenchyma. It is widely believed that Aß peptides trigger synaptic dysfunction by interfering with the synaptic vesicular fusion and the release of neurotransmitters, primarily facilitated by the SNARE protein complexes formed by VAMP-2, SNAP-25, and syntaxin-1. However, Aß interactions with SNARE proteins to ultimately disrupt synaptic vesicular fusion are not well understood. OBJECTIVE: Our objective is to elucidate mechanisms by which Aß peptides perturb SNARE complexes. METHODS: Intensity (qualitative) and lifetime (quantitative) based measurements involving Forster (fluorescence) resonance energy transfer (FRET) followed by fluorescence lifetime imaging microscopy (FLIM) were employed to investigate the effect of Aß peptides on dynamic interactions between VAMP-2, labeled with cerulean (Cer) at the N-terminus (FRET donor), and SNAP-25 labeled with citrine (Cit) on the N-terminus (FRET acceptor). The FRET and FLIM interactions at the exocytosis locations on the pre-synaptic membrane were recorded under spontaneous and high potassium evoked conditions. Moreover, cellular accumulation of fluorescein labeled Aß (F-Aß) peptides and their co-localization with Cer-VAMP2 was investigated by confocal microscopy. RESULTS: The F-Aß40 and F-Aß42 are internalized by differentiated N2A cells, where they colocalize with Cer-VAMP2. Both Aß40 and Aß42 decrease interactions between the N-termini of Cer-VAMP2 and Cit-SNAP25 in N2A cells, as determined by FRET/FLIM. CONCLUSION: By perturbing the N-terminal interactions between VAMP-2 and SNAP-25, Aß40 and Aß42, can directly interfere with the SNARE complex formation, which is critical for the docking and fusion of synaptic vesicles.
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Peptídeos beta-Amiloides/toxicidade , Transferência Ressonante de Energia de Fluorescência/métodos , Neurônios/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Linhagem Celular Tumoral , Humanos , Microscopia de Fluorescência/métodos , Neurônios/química , Neurônios/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Proteína 25 Associada a Sinaptossoma/análise , Proteína 2 Associada à Membrana da Vesícula/análiseRESUMO
Synapse loss and dendritic damage correlate with cognitive decline in many neurodegenerative diseases, underlie neurodevelopmental disorders, and are associated with environmental and drug-induced CNS toxicities. However, screening assays designed to measure loss of synaptic connections between live cells are lacking. Here, we describe the design and validation of automated synaptic imaging assay (ASIA), an efficient approach to label, image, and analyze synapses between live neurons. Using viral transduction to express fluorescent proteins that label synapses and an automated computer-controlled microscope, we developed a method to identify agents that regulate synapse number. ASIA is compatible with both confocal and wide-field microscopy; wide-field image acquisition is faster but requires a deconvolution step in the analysis. Both types of images feed into batch processing analysis software that can be run on ImageJ, CellProfiler, and MetaMorph platforms. Primary analysis endpoints are the number of structural synapses and cell viability. Thus, overt cell death is differentiated from subtle changes in synapse density, an important distinction when studying neurodegenerative processes. In rat hippocampal cultures treated for 24 h with 100 µM 2-bromopalmitic acid (2-BP), a compound that prevents clustering of postsynaptic density 95 (PSD95), ASIA reliably detected loss of postsynaptic density 95-enhanced green fluorescent protein (PSD95-eGFP)-labeled synapses in the absence of cell death. In contrast, treatment with 100 µM glutamate produced synapse loss and significant cell death, determined from morphological changes in a binary image created from co-expressed mCherry. Treatment with 3 mM lithium for 24 h significantly increased the number of fluorescent puncta, showing that ASIA also detects synaptogenesis. Proof of concept studies show that cell-specific promoters enable the selective study of inhibitory or principal neurons and that alternative reporter constructs enable quantification of GABAergic or glutamatergic synapses. ASIA can also be used to study synapse loss between human induced pluripotent stem cell (iPSC)-derived cortical neurons. Significant synapse loss in the absence of cell death was detected in the iPSC-derived neuronal cultures treated with either 100 µM 2-BP or 100 µM glutamate for 24 h, while 300 µM glutamate produced synapse loss and cell death. ASIA shows promise for identifying agents that evoke synaptic toxicities and screening for compounds that prevent or reverse synapse loss.
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In the version of this paper originally published, Figure 4a contained errors that were introduced during typesetting. The bottom 11° ThunderSTORM image is an xz view but was incorrectly labeled as xy, and the low x-axis value in the four line profiles was incorrectly set as -60 instead of -50. These errors have been corrected in the PDF and HTML versions of the paper.
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With the widespread uptake of two-dimensional (2D) and three-dimensional (3D) single-molecule localization microscopy (SMLM), a large set of different data analysis packages have been developed to generate super-resolution images. In a large community effort, we designed a competition to extensively characterize and rank the performance of 2D and 3D SMLM software packages. We generated realistic simulated datasets for popular imaging modalities-2D, astigmatic 3D, biplane 3D and double-helix 3D-and evaluated 36 participant packages against these data. This provides the first broad assessment of 3D SMLM software and provides a holistic view of how the latest 2D and 3D SMLM packages perform in realistic conditions. This resource allows researchers to identify optimal analytical software for their experiments, allows 3D SMLM software developers to benchmark new software against the current state of the art, and provides insight into the current limits of the field.
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Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Imagem Individual de Molécula/métodos , Software , AlgoritmosRESUMO
Naive CD4+ T lymphocytes differentiate into various Th cell subsets following TCR binding to microbial peptide:MHC class II (p:MHCII) complexes on dendritic cells (DCs). The affinity of the TCR interaction with p:MHCII plays a role in Th differentiation by mechanisms that are not completely understood. We found that low-affinity TCRs biased mouse naive T cells to become T follicular helper (Tfh) cells, whereas higher-affinity TCRs promoted the formation of Th1 or Th17 cells. We explored the basis for this phenomenon by focusing on IL-2R signaling, which is known to promote Th1 and suppress Tfh cell differentiation. SIRPâº+ DCs produce abundant p:MHCII complexes and consume IL-2, whereas XCR1+ DCs weakly produce p:MHCII but do not consume IL-2. We found no evidence, however, of preferential interactions between Th1 cell-prone, high-affinity T cells and XCR1+ DCs or Tfh cell-prone, low-affinity T cells and SIRPâº+ DCs postinfection with bacteria expressing the peptide of interest. Rather, high-affinity T cells sustained IL-2R expression longer and expressed two novel Th cell differentiation regulators, Eef1e1 and Gbp2, to a higher level than low-affinity T cells. These results suggest that TCR affinity does not influence Th cell differentiation by biasing T cell interactions with IL-2-consuming DCs, but instead, directly regulates genes in naive T cells that control the differentiation process.