Advanced Imaging Core

Advanced Imaging Core – Abstract The overall goal of the Advanced Imaging Core is to provide DDRCC investigators with sophisticated technologies, methods, and training required for successful, high-end cell- and tissue-based imaging and analysis, including confocal, multiphoton, and super-resolution microscopy, intravital imaging, and automated imaging of multi-well plates. Specific Aims are as follows: 1) To provide DDRCC investigators with state-of-the- art capabilities in optical microscopy; 2) To develop novel imaging applications that address the emerging scientific needs of DDRCC members; and 3) To support Members with expert mentoring, education, training, and consultation on sophisticated optical imaging technologies that enable scientific discovery. The Core houses the following major imaging systems: 1) Leica Stimulated Emission Depletion (STED) super-resolution microscope to be installed in the summer/fall of 2024; 2) Zeiss LSM 880 NLO multiphoton/confocal system equipped with Quasar spectral detection and Fast Airyscan super-resolution capability; 3) Olympus Fluoview FV1200 multiphoton microscope with silicone oil optics for intravital imaging; 4) Olympus Fluoview FV 10i LIV live cell confocal microscope with water immersion optics; 5) BD BioSciences CARV II spinning disk confocal microscope for video-rate imaging (to be replaced with a new Andor BC43 spinning disk microscope in 2024, 2024); 6) Leica TCS SP8 laser scanning confocal microscope; 7) Zeiss LSM 510 META confocal microscope; 8) Zeiss Axiovert 200M widefield fluorescence microscope; and 9) Agilent Cytation 5 Cell Imaging Multi-Mode Reader. All microscopes are equipped with environmental chambers for temperature and gas phase control to allow nondestructive 3D imaging of living cells, tissues, and organisms. Major applications include 1) 2D and 3D super-resolution imaging of both fixed and living specimens to a lateral resolution of ~20 nm; 2) live cell imaging of parameter-indicating fluorophores to monitor ions, electrical potentials, radical generation, pyridine nucleotide reduction, membrane permeability, cell viability (apoptosis and necrosis), and the submicron distribution of fluorescent proteins and other fluorescent reporters; 3) imaging of tissue sections; 4) fluorescence resonance energy transfer (FRET) and DuoLink to quantify interactions between specific molecules; 4) intravital microscopy to monitor microcirculation, leukocyte margination, mitochondrial polarization and permeability, radical generation, gene expression, and other parameters in living animals; and 5) automated, high throughput fluorescence, brightfield, and phase contrast imaging of specimens in multi-well format. Computer workstations provide offline image processing/analysis (Bitplane Imaris, Huygens, ImageJ FIJI, Metamorph, IPLab and others). Hands-on training in current and next-generation imaging approaches are also provided by seminars, demos, and a biennial 1-week Charleston Workshop on Light Microscopy for the Biosciences.