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CVM Imaging Core

The CVM Imaging Core provides instrumentation and assistance for multi-parameter live cell microscopy experiments and cell/tissue section image acquisition. It is open for use by all NCSU/CVM researchers, and operates as a by appointment, fee for service facility. Although the facility is intended primarily for local users who need live cell imaging services (i.e., those who cannot run all over town and beyond with their delicate live cells), a variety of fixed cell/tissue section imaging services are also available to all NCSU researchers. For researchers who are more infrequent, less proficient users of microscopy techniques, the facility provides a completely assisted technical support service; this is an attractive option for researchers who need timely results for publications and funding applications but do not have the time to dedicate to learning the intricacies of imaging. The facility also offers consultation services for imaging experimental design, sample preparation, labeling of cells/tissues with fluorescent proteins, dyes and antibodies, and troubleshooting. Like other core services, user fees are charged to offset the cost of service contracts, maintenance, consumables and facility staff, all of which are needed to keep the equipment and the facility in good working order.

CVM Imaging Core images

Services Available

The CVM Imaging Core offers the following services:

  • Staff assisted image acquisition and processing
  • Image data transfer
  • Experimental planning consultation
  • Troubleshooting

Applications

Cells are dynamic entities. They’re not just sitting there on the culture vessel while you observe them during routine cell culture! What are your cells up to when you’re not watching? Ever wondered how fast they’re dividing? How they move around as they grow and divide? How their organelles move? How their protein expression or phenotype changes in response to experimental treatments over time?

These and many other scientific questions can be answered using live cell microscopy. Instead of looking at single snapshots of cells, a live cell microscopy system can record images at predetermined intervals over an extended period of time as the cells are maintained under optimal environmental conditions of temperature, humidity, and CO2 control. The resulting images are then combined to produce videos that can reveal many secrets of cell division, migration, growth, and movement as well as organelle behavior, protein expression, and changes in cell phenotype in response to applied stimuli. Contrast imaging modes, such has differential interference contrast (DIC) can be used in conjunction with fluorescence imaging to yield additional structural data. The CVM Imaging Core facility has resources available to run many types of live cell imaging experiments, but the system can also be used for fixed cell and tissue imaging applications.

Listed below are some exciting examples:

Live cell examples:

  • Cell division dynamics- rate of division, perturbations to mitosis
  • Cell migration dynamics- how fast cells move in response to stimuli, e.g., wounding/scratch assays
  • Chemotaxis- movement of cells in response to chemical stimuli
  • Intracellular dynamics- fluorescent protein targeting/sorting, organelle dynamics
  • Total Internal Reflection Fluorescence (TIRF)- studying events at high resolution in close proximity to the cell membrane (approximately 100-300 nm in from the coverglass) such as vesicle movement, exocytosis, endocytosis, cell adhesion dynamics, cell migration, receptor dynamics, etc.
  • Translocation of proteins between the cytoplasm and the nucleus or other organelles
  • Changes in protein expression and localization dynamics in response to treatments- pharmacological agents, biological/biochemical factors, hypoxia, etc.

Fixed cell examples:

  • Multiple color fluorescent imaging of antibody-labeled specimens
  • Analysis of fluorescent fusion protein expression (GFP, RFP, etc.)
  • Color camera imaging of samples treated with histological stains and colorimetric immunohistochemical labeling (IHC)
  • Z-sectioning, deconvolution and 3D reconstruction of any labeled cell or group of cells/tissue sections
  • Image stitching for groups of cells or whole tissue sections

