Today is Friday, Feb. 21, 2020

Department of Environmental Health

Center for Environmental Genetics

 

Confocal Imaging

Shared Equipment and Services: The CEG owns and manages equipment including flow cytometry, confocal microscopy, versatile imaging systems‎, and single cell sequencing. Access to equipment and services is free of charge for CEG members. Access requires pre-schedule and sign-up (registration link under development). See the appropriate contact persons listed below for special requests/questions regarding each service or type of equipment. (The flow cytometer and confocal microscope are no longer eligible for ITS subsidy unless their usage involves new technology development. Please contact ITS Core director Ying Xia, Ph.D., if you have any questions about this change in ITS matching funds policy).

Confocal Imaging:  The Confocal Imaging service is equipped with “Carl Zeiss Confocal Laser Scanning Microscopy” LSM700.

Confocal Laser Scanning Microscopy combines high-resolution and high-contrast optical imaging with depth selectivity allowing visual sectioning of tiny structures and constructing three-dimensional (3D) structures from the obtained images. It is broadly used to resolve the detailed structure of specific objects within the cell; it also enables the creation of sharp images of the exact plan of focus without any disturbing fluorescent light from the background or other region of specimen. Stacking several images from different optical plans allows visualization and analyses of 3D structures. However, due to limited depth of sample penetration, the thickness of specimen is an important consideration for experimental success.  Use of confocal microscopy in the biomedical sciences includes the imaging of multiple labeled specimens and the measurement of physiological events in living cells. Visualization of sufficient variants within both cell and tissue context will allow spatial and dynamic analyses of cell biology and may lead to advancement in understanding of biological processes.

Brochure:https://www.zeiss.com/microscopy/int/solutions/reference/all-tutorials/spectral-imaging/lsm700-light-pathways.html

Contact: Chia-I Ko, Ph.D. (koci@ucmail.uc.edu)

Location: Kettering laboratory complex, Room 443 (4th floor, Kettering Addition).

Instruction: Microscope use is free of charge only for CEG members. The sign-up/reservation form is mandatory for each use (link under development; in the interim, please contact Dr. Ko). Individuals who have received the microscope training are allowed and will be approved to schedule a reservation. New users should contact Dr. Ko to schedule an initial training before planning for experiments. Other services such as sample preparation instructions and troubleshooting for image-taking can be provided upon request; however, we do not guarantee for expected results.  In case system failure is detected due to carelessness and mishandling, users will be charged for repair costs.

Sign-up/reservation form (link under development)

Odyssey imaging Systems: The Odyssey CLx is the next generation multifunctional imaging platform that can provide a wide range of applications. The Odyssey CLx platform uses infrared laser excitation that out-performs LED and visible white light systems for Western blots. This increased sensitivity offers a clear image of your data that is unmatched by other digital imaging systems. Two separate lasers and detectors simultaneously detect both fluorescent signals. The optical system employs diode lasers and solid-state detectors with long lifetimes and very low maintenance requirements. Infrared laser excitation outperforms systems that use white light, LED light sources, and filter wheels by delivering higher intensity excitation light to the fluorophore. A variety of fluorescent dyes and stains are compatible with the 700 nm and 800 nm excitation wavelengths of the two diode lasers in the Odyssey CLx. Spectral overlap is minimized by a 100 nm separation of the two detection channels, and optical filtering ensures that each detector measures fluorescence from only one of the infrared dyes

Major advantages (https://www.licor.com/bio/odyssey-clx/):

See your bands reliably with stable fluorescent signals.  Fluorescent signals are stable and unaffected by timing, so you can compare band intensities with confidence. You can even conveniently re-image the same blot on the Odyssey CLx later and see the same results.

See multiple targets on the same blot. With multiplex fluorescence, you can detect two protein targets in each sample lane, with great sensitivity in both fluorescence channels.

See your strong bands accurately and immediately. Get deeper data capacity with digital fluorescence and the unprecedented linear dynamic range of the Odyssey CLx, and never lose data because of image saturation.

Get better sensitivity with low image background. Powerful, precise laser excitation and specialized optics enable the Odyssey CLx to provide high signal-to-noise ratios and outstanding image quality.

See the full story in a single image. See both strong and faint bands clearly in a single image acquired on the Odyssey CLx, with great sensitivity and no image saturation. Multiplex with two fluorescent colors to see even more data.

Do more with digital fluorescence. Get consistent, accurate digital images, without the hassles and unpredictability of film – so you can analyze your data right away and plan your next experiment.

Stable NIR fluorescence and detection of multiple targets in the same lane. Get the most accurate results with over 6 logs of linear dynamic range, so you can make more discoveries.

In-cell western capability present. However, user will have to develop protocol and initiate use of this facility.

Training: Training will mainly involve basic instrument and software manipulation to allow new users to start taking western-blot images independently. At least 10 min instrument demonstration and first time guided hands-on software running are required prior to independent usage. For Odyssey-CLx knowledge, experimental design, data interpretation, and result presentation, users should refer to tutorials available on request. Also refer to https://www.licor.com/bio/webinars/ , https://www.licor.com/bio/odyssey-clx/resources  before  starting experiment.

Scheduling: After being trained and able to perform assays independently, users can contact our staff for scheduling. Usage slots of more than one hour must be justified.  Users are also required to sign on the usage page and provide project information for CEG.

Sample format: Western blot membranes and reagents should be provided by users. First time users of this NIR western blotting technique can request use of secondary antibodies and blocking solutions from Dr. Tarapore.

Fees: Available to CEG members without charge

Data Storage: A project drive, called “CEG_Users”, has been set up for temporary storage. If the computer space is limited, the stored data will be removed after 6-month from initial acquisition.

Data Analysis: Data can be analyzed with the software Image Studio provided by the manufacturer. See Dr. Pheruza Tarapore for details (tarapopp@ucmail.uc.edu)