MitoCare Service Centers
The MitoCare Service Centers are comprised of the following areas and detailed below
- Confocal, Multiphoton and Super-resolution Imaging
- Electron Microscopy & Tomography
- Seahorse XF24
Mission, Goals, Capabilities
The confocal, multiphoton and super-resolution imaging facility will provide services particularly focused on high resolution fluorescence visualization. The available confocal/multiphoton system provides for a range of experiments from sub-cellular localization to mapping of large tissue sections. The super-resolution capability utilizes single molecule localization by stochastic imaging (StORM/PALM) to circumvent the diffraction limit of optical microscopy and provide resolution at < 50 nm. The facility will provide access to these instruments for researchers, training on their use and consultation on experimental design and analysis.
Zeiss LSM780 NLO confocal/multiphoton microscope: 405, 457, 488, 514, 561, 594 and 633 nm laser lines for single photon excitation. Chameleon Vision II system (Coherent) for MP illumination. The confocal scanner is coupled to a Zeiss inverted microscope (Axio Observer). Three internal detectors including Meta detector for spectral imaging. Two external, non-descanned detectors for multi-photon imaging. Transmitted light detector for DIC/phase contrast. 10x, 20x, 25x, 40x, 63x and 100x objectives. Targeted illumination capability for photo-bleaching/activation; motorized focus for z-stacks; encoded, motorized stage; temperature and humidity controlled incubator with CO2 regulation. Zen acquisition and analysis software.
Bruker Vutara 352 super-resolution microscope: StORM/PALM type super-resolution with 488, 561, 639 and 750nm lasers for excitation and 405nm for photoswitching. Detection via Quad-Field projection onto a sCMOS camera (Hamamatsu Orca-Flash4.0 V2) allows simultaneous imaging of two colors in two focal planes or up to four colors in a single-plane. Includes a motorized TIRF module and temperature-controlled incubation for live cell imaging. Includes software for analysis of single-molecule localization and quantitative analysis of super-resolution experiments and/or single particle tracking.
- High resolution, 3D imaging of live and fixed specimens
- Tile-scan images for large areas
- Imaging in multi-well formats
- Photo-activation, bleaching or other optogenetic techniques
- Tissue imaging with multi-photon excitation
- Sub-diffraction-limit molecular localization microscopy.
- Training on microscope use and data analysis
- Consultation on experiment design.
Gyorgy Hajnoczky, M.D. Ph.D. & David Weaver
Contact – David.Weaver@jefferson.edu
Mission, Goals, Capabilities
The Research Electron Microscopy Laboratory in the Department of Pathology, Anatomy and Cell Biology aims to provide researchers with ultrastructure in specimen prepared from tissue, cellular or subcellular preparations. We are also open (on case by case base) to collaborative consultations to assist users in TEM related experimental design and analysis. Current capabilities allow 2D transmission electron microscopy. Tilt-angle tomography of ~150 nm thick sections of resin-embedded samples (or negative-stained particles) will be available* for limited collaborations.
The core instrument is an FEI Tecnai 12 120 keV digital TEM, equipped with an AMT XR111 CCD camera and upgraded with a tomography package that uses the FEI Xplore 3D Tomography software. Addition of a high field of view tomography holder and Xplore3D retrofit package allows acquisition of electron tomograms at high tilt angles. The sample preparation laboratory has two Leica UCT ultramicrotomes.
Services available include
- (20% technician/operator effort, ~1 day scope time/week). Providing training and supervision for user to obtain images with the scope. Providing instructions/demo for routine and non-routine fixation and embedding. Compact scoping assignments.
- (40% technician/operator effort ~2 days scope time/week) in addition to I, extended limited scoping assignments, electron tomography (limited collaborations). Resin embedding. Ultramicrotomy and (post)staining of thin and thick sections.
*: Tilt-angle tomography requires a currently missing part, the tomography sample holder, for which a capital budget request is submitted.
Gyorgy Csordas, MD
Contact: TBN Tech
Mission, Goals, Capabilities
The Seahorse XF24 instrument is a sensitive, high-throughput (24-well) easy-to-use technology to measure changes in O2 tension and proton concentration in the medium, as a function of time (~10 second resolution). The typical application is to interrogate mitochondrial bioenergetics in adherent preparations (intact or permeabilized adherent cells, or isolated mitochondria electrostatically adhered to plastic). The technology is endorsed by the mitochondrial biology community, and has been used in 100s of publications in multiple areas of biology).
The service center will 1) provide access to the Seahorse XF24, 2) provide basic training, and 3) ensure the functionality of the XF24.
The XF24 is located within the MitoCare Center, JAH 528, at the “Seifert lab” space.
Seahorse XF24 Instrument; PC with acquisition software and Excel for initial analysis of data; 37ºC, ambient CO2 incubator.
To provide access to the Seahorse instrument, as well as to provide 1) training for 1st time users in instrument use and navigating the data output file, and refresher training if needed for returning users, 2) basic information about cell seeding density, concentrations of uncoupler and substrate conditions, 3) access to an incubator for overnight hydration of the cartridge, 4) access to bench space for plate preparation, 5) access to Excel for initial data analysis, 6) oversight of instrument scheduling, 7) leadership to ensure the functionality of the instrument, by regular inspection of instrument function, by arranging the purchase of the service contract, scheduling annual on-sight preventative maintenance and any additional maintenance.
Not provided: consumables (e.g. custom culture plates, cartridges, assay media, permeabilizing reagents, cartridge hydration solution), pipettors, assistance with experimental design and data interpretation, anything related to and including an actual seahorse.
Erin Seifert, PhD