Instrumentation

The CVM Imaging Core Facility houses a Leica AF7000 inverted microscope with a motorized stage, a stage-mounted incubation chamber capable of maintaining stable temperature, 3 heated sample holders; one for 60 mm and 35 mm coverglass-bottom dishes, one for coverglass-bottom chamber slides, and one for 12 well glass-bottom plates. Covers for these heated sample holders supply humidified gases to create mini on-stage incubators for maintaining stable CO2 and O2 concentrations for animal cell culture. A perfusion system is also available to bathe cells with different media or to add treatments to the cell medium. The microscope is equipped with optics for widefield epi-fluorescence, differential interference contrast (DIC), polarized light, and brightfield imaging. The system is also equipped with a 4 diode laser total internal reflection fluorescence (TIRF) system. Objectives range from 5x to 100x oil immersion. Images can be recorded with a high resolution Andor Clara CCD camera, a Photometrics Evolve 512 EMCCD camera for detecting low light levels, or a Leica color camera for colorimetric IHC or pathology imaging. The motorized stage and Leica microscope automation software allow multi-position, time lapse image collection in x, y and z dimensions to enable live cell video time course experiments, deconvolution and 3D reconstruction modules, and image stitching. Images can be taken of multiple samples (e.g., in separate treatment wells) in a single time course. Time between exposures can be user-defined from milliseconds to hours.

Available Fluorescence Filter Sets

Filter set Excitation Dichroic Emission
Triple cube VBG-T/ET
No : 11523031
BP 490/20

BP 422/44

BP 552/24

435

505

550

BP 465/45

BP 545/55

BP 610/65

GFP-T/ET
No : 11523032
BP 490/20 500 BP 525/50
CFP/YFP+AR+/ET
No : 11523025
BP 490/20

BP 405/12

505

435

BP 545/55

BP 465/45

DsRed-T/ET "RFP-T"
No : 11523028
BP 552/24 570 BP 605/65
DAPI, HP, size:"K"
No : 11600232
BP 360/40 400 BP 470/40

We have 4 diode lasers on the TIRF system:

405 nm
488 nm
561 nm
635 nm

Software

The microscope is equipped with Leica LAS AF software that includes 2D and 3D deconvolution and 3D rendering/rotation software modules. Experiments are stored in the Leica proprietary .lif file format, and single images/movies can be exported as TIFFs, JPEG, MPEG-4, etc. The freeware Leica LAS AF lite is also available for all users to export whole data sets from the microscope computer as .lif files and to view them in the Leica software for cataloging and further analysis.

Rates

  • Initial consultation for study feasibility: first 2 hours at no charge
  • Basic experiment setup and use of the system: $55/hour. Minimum of 1 hour; additional use is charged in 15 minute increments.
  • Time lapse experiments: $55 per hour for experiment set up; for longer-term time lapse experiments (e.g., overnight), reduced rates will be available, TBD
  • Additional consultation/troubleshooting, image analysis services: $55/hour

It is up to the individual to establish an account with the facility when becoming a new user. It is also the responsibility of the user to update any funding information.

Policies

  • New users are requested to make an appointment with the facility manager, Steven Nagar, to discuss the proposed experiment to ensure the facility has the capabilities to perform the desired imaging experiment.
  • Users must schedule imaging appointments directly with the facility manager via email as far in advance as possible.
  • Users will be given access to an online Google calendar system where they can monitor system use and schedule their appointments.
  • All appointments require 24 hour notice for cancellations. If you do not notify the facility manager in writing, you may be charged.
  • Bills are broken down by specific user, per date, and are sent out monthly.
  • Users of the facility will automatically be added to a CVM Imaging Core listserv that will keep users up to date with the state of the facility.
  • Image data will be stored locally on the microscope computer, and backed up on an external hard drive. We strongly suggest you remove important data in a timely manner. Notice will be sent out before any data is permanently removed from the microscope computer.

Dr. Steven Nagar has over 25 years of experience with a wide variety of light microscopy techniques, and over the years has consulted with numerous members of the NCSU community, from students to faculty, including researchers at the College of Agricultural and Life Sciences (CALS), the College of Natural Resources (CNR), and the College of Veterinary Medicine (CVM), to design and troubleshoot immunolocalization, in situ hybridization, fluorescent fusion protein-transfected live-cell, and other cell biology imaging experiments. Steve has worked with a wide variety of samples, from plant cells and tissues to mammalian cells and tissues. Drawing from his experience and his passion for all things microscopy and for helping others, Steve is dedicated to sharing his knowledge with other researchers who need assistance with microscopy experiments.

Dr. Steven Nagar

steve_nagar@ncsu.edu

Microscope facility: Research Building Room 327

Office: Research Building Room 382