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Journal of Visualized Experiments : JoVE Apr 2024Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the tumor...
Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the tumor microenvironment and its response to therapy. These imaging modalities have micron-scale resolution but have limited use in the clinic due to their shallow penetration depth into tissue. More clinically applicable imaging modalities such as CT, MRI, and PET have much greater penetration depth but have comparatively lower spatial resolution (mm scale). To translate preclinical intravital imaging findings into the clinic, new methods must be developed to bridge this micro-to-macro resolution gap. Here we describe a dorsal skinfold window chamber tumor mouse model designed to enable preclinical intravital and clinically applicable (CT and MR) imaging in the same animal, and the image analysis platform that links these two disparate visualization methods. Importantly, the described window chamber approach enables the different imaging modalities to be co-registered in 3D using fiducial markers on the window chamber for direct spatial concordance. This model can be used for validation of existing clinical imaging methods, as well as for the development of new ones through direct correlation with "ground truth" high-resolution intravital findings. Finally, the tumor response to various treatments-chemotherapy, radiotherapy, photodynamic therapy-can be monitored longitudinally with this methodology using preclinical and clinically applicable imaging modalities. The dorsal skinfold window chamber tumor mouse model and imaging platforms described here can thus be used in a variety of cancer research studies, for example, in translating preclinical intravital microscopy findings to more clinically applicable imaging modalities such as CT or MRI.
Topics: Animals; Mice; Intravital Microscopy; Magnetic Resonance Imaging; Translational Research, Biomedical; Disease Models, Animal; Female
PubMed: 38682919
DOI: 10.3791/66383 -
Journal of Visualized Experiments : JoVE Apr 2024Neural stem cells (NSCs) divide and produce newborn neurons in the adult brain through a process called adult neurogenesis. Adult NSCs are primarily quiescent, a...
Neural stem cells (NSCs) divide and produce newborn neurons in the adult brain through a process called adult neurogenesis. Adult NSCs are primarily quiescent, a reversible cell state where they have exited the cell cycle (G0) yet remain responsive to the environment. In the first step of adult neurogenesis, quiescent NSCs (qNSCs) receive a signal and activate, exiting quiescence and re-entering the cell cycle. Thus, understanding the regulators of NSC quiescence and quiescence exit is critical for future strategies targeting adult neurogenesis. However, our understanding of NSC quiescence is limited by technical constraints in identifying quiescent NSCs (qNSCs) and activated NSCs (aNSCs). This protocol describes a new approach to identify and enrich qNSCs and aNSCs generated in in vitro cultures by imaging NSC autofluorescence. First, this protocol describes how to use a confocal microscope to identify autofluorescent markers of qNSCs and aNSCs to classify NSC activation state using autofluorescence intensity. Second, this protocol describes how to use a fluorescent activated cell sorter (FACS) to classify NSC activation state and enrich samples for qNSCs or aNSCs using autofluorescence intensity. Third, this protocol describes how to use a multiphoton microscope to perform fluorescence lifetime imaging (FLIM) at single-cell resolution, classify NSC activation state, and track the dynamics of quiescent exit using both autofluorescence intensities and fluorescence lifetimes. Thus, this protocol provides a live-cell, label-free, single-cell resolution toolkit for studying NSC quiescence and quiescence exit.
Topics: Neural Stem Cells; Animals; Mice; Microscopy, Confocal; Flow Cytometry; Optical Imaging; Neurogenesis
PubMed: 38682901
DOI: 10.3791/63110 -
PloS One 2024The three-dimensional swimming tracks of motile microorganisms can be used to identify their species, which holds promise for the rapid identification of bacterial...
The three-dimensional swimming tracks of motile microorganisms can be used to identify their species, which holds promise for the rapid identification of bacterial pathogens. The tracks also provide detailed information on the cells' responses to external stimuli such as chemical gradients and physical objects. Digital holographic microscopy (DHM) is a well-established, but computationally intensive method for obtaining three-dimensional cell tracks from video microscopy data. We demonstrate that a common neural network (NN) accelerates the analysis of holographic data by an order of magnitude, enabling its use on single-board computers and in real time. We establish a heuristic relationship between the distance of a cell from the focal plane and the size of the bounding box assigned to it by the NN, allowing us to rapidly localise cells in three dimensions as they swim. This technique opens the possibility of providing real-time feedback in experiments, for example by monitoring and adapting the supply of nutrients to a microbial bioreactor in response to changes in the swimming phenotype of microbes, or for rapid identification of bacterial pathogens in drinking water or clinical samples.
