fluorescent markers
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2022 ◽  
Author(s):  
Sara Caviglia ◽  
Iris A Unterweger ◽  
Akvile Gasiunaite ◽  
Alexandre E Vanoosthuyse ◽  
Francesco Cutrale ◽  
...  

Visualizing cell shapes, interactions and lineages of differentiating cells is instrumental for understanding organ development and repair. Across species, strategies for stochastic multicolour labelling have greatly facilitated tracking cells in in vivo and mapping neuronal connectivity. Nevertheless, integrating multi-fluorophore information into the context of developing tissues in zebrafish is challenging given their cytoplasmic localization and spectral incompatibility with commonly used fluorescent markers. Here, we developed FRaeppli (Fish-Raeppli) expressing bright membrane- or nuclear-targeted fluorescent proteins for efficient cell shape analysis and tracking. High spatiotemporal activation flexibility is provided by the Gal4/UAS system together with Cre/lox and/or PhiC31integrase. The distinct spectra of the FRaeppli fluorescent proteins allow simultaneous imaging with GFP and infrared subcellular reporters or tissue landmarks. By tailoring hyperspectral protocols for time-efficient acquisition, we demonstrate FRaeppli s suitability for live imaging of complex internal organs, like the liver. Combining FRaeppli with polarity markers revealed previously unknown canalicular topologies between differentiating hepatocytes, reminiscent of the mammalian liver, suggesting shared developmental mechanisms. The multispectral FRaeppli toolbox thus enables the comprehensive analysis of intricate cellular morphologies, topologies and tissue lineages at single-cell resolution in zebrafish.


2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Yuri M. Efremov ◽  
Daniel M. Suter ◽  
Peter S. Timashev ◽  
Arvind Raman

AbstractRecent developments such as multi-harmonic Atomic Force Microscopy (AFM) techniques have enabled fast, quantitative mapping of nanomechanical properties of living cells. Due to their high spatiotemporal resolution, these methods provide new insights into changes of mechanical properties of subcellular structures due to disease or drug response. Here, we propose three new improvements to significantly improve the resolution, identification, and mechanical property quantification of sub-cellular and sub-nuclear structures using multi-harmonic AFM on living cells. First, microcantilever tips are streamlined using long-carbon tips to minimize long-range hydrodynamic interactions with the cell surface, to enhance the spatial resolution of nanomechanical maps and minimize hydrodynamic artifacts. Second, simultaneous Spinning Disk Confocal Microscopy (SDC) with live-cell fluorescent markers enables the unambiguous correlation between observed heterogeneities in nanomechanical maps with subcellular structures. Third, computational approaches are then used to estimate the mechanical properties of sub-nuclear structures. Results are demonstrated on living NIH 3T3 fibroblasts and breast cancer MDA-MB-231 cells, where properties of nucleoli, a deep intracellular structure, were assessed. The integrated approach opens the door to study the mechanobiology of sub-cellular structures during disease or drug response.


Pharmaceutics ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2073
Author(s):  
Nahid S. Awad ◽  
Mohamed Haider ◽  
Vinod Paul ◽  
Nour M. AlSawaftah ◽  
Jayalakshmi Jagal ◽  
...  

Quantum dots (QDs) are a promising tool to detect and monitor tumors. However, their small size allows them to accumulate in large quantities inside the healthy cells (in addition to the tumor cells), which increases their toxicity. In this study, we synthesized stealth liposomes encapsulating hydrophilic graphene quantum dots and triggered their release with ultrasound with the goal of developing a safer and well-controlled modality to deliver fluorescent markers to tumors. Our results confirmed the successful encapsulation of the QDs inside the core of the liposomes and showed no effect on the size or stability of the prepared liposomes. Our results also showed that low-frequency ultrasound is an effective method to release QDs encapsulated inside the liposomes in a spatially and temporally controlled manner to ensure the effective delivery of QDs to tumors while reducing their systemic toxicity.


2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi20-vi20
Author(s):  
Eunhee Yi ◽  
Amit Gujar ◽  
Molly Guthrie ◽  
Hoon Kim ◽  
Dacheng Zhao ◽  
...  

