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Pancreatic ductal adenocarcinoma (PDAC) is characterized by increasing fibrosis, which can enhance tumor progression and spread. Here, we undertook an unbiased temporal assessment of the matrisome of the highly metastatic KPC (Pdx1-Cre, LSL-KrasG12D/+, LSL-Trp53R172H/+) and poorly metastatic KPflC (Pdx1-Cre, LSL-KrasG12D/+, Trp53fl/+) genetically engineered mouse models of pancreatic cancer using mass spectrometry proteomics. Our assessment at early-, mid-, and late-stage disease reveals an increased abundance of nidogen-2 (NID2) in the KPC model compared to KPflC, with further validation showing that NID2 is primarily expressed by cancer-associated fibroblasts (CAFs). Using biomechanical assessments, second harmonic generation imaging, and birefringence analysis, we show that NID2 reduction by CRISPR interference (CRISPRi) in CAFs reduces stiffness and matrix remodeling in three-dimensional models, leading to impaired cancer cell invasion. Intravital imaging revealed improved vascular patency in live NID2-depleted tumors, with enhanced response to gemcitabine/Abraxane. In orthotopic models, NID2 CRISPRi tumors had less liver metastasis and increased survival, highlighting NID2 as a potential PDAC cotarget.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Proteómica , Animales , Humanos , Ratones , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/genética , Moléculas de Adhesión Celular , Línea Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Modelos Animales de Enfermedad , Fibrosis , Gemcitabina , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/genética , Proteómica/métodosRESUMEN
Large-language models (LLMs) such as GPT-4 caught the interest of many scientists. Recent studies suggested that these models could be useful in chemistry and materials science. To explore these possibilities, we organized a hackathon. This article chronicles the projects built as part of this hackathon. Participants employed LLMs for various applications, including predicting properties of molecules and materials, designing novel interfaces for tools, extracting knowledge from unstructured data, and developing new educational applications. The diverse topics and the fact that working prototypes could be generated in less than two days highlight that LLMs will profoundly impact the future of our fields. The rich collection of ideas and projects also indicates that the applications of LLMs are not limited to materials science and chemistry but offer potential benefits to a wide range of scientific disciplines.
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Aberrant AKT activation occurs in a number of cancers, metabolic syndrome, and immune disorders, making it an important target for the treatment of many diseases. To monitor spatial and temporal AKT activity in a live setting, we generated an Akt-FRET biosensor mouse that allows longitudinal assessment of AKT activity using intravital imaging in conjunction with image stabilization and optical window technology. We demonstrate the sensitivity of the Akt-FRET biosensor mouse using various cancer models and verify its suitability to monitor response to drug targeting in spheroid and organotypic models. We also show that the dynamics of AKT activation can be monitored in real time in diverse tissues, including in individual islets of the pancreas, in the brown and white adipose tissue, and in the skeletal muscle. Thus, the Akt-FRET biosensor mouse provides an important tool to study AKT dynamics in live tissue contexts and has broad preclinical applications.
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Técnicas Biosensibles , Proteínas Proto-Oncogénicas c-akt , Ratones , Animales , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Técnicas Biosensibles/métodosRESUMEN
The application of statistical analysis software programs has proven useful for the investigation of rockfall runout distance along a designed slope. Rockfall modeling programs are continually being upgraded with more sophisticated analysis tools, such as the use of the rigid body versus lump mass models. Engineers at mine sites utilizing the software may have varied experience related to these models, their associated input parameters, and how to interpret the generated results. To address this concern, researchers at the Spokane Mining Research Division (SMRD) of the U.S. National Institute for Occupational Safety and Health (NIOSH) investigated the influence of slope height, slope angle, slope material, and rock size for both rigid body and lump mass models in a 2-D statistical analysis program. Based on a literature search and industry input, specific ranges common to that of an open pit mining environment were chosen for each of the input parameters to determine 90% rock runout distance as well as their sensitivity to change. Data collected from this numerical analysis and simulation will be compared to empirical rockfall data gathered through the duration of the Highwall Safety project conducted by NIOSH from 2022-2026.