Topics: Holography; Deep Learning; Microscopy; Imaging, Three-Dimensional; Bacteria; Quantitative Phase Imaging
PubMed: 38669245
DOI: 10.1371/journal.pone.0301182 -
Heliyon Apr 2024Additive manufacturing technology has advanced beyond creating optimized features, from strengthening materials to make them lightweight to fabricating multi-material...
Additive manufacturing technology has advanced beyond creating optimized features, from strengthening materials to make them lightweight to fabricating multi-material combinations that offer functionalities beyond the capabilities of individual materials. In this study, a lamination method for laser-directed energy deposition (LDED) is developed to achieve dense multi-material features, and a design that combines different and dissimilar materials is developed. To evaluate these novel developments, two materials-AISI 316L stainless steel and Inconel 625-are introduced. Tensile specimens, fabricated via multi-material additive manufacturing using LDED, are subjected to tensile tests that are recorded on video for digital image correlation. After the tests, fracture surface analyses of the fractured specimens are performed via scanning electron microscopy, and optical monitoring analyses are performed on the specimens that are not subjected to the tensile tests. The results indicate that the specimens demonstrate varied mechanical properties due to the influence of lamination direction and order, which affect the formation of critical cracks and pores.
PubMed: 38655361
DOI: 10.1016/j.heliyon.2024.e29666 -
Clinical Oral Investigations Apr 2024Confocal laser endomicroscopy (CLE) is an optical method that enables microscopic visualization of oral mucosa. Previous studies have shown that it is possible to...
OBJECTIVES
Confocal laser endomicroscopy (CLE) is an optical method that enables microscopic visualization of oral mucosa. Previous studies have shown that it is possible to differentiate between physiological and malignant oral mucosa. However, differences in mucosal architecture were not taken into account. The objective was to map the different oral mucosal morphologies and to establish a "CLE map" of physiological mucosa as baseline for further application of this powerful technology.
MATERIALS AND METHODS
The CLE database consisted of 27 patients. The following spots were examined: (1) upper lip (intraoral) (2) alveolar ridge (3) lateral tongue (4) floor of the mouth (5) hard palate (6) intercalary line. All sequences were examined by two CLE experts for morphological differences and video quality.
RESULTS
Analysis revealed clear differences in image quality and possibility of depicting tissue morphologies between the various localizations of oral mucosa: imaging of the alveolar ridge and hard palate showed visually most discriminative tissue morphology. Labial mucosa was also visualized well using CLE. Here, typical morphological features such as uniform cells with regular intercellular gaps and vessels could be clearly depicted. Image generation and evaluation was particularly difficult in the area of the buccal mucosa, the lateral tongue and the floor of the mouth.
CONCLUSION
A physiological "CLE map" for the entire oral cavity could be created for the first time.
CLINICAL RELEVANCE
This will make it possible to take into account the existing physiological morphological features when differentiating between normal mucosa and oral squamous cell carcinoma in future work.
Topics: Humans; Microscopy, Confocal; Mouth Mucosa; Male; Female; Middle Aged; Mouth Neoplasms
PubMed: 38652317
DOI: 10.1007/s00784-024-05664-9 -
Journal of Visualized Experiments : JoVE Apr 2024Radiation therapy (RT) is one of the mainstays of modern clinical cancer management. However, not all cancer types are equally sensitive to irradiation, often (but not...