Abstract Oncogenic extrachromosomal DNA elements (ecDNAs) promote intratumoral heterogeneity, creating a barrier for successful cancer treatments. The underlying mechanisms are poorly understood and studies are hampered in part by a lack of adequate tools enabling studies of ecDNA behavior. Here, we show that single-cell ecDNA copy numbers greatly vary between tumor cells, both in vitro and in patient glioblastoma specimens, suggesting uneven ecDNA segregation during mitosis. We established a CRISPR-based approach which leverages unique ecDNA breakpoint sequences to tag ecDNA with fluorescent markers in living cells. Applying this method during mitosis revealed disjointed ecDNA inheritance patterns, providing an explanation for rapid ecDNA accumulation in cancer. Post-mitosis, ecDNAs tended to cluster and clustered ecDNAs colocalized with RNA polymerase II, promoting transcription of cargo oncogenes. Our observations provide direct evidence for uneven segregation of ecDNA and sheds new light on mechanisms through which ecDNAs contribute to oncogenesis.


2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Eric B. Miller ◽  
Sarah J. Karlen ◽  
Kaitryn E. Ronning ◽  
Marie E. Burns

Abstract Background The ability to track individual immune cells within the central nervous system has revolutionized our understanding of the roles that microglia and monocytes play in synaptic maintenance, plasticity, and neurodegenerative diseases. However, distinguishing between similar subpopulations of mobile immune cells over time during episodes of neuronal death and tissue remodeling has proven to be challenging. Methods We recombineered a photoconvertible fluorescent protein (Dendra2; D2) downstream of the Cx3cr1 promoter commonly used to drive expression of fluorescent markers in microglia and monocytes. Like the popular Cx3cr1–GFP line (Cx3cr1+/GFP), naïve microglia in Cx3cr1–Dendra2 mice (Cx3cr1+/D2) fluoresce green and can be noninvasively imaged in vivo throughout the CNS. In addition, individual D2-expressing cells can be photoconverted, resulting in red fluorescence, and tracked unambiguously within a field of green non-photoconverted cells for several days in vivo. Results Dendra2-expressing retinal microglia were noninvasively photoconverted in both ex vivo and in vivo conditions. Local in vivo D2 photoconversion was sufficiently robust to quantify cell subpopulations by flow cytometry, and the protein was stable enough to survive tissue processing for immunohistochemistry. Simultaneous in vivo fluorescence imaging of Dendra2 and light scattering measurements (Optical Coherence Tomography, OCT) were used to assess responses of individual microglial cells to localized neuronal damage and to identify the infiltration of monocytes from the vasculature in response to large scale neurodegeneration. Conclusions The ability to noninvasively and unambiguously track D2-expressing microglia and monocytes in vivo through space and time makes the Cx3cr1–Dendra2 mouse model a powerful new tool for disentangling the roles of distinct immune cell subpopulations in neuroinflammation.


Photonics ◽  
2021 ◽  
Vol 8 (9) ◽  
pp. 383
Author(s):  
Mariana Potcoava ◽  
Jonathan Art ◽  
Simon Alford ◽  
Christopher Mann

Stimuli to excitable cells and various cellular processes can cause cell surface deformations; for example, when excitable cell membrane potentials are altered during action potentials. However, these cellular changes may be at or below the diffraction limit (in dendrites the structures measured are as small as 1 µm), and imaging by traditional methods is challenging. Using dual lenses incoherent holography lattice light-sheet (IHLLS-2L) detection with holographic phase imaging of selective fluorescent markers, we can extract the full-field cellular morphology or structural changes of the object’s phase in response to external stimulus. This approach will open many new possibilities in imaging neuronal activity and, overall, in light sheet imaging. In this paper, we present IHLLS-2L as a well-suited technique for quantifying cell membrane deformation in neurons without the actuation of a sample stage or detection microscope objective.


Sensors ◽  
2021 ◽  
Vol 21 (15) ◽  
pp. 5104
Author(s):  
Jerzy Chrząszcz

Fluorescent markers are widely used to protect banknotes, passports, and other documents. Verification of such documents relies upon visual assessment of the markers revealed by ultraviolet (UV) radiation. However, such an explicit approach is inappropriate in certain circumstances, e.g., when discretely checking people for marks left by a pepper gel thrower. The UV light and fluorescent light must not be visible in such applications, yet reliable detection of the markers must still be performed. This problem was successfully resolved using TRIZ methodology, which led to a patent application. The main idea of the solution is to use low-intensity time-variable UV light for illuminating an object and process the image of the object acquired by a camera to detect colour changes too small to be noticed with the naked eye. This paper describes how popular graphics editors such as Adobe Photoshop Elements were used to validate the system concept devised. Simulation experiments used images taken in both visible and UV light to assess the effectiveness and perceptibility of the detection process. The advantage of such validation comes from using commodity software and performing the experiments without access to a laboratory and without physical samples, which makes this approach especially suitable in pandemic times.