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Assessing drug response within live native tissue provides increased fidelity with regards to optimizing efficacy while minimizing off-target effects. Here, using longitudinal intravital imaging of a Rac1-Förster resonance energy transfer (FRET) biosensor mouse coupled with in vivo photoswitching to track intratumoral movement, we help guide treatment scheduling in a live breast cancer setting to impair metastatic progression. We uncover altered Rac1 activity at the center versus invasive border of tumors and demonstrate enhanced Rac1 activity of cells in close proximity to live tumor vasculature using optical window imaging. We further reveal that Rac1 inhibition can enhance tumor cell vulnerability to fluid-flow-induced shear stress and therefore improves overall anti-metastatic response to therapy during transit to secondary sites such as the lung. Collectively, this study demonstrates the utility of single-cell intravital imaging in vivo to demonstrate that Rac1 inhibition can reduce tumor progression and metastases in an autochthonous setting to improve overall survival.
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Técnicas Biosensibles/métodos , Neoplasias de la Mama/patología , Proteína de Unión al GTP rac1/metabolismo , Aminoquinolinas/farmacología , Animales , Neoplasias de la Mama/diagnóstico por imagen , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Femenino , Transferencia Resonante de Energía de Fluorescencia , Humanos , Neoplasias Pulmonares/diagnóstico por imagen , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Ratones , Ratones Endogámicos BALB C , Pirimidinas/farmacología , Resistencia al Corte , Transducción de Señal , Proteína de Unión al GTP rac1/antagonistas & inhibidoresRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic, chemoresistant malignancy and is characterized by a dense, desmoplastic stroma that modulates PDAC progression. Here, we visualized transient manipulation of focal adhesion kinase (FAK), which integrates bidirectional cell-environment signaling, using intravital fluorescence lifetime imaging microscopy of the FAK-based Förster resonance energy transfer biosensor in mouse and patient-derived PDAC models. Parallel real-time quantification of the FUCCI cell cycle reporter guided us to improve PDAC response to standard-of-care chemotherapy at primary and secondary sites. Critically, micropatterned pillar plates and stiffness-tunable matrices were used to pinpoint the contribution of environmental cues to chemosensitization, while fluid flowinduced shear stress assessment, patient-derived matrices, and personalized in vivo models allowed us to deconstruct how FAK inhibition can reduce PDAC spread. Last, stratification of PDAC patient samples via Merlin status revealed a patient subset with poor prognosis that are likely to respond to FAK priming before chemotherapy.
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The concept that extracellular vesicles (EVs) from the diet can be absorbed by the intestinal tract of the consuming organism, be bioavailable in various organs, and in-turn exert phenotypic changes is highly debatable. Here, we isolate EVs from both raw and commercial bovine milk and characterize them by electron microscopy, nanoparticle tracking analysis, western blotting, quantitative proteomics and small RNA sequencing analysis. Orally administered bovine milk-derived EVs survive the harsh degrading conditions of the gut, in mice, and is subsequently detected in multiple organs. Milk-derived EVs orally administered to mice implanted with colorectal and breast cancer cells reduce the primary tumor burden. Intriguingly, despite the reduction in primary tumor growth, milk-derived EVs accelerate metastasis in breast and pancreatic cancer mouse models. Proteomic and biochemical analysis reveal the induction of senescence and epithelial-to-mesenchymal transition in cancer cells upon treatment with milk-derived EVs. Timing of EV administration is critical as oral administration after resection of the primary tumor reverses the pro-metastatic effects of milk-derived EVs in breast cancer models. Taken together, our study provides context-based and opposing roles of milk-derived EVs as metastasis inducers and suppressors.