Radiation therapy (RT) is one of the mainstays of modern clinical cancer management. However, not all cancer types are equally sensitive to irradiation, often (but not always) because of differences in the ability of malignant cells to repair oxidative DNA damage as elicited by ionizing rays. Clonogenic assays have been employed for decades to assess the sensitivity of cultured cancer cells to ionizing irradiation, largely because irradiated cancer cells often die in a delayed manner that is difficult to quantify with short-term flow cytometry- or microscopy-assisted techniques. Unfortunately, clonogenic assays cannot be employed as such for more complex tumor models, such as patient-derived tumor organoids (PDTOs). Indeed, irradiating established PDTOs may not necessarily abrogate their growth as multicellular units, unless their stem-like compartment is completely eradicated. Moreover, irradiating PDTO-derived single-cell suspensions may not properly recapitulate the sensitivity of malignant cells to RT in the context of established PDTOs. Here, we detail an adaptation of conventional clonogenic assays that involves exposure of established PDTOs to ionizing radiation, followed by single-cell dissociation, replating in suitable culture conditions and live imaging. Non-irradiated (control) PDTO-derived stem-like cells reform growing PDTOs with a PDTO-specific efficiency, which is negatively influenced by irradiation in a dose-dependent manner. In these conditions, PDTO-forming efficiency and growth rate can be quantified as a measure of radiosensitivity on time-lapse images collected until control PDTOs achieve a predefined space occupancy.
Topics: Humans; Organoids; Radiation Tolerance; Neoplasms
PubMed: 38647326
DOI: 10.3791/66680 -
Journal of Visualized Experiments : JoVE Apr 2024Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. However, the...
Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. However, the commonly used methods to purify AAVs make use of cesium chloride or iodixanol density gradient ultracentrifugation. Despite their advantages, these methods are time-consuming, have limited scalability, and often result in vectors with low purity. To overcome these constraints, researchers are turning their attention to chromatography techniques. Here, we present an optimized heparin-based affinity chromatography protocol that serves as a universal capture step for the purification of AAVs. This method relies on the intrinsic affinity of AAV serotype 2 (AAV2) for heparan sulfate proteoglycans. Specifically, the protocol entails the co-transfection of plasmids encoding the desired AAV capsid proteins with those of AAV2, yielding mosaic AAV vectors that combine the properties of both parental serotypes. Briefly, after the lysis of producer cells, a mixture containing AAV particles is directly purified following an optimized single-step heparin affinity chromatography protocol using a standard fast protein liquid chromatography (FPLC) system. Purified AAV particles are subsequently concentrated and subjected to comprehensive characterization in terms of purity and biological activity. This protocol offers a simplified and scalable approach that can be performed without the need for ultracentrifugation and gradients, yielding clean and high viral titers.
Topics: Dependovirus; Chromatography, Affinity; Heparin; Genetic Vectors; Humans; HEK293 Cells
PubMed: 38647283
DOI: 10.3791/66550 -
Journal of Visualized Experiments : JoVE Apr 2024Organoid cell culture systems can recapitulate the complexity observed in tissues, making them useful in studying host-pathogen interactions, evaluating drug efficacy...
Organoid cell culture systems can recapitulate the complexity observed in tissues, making them useful in studying host-pathogen interactions, evaluating drug efficacy and toxicity, and tissue bioengineering. However, applying these models for the described reasons may be limited because of the three-dimensional (3D) nature of these models. For example, using 3D enteroid culture systems to study digestive diseases is challenging due to the inaccessibility of the intestinal lumen and its secreted substances. Indeed, stimulation of 3D organoids with pathogens requires either luminal microinjection, mechanical disruption of the 3D structure, or generation of apical-out enteroids. Moreover, these organoids cannot be co-cultured with immune and stromal cells, limiting in-depth mechanistic analysis into pathophysiological dynamics. To circumvent this, we optimized a bovine primary cell two-dimensional (2D) enteroid-derived monolayer culture system, allowing co-culture with other relevant cell types. Ileal crypts isolated from healthy adult cattle were cultured to generate 3D organoids that were cryopreserved for future use. A 2D monolayer was created using revived 3D enteroids that were passaged and disrupted to yield single cells, which were seeded on basement membrane extract-coated transwell cell culture inserts, thereby exposing their apical surface. The intestinal monolayer polarity, cellular differentiation, and barrier function were characterized using immunofluorescence microscopy and measuring transepithelial electrical resistance. Stimulation of the apical surface of the monolayer revealed the expected functionality of the monolayer, as demonstrated by cytokine secretion from both apical and basal compartments. The described 2D enteroid-derived monolayer model holds great promise in investigating host-pathogen interactions and intestinal physiology, drug development, and regenerative medicine.