Author(s):  
Jennifer L. Cadnum ◽  
Basya S. Pearlmutter ◽  
Annette L. Jencson ◽  
Hanan Haydar ◽  
Michelle T. Hecker ◽  
...  

Abstract Objective: To investigate the frequency of environmental contamination in hospital areas outside patient rooms and in outpatient healthcare facilities. Design: Culture survey. Setting: This study was conducted across 4 hospitals, 4 outpatient clinics, and 1 surgery center. Methods: We conducted 3 point-prevalence culture surveys for methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, Clostridioides difficile, Candida spp, and gram-negative bacilli including Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter baumanii, and Stenotrophomonas maltophilia in each facility. In hospitals, high-touch surfaces were sampled from radiology, physical therapy, and mobile equipment and in emergency departments, waiting rooms, clinics, and endoscopy facilities. In outpatient facilities, surfaces were sampled in exam rooms including patient and provider areas, patient bathrooms, and waiting rooms and from portable equipment. Fluorescent markers were placed on high-touch surfaces and removal was assessed 1 day later. Results: In the hospitals, 110 (9.4%) of 1,195 sites were positive for 1 or more bacterial pathogens (range, 5.3%–13.7% for the 4 hospitals) and 70 (5.9%) were positive for Candida spp (range, 3.7%–5.9%). In outpatient facilities, 31 of 485 (6.4%) sites were positive for 1 or more bacterial pathogens (range, 2% to 14.4% for the 5 outpatient facilities) and 50 (10.3%) were positive for Candida spp (range, 3.9%–23.3%). Fluorescent markers had been removed from 33% of sites in hospitals (range, 28.4%–39.7%) and 46.3% of sites in outpatient clinics (range, 7.4%–82.8%). Conclusions: Surfaces in hospitals outside patient rooms and in outpatient facilities are frequently contaminated with healthcare-associated pathogens. Improvements in cleaning and disinfection practices are needed to reduce contamination.


2021 ◽  
Author(s):  
Daniel S Levic ◽  
Naoya Yamaguchi ◽  
Siyao Wang ◽  
Holger Knaut ◽  
Michel Bagnat

Zebrafish provide an excellent model for in vivo cell biology studies due to their amenability to live imaging. Protein visualization in zebrafish has traditionally relied on overexpression of fluorescently tagged proteins from heterologous promoters, making it difficult to recapitulate endogenous expression patterns and protein function. One way to circumvent this problem is to tag the proteins by modifying their endogenous genomic loci. Such an approach is not widely available to zebrafish researchers due to inefficient homologous recombination and the error-prone nature of targeted integration in zebrafish. Here, we report a simple approach for tagging proteins in zebrafish on their N- or C termini with fluorescent markers by inserting PCR-generated donor amplicons into non-coding regions of the corresponding genes. Using this approach, we generated endogenously tagged alleles for several genes critical for epithelial biology and organ development including the tight junction components ZO-1 and Cldn15la, the trafficking effector Rab11a, and the ECM receptor β1 integrin. Our approach facilitates the generation of knock-in lines in zebrafish, opening the way for accurate quantitative imaging studies.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kazuhiko Ishigaki ◽  
Keiki Kumano ◽  
Kyohei Fujita ◽  
Hiroo Ueno

AbstractAlthough the physiological function of the omentum remains elusive, it has been proposed that it plays an important role in fat storage, immune regulation, and regeneration of injured tissues and organs. Although the omentum undergoes expansion upon activation, reports on the accurate assessment of increased cell types and the origin of the increased cells remain limited. To investigate this aspect, the omenta of parabiotic mice were subjected to activation using distinct fluorescent markers and single-cell RNA sequencing (scRNA-seq) was performed to identify circulation-derived omental cells. We found that a considerable number of circulating cells contributed to the activation of the omentum. The omental cells derived from circulating cells exhibited morphological features similar to those of fibroblasts. scRNA-seq revealed the existence of a novel cell population that co-expressed macrophage and fibroblast markers in the activated omentum, suggesting that it corresponded to circulating macrophage-derived fibroblast-like cells. Lineage tracing experiments revealed that most fibroblasts in the activated omentum were not derived from WT1-positive mesenchymal progenitors. The cell cluster also expressed various chemokine genes, indicating its role in the activation and recruitment of immune cells. These results provide important information regarding the activation of the omentum.


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