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Vesículas Extracelulares , Leche/citología , Neoplasias Experimentales/patología , Administración Oral , Animales , Disponibilidad Biológica , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Bovinos , Línea Celular Tumoral , Proliferación Celular , Transición Epitelial-Mesenquimal , Vesículas Extracelulares/química , Vesículas Extracelulares/genética , Femenino , Humanos , Neoplasias Hepáticas Experimentales/patología , Neoplasias Hepáticas Experimentales/secundario , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Ratones Endogámicos BALB C , Neoplasias Experimentales/terapia , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/terapia , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Osteoclasts are large multinucleated bone-resorbing cells formed by the fusion of monocyte/macrophage-derived precursors that are thought to undergo apoptosis once resorption is complete. Here, by intravital imaging, we reveal that RANKL-stimulated osteoclasts have an alternative cell fate in which they fission into daughter cells called osteomorphs. Inhibiting RANKL blocked this cellular recycling and resulted in osteomorph accumulation. Single-cell RNA sequencing showed that osteomorphs are transcriptionally distinct from osteoclasts and macrophages and express a number of non-canonical osteoclast genes that are associated with structural and functional bone phenotypes when deleted in mice. Furthermore, genetic variation in human orthologs of osteomorph genes causes monogenic skeletal disorders and associates with bone mineral density, a polygenetic skeletal trait. Thus, osteoclasts recycle via osteomorphs, a cell type involved in the regulation of bone resorption that may be targeted for the treatment of skeletal diseases.
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Resorción Ósea/patología , Osteoclastos/patología , Ligando RANK/metabolismo , Animales , Apoptosis , Resorción Ósea/metabolismo , Fusión Celular , Células Cultivadas , Humanos , Macrófagos/citología , Ratones , Osteocondrodisplasias/tratamiento farmacológico , Osteocondrodisplasias/genética , Osteocondrodisplasias/metabolismo , Osteocondrodisplasias/patología , Osteoclastos/metabolismo , Transducción de SeñalRESUMEN
In microorganisms, evolutionarily conserved mechanisms facilitate adaptation to harsh conditions through stress-induced mutagenesis (SIM). Analogous processes may underpin progression and therapeutic failure in human cancer. We describe SIM in multiple in vitro and in vivo models of human cancers under nongenotoxic drug selection, paradoxically enhancing adaptation at a competing intrinsic fitness cost. A genome-wide approach identified the mechanistic target of rapamycin (MTOR) as a stress-sensing rheostat mediating SIM across multiple cancer types and conditions. These observations are consistent with a two-phase model for drug resistance, in which an initially rapid expansion of genetic diversity is counterbalanced by an intrinsic fitness penalty, subsequently normalizing to complete adaptation under the new conditions. This model suggests synthetic lethal strategies to minimize resistance to anticancer therapy.
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Adaptación Fisiológica/genética , Antineoplásicos/farmacología , Resistencia a Antineoplásicos/genética , Mutagénesis , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Serina-Treonina Quinasas TOR/metabolismo , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Reparación del ADN/genética , Aptitud Genética , Estudio de Asociación del Genoma Completo , Humanos , Selección Genética , Transducción de Señal , Serina-Treonina Quinasas TOR/genéticaRESUMEN
Metastasis is a dynamic succession of events involving the dissemination of tumour cells to distant sites within the body, ultimately reducing the survival of patients with cancer. To colonize distant organs and, therefore, systemically disseminate within the organism, cancer cells and associated factors exploit several bodily fluid systems, which provide a natural transportation route. Indeed, the flow mechanics of the blood and lymphatic circulatory systems can be co-opted to improve the efficiency of cancer cell transit from the primary tumour, extravasation and metastatic seeding. Flow rates, vessel size and shear stress can all influence the survival of cancer cells in the circulation and control organotropic seeding patterns. Thus, in addition to using these fluids as a means to travel throughout the body, cancer cells exploit the underlying physical forces within these fluids to successfully seed distant metastases. In this Review, we describe how circulating tumour cells and tumour-associated factors leverage bodily fluids, their underlying forces and imposed stresses during metastasis. As the contribution of bodily fluids and their mechanics raises interesting questions about the biology of the metastatic cascade, an improved understanding of this process might provide a new avenue for targeting cancer cells in transit.
