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In Laser Scanning Confocal Microscopy, a laser beam is scanned across the specimen and the emitted fluorescent light passes through a pinhole aperture, so that the fluorescence from points on the specimen that are not within the focal plane will be obstructed by the pinhole. This nearly eliminates out-of-focus information, improving resolution and background discrimination. This 'optical sectioning' is especially useful when viewing thick specimens. A series of sections can be obtained automatically and a 2-D or 3-D reconstruction created. Signals from 3 different fluorescent dyes or 2 dyes and a transmitted light image can be obtained simultaneously and the images merged to determine co-localization. In Multiphoton Microscopy, a more powerful laser is used to provide simultaneous excitation with 2 or three photons of low energy, exciting the fluorophore to the same level as one high energy photon. The use of lower energy (red wavelengths) reduces damage to the specimen, which is especially important in living tissue. These wavelengths also penetrate deeper into the tissue. Multiphoton excitation occurs only at the focal plane and tissue above and below is not affected so there is no need for pinhole apertures. In confocal a larger volume if tissue is excited but only light from the focal plane is collected. Our confocal system is a Bio-Rad MRC-1024, equipped with an argon-krypton laser for excitation at 488, 568 and 647 nm, three fluorescence emission detectors, a transmitted light detector, and image analysis software. It is attached to an Olympus IX70 inverted microscope, with 10x, 40x, and 100x high numeric aperture objectives and DIC optics. The multiphoton system uses a Ti:Sapphire mode-locked laser which provides 100 fs pulses at 80 MHz and is tunable to wavelengths from 685 nm to 1050 nm for excitation of UV and visible absorbing fluorophores. It is aligned with the confocal scanning system and one can switch easily between them. Applications Confocal and multiphoton microscopy can be used to observe any fluorescent specimen, either biological or non-biological, for qualitative observation or quantitative analysis. Since fluorescence utilizes reflected light, opaque samples may be imaged. The inverted microscope allows easy access to the specimen for physiological experiments.

The confocal microscope is located in D21 Clark Hall and the computer workstation is in D22. For More Information contact:
Carol Bayles
Tel: 607-254-4860
email: mifbiotech@cornell.edu

View or download user guides (in pdf)
Intro to NBTC Fluorescence Microscopy
NBTC Confocal Facility
Confocal with LaserSharp
Multiphoton with LaserSharp
Microfabrication with MP
Olympus IX70

Trained and authorized users can schedule time on the web.

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This material is based upon work supported in part by the STC Program of the National Science Foundation under Agreement No. ECS-9876771. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.

Any opininons, findings, conclusions or recommendations expressed are those of the author(s) and do not necessarily reflect the views of the New York State Office of Science, Technology and Academic Research.

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