Cell Biology and Bioimaging Core

Faculty

David Burk, PhD

David Burk, PhD

About this Core

The mission of the Cell Biology and Bioimaging Core is to provide investigators access to imaging, flow cytometry, and specimen preparation related instrumentation, training, and expertise and to promote and facilitate research on obesity, diabetes, nutrition, and metabolic disease.

The Cell Biology and Bioimaging core provides investigators the opportunity to use equipment dedicated to histology, flow cytometry, and optical imaging techniques. Located centrally in the Pennington Biomedical Basic Sciences Building, the CBBC occupies rooms L4076, L4016, and L4011, with separate cell culture and dissociation equipment in C1015. 

The imaging equipment in L4076 and L4011 include a wide variety of optical imaging platforms including widefield fluorescence and transmitted imaging (Zeiss Axioskop 40, Zeiss Axiovert 40), semiautomated imaging (BioTek Lionheart FX), whole slide scanning (Hamamatsu Nanozoomer, Zeiss Axioscan 7), laser microdissection (Leica LMD6000), total internal reflection fluorescence (Leica TIRF), light sheet microscopy (LaVision BioTec Ultramicroscope II), and confocal (Leica SP5). Analytical FACS instrumentation is located in L4076 and include a 4-channel BD FACSCalibur and >30-color-capable Cytek Aurora 5-laser analyzer.

The CBBC also houses the necessary equipment for tissue processing, staining, and sectioning, including cryostats, rotary microtomes, a sliding microtome, compresstome, autostainer, and coverslipper. Ancillary space is available for conducting tissue clearing, RNAScope in situ staining, cell culture, and tissue dissociation via a Miltenyi Biotec gentleMACS Octo dissociator with heaters.   

