The FCCIC maintains three primary instruments: A Zeiss LSM880 laser scanning confocal microscope, a Leica TCS SP8 DIVE multiphoton microscope, and a Zeiss wide-field microscope. The Zeiss confocal microscope provides high resolution images, whereas the Leica multiphoton system provides superior imaging of thick specimens and sections. The core also maintains an Axio-Observer wide-field epifluorescence microscope appropriately configured for time-lapse imaging applications. Additional core resources include Bitplane’s Imaris image processing software enabling 3D and 4D image reconstructions and advanced qualitative and quantitative image analysis. Core personnel are experts in immunofluorescence staining and tissue preparative protocols, the analyses of cellular and subcellular distribution of proteins, tissue architecture, UV DNA damage experiments, live-cell imaging, 3D-reconstructions of z-stack images and quantitative and cell tracking analyses. Core staff can guide users to optimize specimen preparation, choice of instrumentation and imaging parameters and develop new applications for investigators. The core also helps users understand and apply image processing software (e.g. Bitplane’s Imaris software), and can train dedicated users to operate the microscopes independently.
Services and Applications
The FCCIC provides assisted use Services (hourly fee) on all instruments, including the LSM880 confocal microscope, TCS SP8 DIVE multiphoton microscope, and Imaris image processing station. Training and unassisted use (at a discounted rate) are also available on the LSM880 confocal microscope and Imaris workstation. Confocal time lapse imaging is available after hours and on weekends at a discounted hourly rate for independent users. Additional Services include: vibratome sectioning, immunofluorescence staining (standard and thick tissue), wide field imaging (assisted, unassisted and training) and Imaris image processing. Service Fees vary and are available via iLab along with our Service Request Form.
Cell Imaging recommends first time users contact Collene Jeter (email@example.com) to schedule a consultation before finalizing any experimental plans. This consultation will allow users and core staff to discuss experimental design, expectations of lab services, and cost of services for the scope of the project.
Consultations also help users select optimal fluorophores for particular applications, choosing the instrument best suited to experimental needs, and guidance for tissue fixation and permeabilization to optimize detection of desired cellular components.
- Multispectral Fluorescence Imaging
- Protein Distribution Analyses
- Colocalization Studies
- 3D Reconstruction & Tissue Architecture
- UV-induced DNA Damage Experiments
- FRET and FRAP
- Live Cell Imaging
- Cell and Tissue Immunofluorescence Staining
- Vibratome Sectioning
- Consultation and Training
Core staff give periodic seminars to keep users up-to-date on new and improved microscopy applications, instruments, and imaging software. They also help new and seasoned users understand the equipment and optimize experiments for the best imaging. Finally they can help users with image processing software (e.g. Bitplane's Imaris software), and can train dedicated users to operate the micrscopes independently.
The Zeiss LSM880 confocal microscope has five lasers for efficient excitation of fluorophores ranging from blue to far red, a 355 nm laser for DNA damage repair studies, an Airyscan detector allowing near super-resolution imaging, a FRET/FRAP module, and an incubator chamber for time-lapse imaging. Airyscan provides improved signal-to-noise ratio (SNR) relative to conventional GaAsP detectors and 1.7 x higher resolution in all spatial dimensions (up to 140 nm laterally and 400 nm axially). FAST Airyscan imaging modality provides a balance between high sensitivity, resolution and speed for the fastest and gentlest imaging of live cells and tissues. In combination with our UVA microirradiation laser investigators can quantify DNA damage and repair by imaging the dynamic interactions between molecules at sites of DNA damage. The photomanipulation (e.g., FRAP and photoactivation/conversion) module permits analysis of dynamic molecular processes and the FRET module permits interrogation of candidate protein-protein interactions. This scope combined with Bitplane's Imaris image processing software will allow for 3D reconstructions of z-stacked images, quantitative analyses, and cell tracking analyses.
The Leica TCS SP8 DIVE can be used to examine thick sections, intact organs, and in vivo processes with maximal imaging depth, minimal light scattering, increased z-resolution, and reduced phototoxicity. The SpectraPhysics Insight X3 with dual laser lines includes a tunable line that can be tuned to between 680 nm and 1300 nm and a 1040 nm fixed line that coupled with variable dichroic-based spectral detection provides spectral separation for simultaneous, multi-channel acquisition of virtually any fluorophore. Coupled with 4 independently tunable high QE non-descanned hybrid (HyD) detectors, this system offers spectral freedom and maximal efficiency in fluorescence detection. An additional detector in the transmitted light path is optimal for second (and third) harmonics. The Leica DIVE beam routing optics with Vario Beam Expander allow dynamic adjustment of the beam diameter and focus to optimize the excitation beam for colocalization of visible and IR wavelengths as well as to tune the excitation spot for maximum depth or maximum resolution depending on the needs of the experiment. The scan optics of the Leica SP8 DIVE MP system are optimized for transmission and chromatic correction from 400 nm to 1300 nm and include a 25X water immersion dipping objective (NA 0.95, w.d. 2.6 mm) and 40X water coverslip corrected objective (NA 1.1, w.d. 0.65 mm). To support multiphoton imaging, the core also maintains a Leica VT1200S automated vibratome to cut thick tissues and a Leica M165FC fluorescence stereomicroscope for microdissections and tissue preparation
Our Axio Observer wide-field epifluorescence microscope is equipped with DIC/Nomarksi optics and a full range of objectives (10X, 20X, 40X, 63X and 100X) to meet imaging needs. In addition, it has an incubator chamber, thus making it available for time-lapse imaging applications.
The core also maintains a Leica VT1200S automated vibratome to cut thick tissues and a microdissection fluorescence microscope for tissue preparation.
Bitplane's Imaris C1 Package image processing software provides tools to qualitatively and quantitatively process confocal images. Functionality includes analysis of 2D, 3D and 4D cell images, cell tracking, colocalization analysis, quantitation and statistical analyses. Modules include: Imaris Core, MeasurementPro, Vantage, Track, Coloc, Cell and XT.
Links and Resources
Before finalizing your experimental plan, please consult with Collene Jeter, PhD (firstname.lastname@example.org), who is also availble for instrument training and new protocol development.
Prescreen all slides for quality prior to adding precious sample and/or reagents.
Always include appropriate controls (e.g. normal antibody, isotype control, secondary antibody only, etc.)
Requests to schedule appointments requiring assistance with the instruments should be made at least 1 week in advance. The appointment will be scheduled the following week, as time is available.
Only trained and authorized users may use the imaging equipment.
Personal data devices such as hard drives and flash drives are not permitted. Please contact core staff for methods to transfer data.
Any and all issues with the equipment should be reported to core personnel promptly.
Zeiss LSM880 Confocal Images
Super-resolution image of a mouse fibroblast cell stained with LAMP1 (green) and LC8 (red) to visualize lysosomes and the dynein motor complex, respectively
Stitched mosaic subset of a 5x5 tile scan showing DNA damage foci - γH2AX (green) and 53BP1 (red) in nuclei (blue)
Multispectral analysis of thymocyte epithelial cell specification during development. Two transcription factors (red and green) specify cell fate
Tile mosaic of four fields of view showing regenerated prostate cells with basal cell (red) and luminal cell (green) characteristics
Stitched mosaic of 25 fields of view showing heterogeneity in the prostate. Basal cells (red), luminal cells (green), nuclei (blue)
Functional analysis of stem cell markers in colon crypts. Stem cells (green), differentiated Paneth cells (red), cell nuclei (blue).
Analysis of post-translational modification of tubulin in germ cell development. Acetylated tubulin (red), nuclei (blue)