Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) and other respiratory pathologies in vivo is difficult due to the limited accessibility and inherent motion artifacts of the lungs. Nonetheless, various approaches have been developed to overcome these challenges. This protocol describes a method for intravital fluorescence microscopy to study real-time leukocyte-endothelial interactions in the pulmonary microcirculation in an experimental model of ALI. An in vivo lung imaging system and 3-D printed intravital microscopy platform are used to secure the anesthetized mouse and stabilize the lung while minimizing confounding lung injury. Following preparation, widefield fluorescence microscopy is used to study leukocyte adhesion, leukocyte rolling, and capillary function. While the protocol presented here focuses on imaging in an acute model of inflammatory lung disease, it may also be adapted to study other pathological and physiological processes in the lung.
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J Vis Exp
January 2025
Institute of Biochemistry and Molecular Biology, Hengyang Medical School, University of South China; National Health Commission Key Laboratory of Birth Defect Research and Preventio, Hunan Provincial Maternal and Child Health Care Hospital;
Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. Conflicts between the replication and transcription machinery, termed transcription-replication conflicts (TRCs), pose a considerable risk to genome stability, a critical factor in cancer development. While several factors regulating these collisions have been identified, pinpointing primary causes remains difficult due to limited tools for direct visualization and clear interpretation.
View Article and Find Full Text PDFMol Biol Cell
January 2025
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147 USA.
The endo-lysosomal system plays a crucial role in maintaining cellular homeostasis and promoting organism fitness. The pH of its acidic compartments is a crucial parameter for proper function, and it is dynamically influenced by both intracellular and environmental factors. Here, we present a method based on fluorescence lifetime imaging microscopy (FLIM) for quantitatively analyzing the pH profiles of acidic endolysosomal compartments in diverse types of primary mammalian cells and in live organism .
View Article and Find Full Text PDFJ Pestic Sci
November 2024
Bacillus Tech LLC.
The Cry1Fa insecticidal protein from (Bt) was expressed on the surface of (Bs) spores to create transgenic Bs spores referred to as Spore-Cry1Fa. Cry1Fa, along with its leader sequence, was connected to the carboxyl end of a Bs spore outercoat protein, CotC, through a flexible linker. The Arg-27 residue of the Cry1Fa protein was mutated to Leu to prevent detachment from the spores due to protease digestion.
View Article and Find Full Text PDFJ Pestic Sci
November 2024
Faculty of Agriculture, Tottori University.
A search for antifungal compounds from the mushroom using a bioassay-guided chromatographic fractionation approach led to the discovery of a novel polyketide harboring a rare 3,3a,9,9a-tetrahydro-1-furo[3,4-]chromen-1-one skeleton. The novel compound was named coprinolide. The inhibitory activity and fungicidal potential of coprinolide were evaluated against five economically important plant-pathogenic fungi.
View Article and Find Full Text PDFHardwareX
March 2025
LIGHT Community, Physics Department, Imperial College London SW7 2AZ, UK.
We recently demonstrated polarisation differential phase contrast microscopy () as a robust, low-cost single-shot implementation of (semi)quantitative phase imaging based on differential phase microscopy. utilises a polarisation-sensitive camera to simultaneously acquire four obliquely transilluminated images from which phase images mapping spatial variation of optical path difference can be calculated. microscopy can be implemented on existing or bespoke microscopes and can utilise radiation at a wide range of visible to near infrared wavelengths and so is straightforward to integrate with fluorescence microscopy.
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