Cellular Imaging
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 and provides assisted use Services (hourly fee) on all instruments as well as a dedicated Imaris image processing workstation. Training and unassisted use (at a discounted rate) are available for the LSM880 confocal microscope and Imaris workstation. Confocal time lapse imaging is available after hours and on weekends for independent users at a discounted hourly rate. 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. Please contact Collene Jeter (cjeter@mdanderson.org) prior to scheduling initial services.
Services and Applications
Confocal Imaging
The Zeiss LSM880 laser scanning confocal microscope permits high resolution, multispectral imaging of cells and tissue sections. Applications include: multispectral imaging, z-stacking and 3D confocal imaging, super resolution imaging, laser microirradiations, live-cell imaging, protein-protein interaction (e.g., FRET, PLA, BiFC) and cell tracking analyses. A 96-well plate adapter provides for high through-put imaging on either the confocal or wide-field systems and both systems are equipped with incubation and CO2 control for live cell imaging. Cell Imaging Services include custom immunofluorescence staining on cultured cells, cryo and FFPE samples. Additional Cell Imaging optical microscopy services include wide field imaging on a Zeiss Axio-Observer epifluorescence microscope and image processing using Bitplane’s Imaris software enabling 3D and 4D image reconstructions and advanced qualitative and quantitative image analysis. For more detailed information, please see Instrumentation.
Multiphoton Imaging
The Leica TCS SP8 DIVE multiphoton (MP) microscope uses infrared excitation light, which provides for deeper penetration into thicker tissues and 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. Z-stacks combined with tile scans can be volumetrically rendered to generate 3D reconstructions and for morphometric analysis.
To support multiphoton imaging, the FCCIC also maintains a Leica VT1200S automated vibratome to cut thick tissues and a Leica M165FC fluorescence stereomicroscope for microdissections and tissue preparation. The FCCIC provides vibratome sectioning and tissue preparative services as well as custom thick tissue immunofluorescence staining services. For more detailed information, please see Instrumentation
Other Services and Microscope Applications
- Multispectral Fluorescence Imaging
- Immunofluorescence Staining
- Imaris (Bitplane) Image Processing
- Protein Distribution & Colocalization Studies
- 3D Reconstruction & Tissue Morphometric Analysis
- UVA Laser Microirradiation DNA Damage-Repair
- FRET and FRAP
- Live Cell Imaging
- Vibratome Sectioning
- Consultation and Training
Training
Cell Imaging recommends first time users contact Collene Jeter (cjeter@mdanderson.org) 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.
The Core recommends that users schedule appointments at least 1 week prior to the date of service. This is particularly crucial for users requiring training and/or staff assistance.
Instrument training is mandatory for any user of the Cell Imaging Core who would perform unassisted confocal imaging. Training is provided by Collene Jeter (cjeter@mdanderson.org) and will encompass blocks of time over multiple days. Upon completing training, a certificate of completion will be awarded.
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), or they can perform analysis for a specific project, as time permits, for an additional fee.
Please note that each user is responsible for archiving his or her own data. Core personnel can assist users in learning this practice.
Instrumentation
Zeiss LSM880 with Airyscan
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.
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 (Courtesy of the Bratton Laboratory).
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 (Courtesy of the Richie Laboratory).
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).
Leica TCS SP8 DIVE Multiphoton Microscope
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 16X mixed immersion (e.g. Clarity) vey long working distance objective (NA 0.8, w.d. 8.1 mm), 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
Leica TCS SP8 DIVE Multiphoton Images
Two-photon image of brain tissue stained for GFAP (green), beta-3 tubulin (red) to visualize astrocytes and neurons, respectively, and DAPI stained nuclei (blue).
Two-photon image of intestinal crypts. Lgr5-GFP (green) marks the crypt stem cells, Ki67 (red) marks proliferating cells and Ecad (grey) marks epithelial cells. (Courtesy of the T. Chen Laboratory)
Two photon image of cardiac vasculature. Imaris 9.1 (Bitplane) software was used to render 3D volumes and visualize vasculature (green, PECAM1) and cardiac tissue (red, phalloidin) channels, repsectively. (Courtesy of the Dent Laboratory).
Zeiss Inverted Axio Observer
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.
Leica VT1200S Vibratome
The core also maintains a Leica VT1200S automated vibratome to cut thick tissues and a microdissection fluorescence microscope for tissue preparation.
Imaris Image Processing Workstation
Bitplane's Imaris C1 Package image processing software enables advanced qualitative and quantitative image analysis of confocal and multiphoton microscope images. Functionality includes analysis of 2D, 3D and 4D cell images including 3D reconstructions, spot/vesicle/organelle quantitation, morphometric analysis, cell tracking, colocalization analysis, quantitation and statistical analyses. Modules include: Imaris Core, MeasurementPro, Vantage, Track, Coloc, Cell and XT.
Links and Resources
General Information
Before finalizing your experimental plan, please consult with Collene Jeter, PhD (cjeter@mdanderson.org), who is also available 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.