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Líquidos Corporales/metabolismo , Modelos Biológicos , Neoplasias/metabolismo , Neoplasias/patología , Microambiente Tumoral , Animales , Biomarcadores , Líquidos Corporales/efectos de los fármacos , Matriz Extracelular/metabolismo , Humanos , Terapia Molecular Dirigida , Metástasis de la Neoplasia , Neoplasias/etiología , Neoplasias/terapia , Células Neoplásicas Circulantes/efectos de los fármacos , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patología , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/inmunologíaRESUMEN
The small GTPase RhoA is a master regulator of signalling in cell-extracellular matrix interactions. RhoA signalling is critical to many cellular processes including migration, mechanotransduction, and is often disrupted in carcinogenesis. Investigating RhoA activity in a native tissue environment is challenging using conventional biochemical methods; we therefore developed a RhoA-FRET biosensor mouse, employing the adaptable nature of intravital imaging to a variety of settings. Mechanotransduction was explored in the context of osteocyte processes embedded in the calvaria responding in a directional manner to compression stress. Further, the migration of neutrophils was examined during in vivo "chemotaxis" in wound response. RhoA activity was tightly regulated during tissue remodelling in mammary gestation, as well as during mammary and pancreatic carcinogenesis. Finally, pharmacological inhibition of RhoA was temporally resolved by the use of optical imaging windows in fully developed pancreatic and mammary tumours in vivo. The RhoA-FRET mouse therefore constitutes a powerful tool to facilitate development of new inhibitors targeting the RhoA signalling axis.
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Técnicas Biosensibles , Transferencia Resonante de Energía de Fluorescencia , Preparaciones Farmacéuticas/química , Proteína de Unión al GTP rhoA/metabolismo , Animales , Ratones , Transducción de SeñalRESUMEN
In 2001, researchers from the National Institute for Occupational Safety and Health (NIOSH) installed instruments at the Turquoise Ridge Mine in cooperation with Placer Dome, Inc. to monitor the geomechanical behavior and stability of a cemented rockfill (CRF) sill and the surrounding host rock during test mining of a large undercut span beneath backfill. Six parallel, adjacent drifts were mined and backfilled to construct a CRF sill, approximately 22.9 m (75 ft) wide by 30.5 m (100 ft) long. The sill was then partially undercut, successfully creating a 13.7-m (45-ft) wide by 30.5-m (100-ft) long span beneath the CRF. Only small vertical displacements were measured in the overlying host rock during mining, with most of the movement occurring at shallow depths in the mine roof. Because the back above the CRF sill remained stable, the majority of the mining-induced stress was transferred to the host rock abutments rather than to the backfilled drifts. During retreat mining of the undercut span, the CRF sill and the mine roof remained stable. Most of the measured vertical displacement was caused by separation of the backfill from the overlying host rock, or deflection of the CRF sill, which was comparable to the deflection of a monolithic, elastic plate having similar dimensions, material properties, and undercut spans. The CRF sill moved in mass as a single unit rather than as individual drift segments, and the vertical cold joints between adjacent backfill drifts did not adversely affect their stability. Additional measurements collected from the instruments have shown that the backfill span is still intact and in stable condition more than 16 years after the completion of undercut mining. Displacements in the mine roof and abutments have stabilized, and vertical stress and deformation within the CRF have generally leveled off or decreased. Although only slight mining-induced loads were transferred to the backfilled drifts, the CRF has confined the abutment ribs and mine roof, thereby improving their long-term stability. Results of compressive and tensile strength tests conducted with CRF samples from the test site indicate that the long-term compressive strength gain for CRF is similar to that of concrete, and that the tensile-to-compressive strength ratio for CRF is about 1/6 rather than 1/10. Assuming the in-place CRF gained strength at the same rate as the lab samples, an analytical analysis of the flexural stability of the CRF undercut span shows that the Factor of Safety for the span should have logically increased over time. By providing a better understanding of the long-term strength properties and geomechanical behavior of CRF, these research findings help improve the methods that are used for designing stable, long-term undercut entries beneath cemented backfill.
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Electrochemical performance of nanostructured carbon electrodes was evaluated using cyclic voltammetry and a simple simulation model. The electrodes were prepared from soluble precursors by anodic electrodeposition of two sizes of graphene quantum dot assemblies (hexabenzocoronene (HBC) and carbon quantum dot (CQD)) onto a conductive support. Experimental and simulated voltammograms enabled the extraction of the following electrode parameters: conductivity of the electrodes (a combination of ionic and electronic contributions), density of available electrode states at different potentials, and tunneling rate constant (Marcus-Gerischer model) for interfacial charge transfer to ferrocene/ferrocenium (Fc/Fc+) couple. The parameters indicate that HBC and CQD have significant density of electronic states at potentials more positive than -0.5 V versus Ag/Ag+. Enabled by these large densities, the electron transfer rates at the Fc/Fc+ thermodynamic potential are several orders of magnitude slower than those commonly observed on other carbon electrodes. This study is expected to accelerate the discovery of improved synthetic carbon electrodes by providing fast screening methodology of their electrochemical behavior.