Resources

  • Leica CM1950
    • Cryostat for frozen tissue
  • Microm 355S with STS and CoolCut and two Leica RM2255
    • Rotary microtomes for sectioning paraffin blocks.
  • Leica RM 2000
    • Sliding microtome for sectioning fixed and flash-frozen
  • Vibratome 1500
    • For sectioning fixed
  • Precisionary Instruments Compresstome VF-310-0Z
    • For sectioning live and fixed
  • Sakura VIP 6
    • Automated tissue processor for tissue fixation, dehydration, and wax
  • Leica ST5020 and CV5030
    • Autostainer/coverslipper provide high throughput standard staining (H&E) as well as special staining of slides.
  • Leica BOND RXm stainer
    • Allows increased throughput and reduced variability in immunohistochemical and in situ staining procedures. The system can process (dewax, antigen retrieval, probe application, wash, etc.) up to 30 slides at a time. Users can develop novel staining protocols that allow for heated staining steps as well as multiple chromogens for detection. The system is compatible with off-the-shelf kits from Akoya, ACD, Ultivue, and others for multiplex RNA or protein detection using immunofluorescence. Acquired through COBRE Administrative Supplement - 2003
  • Sanyo VIP and Thermo Revco -80°C Freezers, Sanyo -30° C Freezer, Sanyo LabCool 4°C Refrigerator
    • For sample and reagent storage.
  • Leica LMD 6000
    • Laser microdissection platform for the isolation of fixed or live cells from specialized culture plates or select areas from histological sections based on their morphology for downstream analysis. Cutting objectives range from 5X to 63X and the system is equipped with a triple band fluorescence cutting cube (DAPI, FITC, Cy3/TxRed). The system can operate as a 5-channel fluorescence imaging platform and is equipped with a color using LAS X software and either a color (Leica DMC4500) or monochrome (Leica K5) camera for capturing brightfield, polarized, or fluorescent images.
  • Miltenyi Biotec gentleMACS Octo Dissociator with Heaters
    • For fully automated and heated dissociation or homogenization of tissues for downstream FACS or genomic analysis.
  • Zeiss Axioskop 40 FL (upright)
    • Manual fluorescence microscope with shared Axiocam HrC camera for brightfield and epi-fluorescence imaging of fixed specimens. Filters are available for DAPI, FITC, and TRITC.
  • Zeiss Axiovert 40 FL (inverted)
    • Manual fluorescence microscope with shared Axiocam HrC camera for brightfield, phase contrast and epi-fluorescence imaging of fixed or live specimens using an X-Cite mini+ LED illumination system. Filters are available for DAPI, FITC, and TRITC.
  • Leica AM TIRF MC
    • Multicolor TIRF/live cell platform allows for the analysis of membrane associated events in live cell cultures as well as studies of receptor signaling and agonist interactions within 70- 250 nm of the glass substrate in which the cells are cultured. Ultra-fast imaging in both TIRF and epi modes is accomplished via a 2048 x 2048-pixel Hamamatsu Flash 4 2 sCMOS camera. The system will also allow live cell experiments to be conducted in epi-fluorescence mode using either arc lamp or laser illumination. The system is equipped with a live cell chamber for temperature, humidity and CO2 control, filter cubes for visualizing DAPI, CFP, GFP, YFP, and RFP.
  • Leica SP5 AOBS confocal
    • Provides researchers with the ability to conduct real time 3D analysis of their living cells. In addition, the instrument can create confocal montages of large samples and is equipped for long term live cell experiments. The system is equipped with lasers emitting at 405, 458, 476, 488, 496, 514, 561, 594, and 633 nm, a super-z galvo stage and can operate in conventional (up to 2800 Hz) or resonant mode (up to 16000 Hz). Spectral detection is possible on this instrument.
  • Hamamatsu NanoZoomer HT
    • Digital slide scanner allows high throughput scanning of whole slides in brightfield and fluorescence modes. Fluorescence imaging is done using either single color cubes and subsequent manual overlay of images or via a multipass filter cube. Digital data can be archived or used for morphometric, intensity specific or stereological analysis on the included workstation with Visiopharm software.
  • Zeiss Axioscan 7
    • Slide scanner outfitted with objectives ranging from 5x to 40x including a dedicated POL 20x for imaging The scanner is equipped with single and multi-band filter sets and uses LED illumination for fluorescence experiments and is capable of 5-color multiplexed imaging (DAPI, 488, 555/546, 647, 750). Acquired through COBRE Administrative Supplement – 2021.
  • BioTek Lionheart FX
    • Automated microscope enables long-term live cell observations in a myriad of formats from T25 flasks to 35 mm dishes, to 1536-well plates. The system is equipped with a laser- based autofocusing module, LED-fluorescence cubes for common fluorophores, and objectives for 4x to 40x observations through plastic or glass. In addition, the system is equipped with a dual channel injector for kinetic assays and a CO2/O2 gas mixer for long-term live cell experiments.
  • LaVision BioTec UltraMicroscope II
    • Light sheet microscope allows imaging of cleared tissues, organs, or small organisms. The system is equipped with 4 solid state laser lines – 488, 561, 639, and 785 nm – and is outfitted with the Super Plan configuration that allows imaging with dedicated 1.1x/0.1 NA and 4x/0.35 NA long working distance objectives (>16 mm). Combined with the automated magnification changer the system has an effective magnification range of 0.66x to 10x.
  • Cytek Aurora spectral flow cytometer
    • Outfitted with five lasers (UV 355, violet 405, blue 488, yellow-green 561, and red 640 nm) and 64 + 3 channel APD This configuration provides optimal flexibility in panel design through full spectrum capture and unmixing using Spectraflow software. Resolution of up to 40 unique fluorescence labels is possible with this system.
  • BD FACSCalibur flow cytometer
    • Equipped with two lasers (488 and 635 nm) and detectors for forward scatter, side scatter, and four fluorescent channels. The system can be equipped with a FACSLoader module for semi-automated analysis of up to 38 samples.
  • BD Biosciences FACS Aria II*
    • Cell sorter is housed in a Baker BioProtect IV, model BP504-5 Class II Biosafety cabinet. The instrument and components are in a BSL-2 Enhanced laboratory and in addition, an aerosol management unit is provided for biohazardous sorts. The Aria II is a three-laser instrument (blue 488 nm, red 633 nm, and violet 405 nm) capable of measuring 9 fluorescence emissions. A single cell sort deposition unit is also The computer interface is an HP system running FACS DIVA software version 8.0.1 (*LSU FlowCore). 
  • Mac Pro workstation
    • (iOS/Win7) with 46 GB
  • Win 10 workstation
    • Equipped with 8 core Xeon processor (3.7GHz Skylake), 256GB DDR4 SDRAM, and NVIDIA Quadro P5000 16 GB video card for confocal and light sheet data rendering and analysis.
  • Visiopharm,
    • Visiopharm A/S: 1x open access; semi-quantitative analysis of 2D and whole slide image data using simple thresholding and machine learning algorithms.
  • Imaris
    • Oxford Instruments; software tool to analyze 2,3, and 4D datasets from Core microscopes with special emphasis on confocal and light sheet data. Includes filament tracer, colocalization, measurement pro, and stitching modules.
  • FlowJo
    • FlowJo, LLC; software for interpretation and analysis of flow cytometry data from Core’s analytical platforms.
  • syGlass
    • io; 3 and 4D data visualization software for virtual reality (VR). The software also provides tools for segmentation, quantification, and data export from confocal and light sheet datasets.
  • Zeiss Zen Blue
    • Carl Zeiss AG; acquisition and analysis suite for 2D and whole slide scan data
  • AutoQuant X3
    • Media Cybernetics; deconvolution software for 2,3, and 4D fluorescent
  • FIJI
    • Distribution of ImageJ including many plugins useful for the analysis of image data as well as the production of publication-quality panels.
  • CellProfiler
    • Open-source software that allows users to design analysis pipelines to identify and measure biological objects and features in data collected on Core microscopes.
  • QuPath
    • Open-source software for the analysis of digital images and whole slide datasets.