Optical Imaging Program

The NeuroDiscovery Optical Imaging Program provides access to advanced confocal, multi-photon and wide field microscopy. The facility specializes in various forms of fluorescence, brightfield, DIC (Differential Interference Contrast) and live cell imaging. We offer users comprehensive training (data acquisition and image analysis), assistance in the design of experimental protocols and ongoing consultation and support.

Questions regarding cell and tissue fixation techniques, fluorophore selection and immunostaining principles are always welcome, as are requests for instruction in live cell and tissue imaging. We offer flexible and customized training and support to both established and new users throughout the NeuroDiscovery community.

Program Resources

Instrumentation

Two Zeiss LSM 510 META upright confocal microscopes are the centerpieces of our facility. Each microscope has three visible wavelength lasers and META spectral emission detectors which can precisely and effectively separate strongly overlapping fluorescence emission spectra. One system has UV capability and the other has two-photon capability.

The UV system features the Coherent Innova® Enterprise™ II ion laser system which offers powerful 80mW multi-color performance in the true UV range at 351nm and 364 nm.

The Two-Photon system features the Coherent Chameleon™ Multi-photon Excitation (MPE) laser tunable from 720nm – 930nm with 4 NLOs (External detectors). Designed specifically for MPE microscopy, the Chameleon™ provides widely tunable femtosecond pulses with more than 1W of average power. The Chameleon can excite all the fluorophores commonly used in MPE. The longer wavelengths of the Chameleon reduce cell photo bleaching, and allow biologists to image deeper into tissue due to less light scatter, with less tissue damage.

The Olympus spinning disk confocal microscope is a motorized inverted scope that offers modest live cell imaging capacity. Multiple stage adapters allow for slide dishes and 96 well plates. Additional features include a motorized xy stage and a color camera for histology sections. Widefield, DIC (differential interference contrast) and confocal imaging are all possible.

The spinning disk is equipped with a Hamamatsu EMCCD Camera. From the manufacturer, with on-chip multiplication technology of the EM-CCD sensor the signal is drastically boosted while readout noise value is kept below 1 electron r.m.s. at high gain mode. The gain factor of the C9100-02 is up to 2000 and this camera is able to grab 30 frames per second keeping 14 bit dynamic range and full resolution of 1000 x 1000 pixel. The C9100-02 supports sub-array and binning modes, which enable high frame rate, 100 frames per second or higher. The C9100-02 is recommended for all applications requiring high gain, speed, good resolution, high dynamics and signal to noise ratio.

Peripheral Equipment

• Motorized XY stage for tiling large tissue sections.
• VWR tissue culture incubator (37°C w/ 5% CO₂) for short-term storage.

Workstations

• Dell Precision 380 3.73 GHz processor, 3.5 GB RAM, and 512 MB Nvidia QuadroFX 4500
• Dell Precision 380 3.00 GHz processor, 3.5 GB RAM, and 512 MB Nvidia QuadroFX 4400
• Fujutsu/Seimens Celsius 600 Xenon 2.4GB processor, 3.2 GB RAM and 128 MB Quadro 4 980XGL

Image Analysis

Imaris Bitplane - this software platform allows investigators to work with 3-D multi-channel images. It can load, process and visualize a wide array of image formats and can display results with an advanced movie generator and image export feature. The software we have includes Imaris Suite 5.5, FilamentTracer 5.5 and AutoAligner 2.0.'

Metamorph Imaging System - once a set of images has been acquired, researchers can use MetaMorph's extensive analytical functions to measure areas and distances, count cells, graph intensity levels over time, deconvolve images, or color combine multi-fluorescence images.

Zeiss LSM Software package - software for creating overlays, projections and for quantification of intensities. Images may be exported to Adobe PhotoShop, Scion Image and other programs accepting TIFF files.

3-D for LSM - high-performance automated measuring functions for the quantitative analysis of 3D image data. Surface and alpha rendering are convenient 3D visualization tools which allow you to change the data of an image sequence into an overall 3D image that can be viewed from any required angle.

IP Lab - IPLab is installed on the DSU scope and acquires, processes, and quantifies your data, captures real time, time lapse, and 3D pictures of live and fixed samples, and controls the camera and microscope hardware. The data collected can saved and opened in a spreadsheet program such as Excel for further analysis.

Viewing Images

Zeiss LSM Files - LSM Image Browser is Windows program that will allow you to view, manipulate and export your Zeiss LSM Files. (Download image browser zip file).  For Mac users, FocalPoint Image Viewer is a thumbnail viewer that supports specialised formats used by various microscope manufacturers (lsm, oif, oib, ipl).

Universal Imaging Program - ImageJ is a free NIH imaging program designed to run on both Macs, PCs and linux. The ImageJ LSM plugin allows you to open LSM images and the IPLab reader plugin allows you to open IPLab images acquired from the Olympus spinning disk microscope. To install plugins copy them to the Program Files\ImageJ\plugins\Input-Output folder.

• ImageJ
• MBF_ImageJ
• ImageJ LSM plugin
• IPLab Reader plugin

Objectives:

Zeiss Objectives

Olympus Objectives  

NOTE: Additional instruments are always under consideration and investigators are encouraged to make recommendations regarding specific equipment that may be useful to the research community. Please contact the This e-mail address is being protected from spam bots, you need JavaScript enabled to view it with any questions or suggestions.

Access

Although the facility primarily supports NeuroDiscovery investigators studying neurodegenerative disease, we welcome inquires from other users. Please contact This e-mail address is being protected from spam bots, you need JavaScript enabled to view it , the optical imaging manager, or This e-mail address is being protected from spam bots, you need JavaScript enabled to view it , the optical imaging technician to discuss your imaging needs.

All users must complete training before access to the facility is granted. For training, you must visit our Members' Directory for NeuroDiscovery membership details, return the completed user form providing a brief research summary of your project, and familiarize yourself with our facility policies, including user fees . After returning the User Form you will be contacted to schedule a training session.

The facility is staffed from Monday-Friday, 8:00 am-5:00 pm. Experienced users are provided access out of the regular hours with consent from the Facility Manager. Only those users with sufficient training on the microscopes can gain access to the facility during off-peak hours.