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Heterogeneous subtypes of cancer-associated fibroblasts (CAFs) coexist within pancreatic cancer tissues and can both promote and restrain disease progression. Here, we interrogate how cancer cells harboring distinct alterations in p53 manipulate CAFs. We reveal the existence of a p53-driven hierarchy, where cancer cells with a gain-of-function (GOF) mutant p53 educate a dominant population of CAFs that establish a pro-metastatic environment for GOF and null p53 cancer cells alike. We also demonstrate that CAFs educated by null p53 cancer cells may be reprogrammed by either GOF mutant p53 cells or their CAFs. We identify perlecan as a key component of this pro-metastatic environment. Using intravital imaging, we observe that these dominant CAFs delay cancer cell response to chemotherapy. Lastly, we reveal that depleting perlecan in the stroma combined with chemotherapy prolongs mouse survival, supporting it as a potential target for anti-stromal therapies in pancreatic cancer.
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Fibroblastos Asociados al Cáncer/patología , Resistencia a Antineoplásicos/genética , Proteoglicanos de Heparán Sulfato/metabolismo , Neoplasias Pancreáticas/patología , Proteína p53 Supresora de Tumor/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Regulación Neoplásica de la Expresión Génica/genética , Ratones , Ratones Endogámicos BALB C , Invasividad Neoplásica/patología , Páncreas/patología , Neoplasias Pancreáticas/genética , Transducción de Señal/fisiología , Proteína p53 Supresora de Tumor/genéticaRESUMEN
We describe an open-source automated multiwell plate fluorescence lifetime imaging (FLIM) methodology to read out Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) labeling endogenous kinetochore proteins (KPs) in live budding yeast cells. The low copy number of many KPs and their small spatial extent present significant challenges for the quantification of donor fluorescence lifetime in the presence of significant cellular autofluorescence and photobleaching. Automated FLIM data acquisition was controlled by µManager and incorporated wide-field time-gated imaging with optical sectioning to reduce background fluorescence. For data analysis, we used custom MATLAB-based software tools to perform kinetochore foci segmentation and local cellular background subtraction and fitted the fluorescence lifetime data using the open-source FLIMfit software. We validated the methodology using endogenous KPs labeled with mTurquoise2 FP and/or yellow FP and measured the donor fluorescence lifetimes for foci comprising 32 kinetochores with KP copy numbers as low as ~2 per kinetochore under an average labeling efficiency of 50%. We observed changes of median donor lifetime ≥250 ps for KPs known to form dimers. Thus, this FLIM high-content analysis platform enables the screening of relatively low-copy-number endogenous protein-protein interactions at spatially confined macromolecular complexes.
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Automatización de Laboratorios/métodos , Transferencia Resonante de Energía de Fluorescencia , Procesamiento de Imagen Asistido por Computador/métodos , Cinetocoros/química , Imagen Óptica/métodos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , División Celular , Proteínas de Saccharomyces cerevisiae/análisis , Coloración y Etiquetado/métodosRESUMEN
Intravital microscopy can provide unique insights into the function of biological processes in a native context. However, physiological motion caused by peristalsis, respiration and the heartbeat can present a significant challenge, particularly for functional readouts such as fluorescence lifetime imaging (FLIM), which require longer acquisition times to obtain a quantitative readout. Here, we present and benchmark Galene, a versatile multi-platform software tool for image-based correction of sample motion blurring in both time resolved and conventional laser scanning fluorescence microscopy data in two and three dimensions. We show that Galene is able to resolve intravital FLIM-FRET images of intra-abdominal organs in murine models and NADH autofluorescence of human dermal tissue imaging subject to a wide range of physiological motions. Thus, Galene can enable FLIM imaging in situations where a stable imaging platform is not always possible and rescue previously discarded quantitative imaging data.