Topics: Animals; Cattle; Organoids; Translational Research, Biomedical; Cell Culture Techniques; Coculture Techniques; Ileum
PubMed: 38647281
DOI: 10.3791/65901 -
Advanced Science (Weinheim,... Jun 2024Single-molecule localization microscopy has proved promising to unravel the dynamics and molecular architecture of thin biological samples down to nanoscales. For...
Single-molecule localization microscopy has proved promising to unravel the dynamics and molecular architecture of thin biological samples down to nanoscales. For applications in complex, thick biological tissues shifting single-particle emission wavelengths to the shortwave infrared (SWIR also called NIR II) region between 900 to 2100 nm, where biological tissues are more transparent is key. To date, mainly single-walled carbon nanotubes (SWCNTs) enable such applications, but they are inherently 1D objects. Here, 0D ultra-small luminescent gold nanoclusters (AuNCs, <3 nm) and ≈25 nm AuNC-loaded-polymeric particles that can be detected at the single-particle level in the SWIR are presented. Thanks to high brightness and excellent photostability, it is shown that the dynamics of the spherical polymeric particles can be followed at the single-particle level in solution at video rates for minutes. We compared single particle tracking of AuNC-loaded-polymeric particles with that of SWCNT diffusing in agarose gels demonstrating the specificity and complementarity of diffusion properties of these SWIR-emitting nano-objects when exploring a complex environment. This extends the library of photostable SWIR emitting nanomaterials to 0D nano-objects of variable size for single-molecule localization microscopy in the second biological window, opening unprecedented possibilities for mapping the structure and dynamics of complex biological systems.
PubMed: 38639398
DOI: 10.1002/advs.202309267 -
Archives of Dermatological Research Apr 2024This paper explores the role of teledermatology (TD) in Mohs micrographic surgery (MMS) at various stages of patient care. The study aims to assess the benefits,... (Review)
Review
This paper explores the role of teledermatology (TD) in Mohs micrographic surgery (MMS) at various stages of patient care. The study aims to assess the benefits, limitations, and patient experiences surrounding TD integration into MMS practices. We conducted a PubMed search using keywords related to TD and MMS, categorizing selected articles into pre-operative, intra-operative, and post-operative stages of MMS. TD reduced waiting times (26.10 days for TD compared to 60.57 days for face-to-face [FTF]) and consultation failure rates (6% for TD vs. 17% for FTF) for MMS preoperative consultations. It also shortened time to treatment by two weeks and led to notable travel savings (162.7 min, 144.5 miles, and $60.00 per person). Telepathology facilitated communication and decision-making during MMS, improving accuracy and efficiency, especially in challenging cases requiring collaboration where physical presence of another surgeon or pathologist is not feasible. Telepathology definitively diagnosed benign lesions and malignant tumors in 81.8% of cases (18/22). Additionally, there was a 95% agreement between conventional light microscopy diagnosis and telepathology in tumors (19/20), and 100% agreement for all 20 Mohs frozen section consultations. For post-operative follow-up, telephone follow-up (TFU) and text messaging proved effective, cost-efficient alternatives with high patient satisfaction (94% in New Zealand and 96% in the U.K.) and early complication identification. This study underscores TD's multifaceted benefits in MMS: enhanced patient experience preoperatively, improved communication during surgery, and cost-effective postoperative follow-up. Limitations include the financial expense and technical issues that can arise with TD (connectivity problems, delays in video/audio transmission, etc.). Further studies are needed to explore emerging TD modalities in post-operative patient management. The integration of TD into MMS signifies a progressive step in dermatological care, offering convenient, cost-effective, and better solutions with the potential to enhance patient experiences and outcomes.
Topics: Humans; Mohs Surgery; Communication; New Zealand; Pathologists; Patient Satisfaction
PubMed: 38625403
DOI: 10.1007/s00403-024-02851-2