Department of Zoology, University of Oklahoma

Neuropeptide producing nerve cells in a fruitfly nervous system (confocal Z-series projection). OU Confocal Microscope Facility

This page summarizes a few of the features and procedures for usage of this facility. For further information on access, training, and usage, please contact Randy Hewes at hewes<<at>>ou.edu.

View the OU confocal usage schedule online.

Overview

Confocal microscopy obtains high resolution, multi-color images from fluorescent samples by generating and digitally sampling fluorescence within a single focal plane while rejecting light coming from above and below the focal plane. The confocal software allows the user to obtain images in multiple focal planes and project the images in 2 or 3 dimensions, in stereo, or as movies. It also provides tools for time-lapse imaging, photobleaching and/or sampling from multiple regions of interest (ROIs), ratiometric imaging of calcium or other elements, fluorescence resonance energy transfer (FRET) experiments, and fluorescence recovery after photobleaching (FRAP) experiments.

The confocal microscope is housed at the Stephenson Research and Technology Center. This is a multiuser core facility for researchers on the Norman campus. It is also available to other Oklahoma researchers on a case-by-case basis. The hourly rates are:

Rate Type	Description	Hourly rate for internal grants and contracts
On-Line Usage* (grant supported research)	Use of entire confocal microscope	$30.00
On-Line Usage*	Use of entire confocal microscope	$43.00
Off-Line Usage	Use of computer and software only	$11.00
Training Rate**	Assisted use	$13.00
* Academic users. The rate for external, federal users is $64/hour. ** In addition to rates for on-line or off-line usage.

There is limited file storage space available on the confocal for use during scanning sessions. Users can save data files to the hard drive during sessions, but no space is available for users to leave files on the system between sessions. Files can be moved from the system by burning them to CD or by sending them over the network. High-capacity network server file space, with automated back-ups, is available to individual users through the Research Campus Computing Center (http://www.rccc.ou.edu/).

The microscope

The OU confocal is an Olympus FluoView 500. This is a fully automated, point-scanning, point-detection, confocal laser scanning microscope system with laser lines in the green (488 nm), red (543 nm), and far-red (633 nm) spectrum. The microscope permits simultaneous collection of up to four fluorescence detection channels and one transmitted light detection channel.

The confocal is configured on an Olympus BX61 motorized research microscope platform. In addition to fluorescence, this microscope is configured for brightfield, phase contrast, or differential interference contrast (DIC) detection. It is equipped with a wide range of high numerical aperture air, oil-immersion, water, and long-working distance water-immersion objectives.

Further technical details of this system are listed below. You can also obtain more technical information (and general tutorials) at www.olympusfluoview.com and at www.olympusmicro.com. For assistance in choosing fluorophores, see www.olympusconfocal.com/java/dualprobes/index.html.

Resolution:
The maximum pixel array size of 2048 x 2048, with each channel digitized to 4096 gray levels (12-bit resolution).

Supported fluorophores:
• FITC (fluorescein), GFP, EGFP, Alexa 488 and other “green” fluorophores – excite with Blue Argon (488 nm) laser
• Rhodamine, TRITC, Texas Red, Cy3, Alexa 568 and other “red” fluorophores – excite with Green Helium Neon (543 nm) laser
• Cy5, Cy7 and other “far-red” fluorophores – excite with Red Helium Neon (633 nm) laser
• An attached cooled CCD camera permits UV excitation (100 Watt) in the blue light spectrum for dyes such as DAPI and Hoechst. The camera is mounted on the confocal microscope and is operated using a second computer with dedicated software, and the resulting conventional fluorescence images can be overlaid with the confocal images.

Scanning modes:
• XY scanning – a single horizontal plane through the sample – this plane can be rotated 360 degrees, and can be sampled as a plane or as a single line
• XYZ scanning – a series of horizontal planes through the vertical thickness of the sample (an XYZ series) – this permits 2D or 3D projections of thick samples
• XZ scanning – a single vertical plane through the sample – this plane can be rotated 360 degrees, and can be sampled as a plane or as a single line
• XYT scanning – a single horizontal plane imaged over time – this permits observation and analysis of live cell properties, such as movements of GFP tagged proteins and changes in intracellular calcium – even faster temporal resolution is obtained with line (XT) or point scanning
• XYZT scanning – an XYZ series sampled over time – this permits observation and analysis of live cell properties in 3D

Sequential Scanning:
Images can be acquired in fully automated line-by-line or frame-by-frame sequential scanning modes to minimize cross-talk among channels in multi-color images.

Objectives
• Air 10X-40X
• Oil-immersion 20X-60X
• Water-immersion 10X-60X
• Long-distance (deep) water-immersion 10X-60X
• Phase-contrast 20X-40X

Scheduling and Contact Information

Randall S. Hewes, Ph.D.
hewes<<at>>ou.edu

Facilities:

Introduction


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