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Imagenología Tridimensional , Microscopía Intravital , Movimiento (Física) , Algoritmos , Animales , Técnicas Biosensibles , Adhesión Celular , Simulación por Computador , Transferencia Resonante de Energía de Fluorescencia , Guanosina Trifosfato/metabolismo , Humanos , Intestinos/fisiología , Ratones , Microscopía Fluorescente , Modelos Biológicos , Metástasis de la Neoplasia , Neuropéptidos/metabolismo , Neoplasias Pancreáticas/patología , Piel/anatomía & histología , Programas Informáticos , Proteína de Unión al GTP rac1/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
Application of advanced intravital imaging facilitates dynamic monitoring of pathway activity upon therapeutic inhibition. Here, we assess resistance to therapeutic inhibition of the PI3K pathway within the hypoxic microenvironment of pancreatic ductal adenocarcinoma (PDAC) and identify a phenomenon whereby pronounced hypoxia-induced resistance is observed for three clinically relevant inhibitors. To address this clinical problem, we have mapped tumor hypoxia by both immunofluorescence and phosphorescence lifetime imaging of oxygen-sensitive nanoparticles and demonstrate that these hypoxic regions move transiently around the tumor. To overlay this microenvironmental information with drug response, we applied a FRET biosensor for Akt activity, which is a key effector of the PI3K pathway. Performing dual intravital imaging of drug response in different tumor compartments, we demonstrate an improved drug response to a combination therapy using the dual mTORC1/2 inhibitor AZD2014 with the hypoxia-activated pro-drug TH-302.
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Resistencia a Antineoplásicos , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal , Animales , Benzamidas , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Quimioterapia Combinada , Femenino , Transferencia Resonante de Energía de Fluorescencia , Humanos , Hipoxia , Microscopía Intravital/métodos , Ratones , Ratones Endogámicos BALB C , Morfolinas/farmacología , Morfolinas/uso terapéutico , Nanopartículas/química , Nitroimidazoles/farmacología , Nitroimidazoles/uso terapéutico , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Mostazas de Fosforamida/farmacología , Mostazas de Fosforamida/uso terapéutico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pirimidinas , Transducción de Señal/efectos de los fármacos , Trasplante Heterólogo , Microambiente TumoralRESUMEN
Molecular mobility, localisation and spatiotemporal activity are at the core of cell biological processes and deregulation of these dynamic events can underpin disease development and progression. Recent advances in intravital imaging techniques in mice are providing new avenues to study real-time molecular behaviour in intact tissues within a live organism and to gain exciting insights into the intricate regulation of live cell biology at the microscale level. The monitoring of fluorescently labelled proteins and agents can be combined with autofluorescent properties of the microenvironment to provide a comprehensive snapshot of in vivo cell biology. In this Review, we summarise recent intravital microscopy approaches in mice, in processes ranging from normal development and homeostasis to disease progression and treatment in cancer, where we emphasise the utility of intravital imaging to observe dynamic and transient events in vivo We also highlight the recent integration of advanced subcellular imaging techniques into the intravital imaging pipeline, which can provide in-depth biological information beyond the single-cell level. We conclude with an outlook of ongoing developments in intravital microscopy towards imaging in humans, as well as provide an overview of the challenges the intravital imaging community currently faces and outline potential ways for overcoming these hurdles.
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Microscopía Intravital/tendencias , Imagen Molecular/tendencias , Neoplasias/genética , Microambiente Tumoral/genética , Progresión de la Enfermedad , Humanos , Neoplasias/ultraestructuraRESUMEN
This paper reports the development, modelling and application of a semi-random multicore fibre (MCF) design for adaptive multiphoton endoscopy. The MCF was constructed from 55 sub-units, each comprising 7 single mode cores, in a hexagonally close-packed lattice where each sub-unit had a random angular orientation. The resulting fibre had 385 single mode cores and was double-clad for proximal detection of multiphoton excited fluorescence. The random orientation of each sub-unit in the fibre reduces the symmetry of the positions of the cores in the MCF, reducing the intensity of higher diffracted orders away from the central focal spot formed at the distal tip of the fibre and increasing the maximum size of object that can be imaged. The performance of the MCF was demonstrated by imaging fluorescently labelled beads with both distal and proximal fluorescence detection and pollen grains with distal fluorescence detection. We estimate that the number of independent resolution elements in the final image - measured as the half-maximum area of the two-photon point spread function divided by the area imaged - to be ~3200.