Core D - Cell & Tissue Imaging & Histology (CTIH)
|MDR 512||Inverted microscope|
|MDR 627||Confocal microscope|
|CII||Two-Photon confocal microscope|
|Core Directors:||Richard Benton, Ph.D.|
|Jay Hoying, Ph.D.|
|Personnel:||Jason Beare, M.S.|
Currently, the capabilities available through the Microscopy Core include:
- In vitro and in vivo brightfield image capture and analysis
- In vitro and in vivo fluoresence image capture and analysis
- Laser confocal fluoresence image capture and analysis
- Motorized stage montaging of brightfield and fluorescence in vitro and in vivo preparations
Capability/value added by the expansion of the Cell and Tissue Imaging Core include:
- In vitro ratio-metric calcium measurements
- In vitro visualization of reactive oxygen species including hydrogen peroxide and superoxide
- In vitro measurement of mitochondrial potential
- Time lapse microscopy studies of process extension/cell movement
A primary reason for the tremendous success of Core D over the last 10 years has been its emphasis on integration with other core laboratories and close interaction with COBRE/KSCIRC PIs to continually develop needed methodologies and procedures. This commitment and emphasis will remain as the new Cell and Tissue Imaging Core is further developed to include new areas of interest recently developed and required by Users. Following are the major areas that will be emphasized in the proposed expansion of capacity/capabilities:
- greater access to image analysis by increasing number of stations
- offering basic histology consult to associated PIs with limited expertise
- assistance with appropriate image optimization through non-biasing image correction
- implementation of cutting-edge capacity for visualization of live cell biology
- development and optimization of new and innovative imaging capacity
These areas of emphasis will provide for the enhanced institutional impact and productivity that will continue to a major goal of the new Cell and Tissue Imaging Core facility.
Complementary integration of these capabilities with existing University resources.
The proposed expanded Cell and Tissue Imaging Core facility will complement existing capabilities within the University. The Core Analytical Microscopy Laboratory (CAML) housed in the Department of Anatomical Sciences & Neurobiology, provides state-of-the-art electron microscopic support. In addition, two-photon microscopy is available within the Cardiovascular Innovation Institute (CII) of the Department of Surgery at the University of Louisville. All COBRE/KSCIRC and associated PIs currently have access to these facilities. The COBRE/KSCIRC PIs determined they would not at present use these UofL facilities enough to justify direct financial support by this application. They will be accessed either by fee-for-service or presently active instrument sharing arrangements. Thus, the expanded Core D facility will provide unique capabilities to the University community, with minimal replication of resources.
As with other COBRE/KSCIRC Cores, training of personnel for the appropriate and efficient use of imaging equipment is central to its effective operation.
- Training: The operating procedures for the Core include mandatory training of every prospective Core User, including PIs whose trainees/staff are the primary Users. The training is performed by Jason Beare (email Jason) -confocal scope, inverted scope, stereology system, or by Jing-Juan Zheng (email Jing-Juan Zheng) -live imaging system. The training will include basic rules of equipment use, application-specific topics, and practical training on the actual equipment.
- Systems: Each authorized User will obtain an individual User account on the requested equipment.
- Scheduling: There is an online signup system. Sign up blocks are 2 hrs (7-9, 9-11, 11-1, 1-3, and 3-5). Users cannot sign up for more than 2 blocks/day with a maximum of 4 blocks/week. Increased usage beyond that will need to be cleared with Jason or Core D Director Dr. Richard Benton. Additional blocks can be reserved on the day of usage, if available. Longer/after hours usage can be discussed with the Core staff. The equipment use will be free to all Core/KSCIRC PIs. Every week the equipment will be inspected by the responsible staff for the proper performance.
Before Users are given access to the facility, their capacity for independent use of Core equipment will be evaluated and assured by the Core personnel. Core D access doors are always locked and access is by fob keys which identify who have entered the facility. In case of equipment damage due to improper use, the PI may be charged for the necessary repairs. If necessary, IBC approval for use of biohazardous materials will be required and applicable standard operating procedures followed.
Users will schedule training sessions by contacting Jason Beare or Jenny Zheng. In addition, routine training/refreshers by the representatives of manufacturers of Core equipment and software will be arranged by the Core personnel and if they deem it appropriate, mandate all Users’ attendance. This will provide current updates and information to promote the most efficient and advanced use of all imaging equipment capabilities.
This is an area of priority for Core D. To ensure the highest possibly quality, validity, and reproducibility of all data generated by this Core, users will be extensively trained, using standardized and optimized protocols whenever possible. As with other Cores, quality control for the tissue preparation, image acquisition, and image analysis will be ensured by the feedback that Users will receive about endpoint analyses in Core D as compared to the known “golden” standards we have developed over the past years of NCRR/COBRE support. The interpretation of the feedback is left up to the Users who can obtain assistance or advice from Core personnel, if necessary.
As with other cores, Core D personnel will on occasion assist new investigators who do not have personnel yet. The purpose of this allowance is to obtain preliminary data for grants. This will be approved by the Core Director in consultation with the Steering Committee after evaluation of the specific aims of the grant proposal to be submitted.
Fig. 1. This figure depicts examples of imaging innovation facilitated by Core D. In panel A, an intravital cocktail of TRITC-LEA (red) and Hypoxyprobe® (green) was used in combination to examine spinal perfusion status relative to tissue hypoxia following focal spinal ischemia. In panel B, example of the use of FITC-LEA intravascularly to determine microvascular perfusion status combined by traditional immunohistochemistry to show clotting potential (vWF, red), countered with a nuclear dye (Hoechst, blue).
Fig. 2. Shown is an example of an fast Fourier transformation of an image generated for a PI by Core D. This method has become a superior standard when dealing with problematic backgrounds due to specific transformation of noise with little signal loss or image biasing.