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Journal of Biomedical Optics

Extended-working-distance multiphoton micromanipulation microscope for deep-penetration imaging in live mice and tissue
Author(s): Milan Makale; Michele McElroy; Peter O'Brien; Robert M. Hoffman; Sharon Guo; Michael Bouvet; Leo Barnes; Elizabeth Ingulli; David Cheresh
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Paper Abstract

We constructed a multiphoton (2-P) microscope with space to mount and operate microphysiology hardware, and still acquire high quality 2-P images of tumor cells deep within tissues of live mice. We reconfigured for nondescanned 2-P imaging, a dedicated electrophysiology microscope, the Nikon FN1. This microscope is compact, with retractable objectives, allowing more stage space. The instrument is fitted with long-working-distance objectives (2.5- to 3.5-mm WD) with a narrow bore, high NA, and efficient UV and IR light transmission. The system is driven by a powerful 3.5-W peak power pulsed Ti-sapphire laser with a broad tuning range. This 2-P system images a fluorescent standard to a depth of 750 to 800 μm, acquires images of murine pancreatic tumors in vivo, and also images fluorescently labeled T-cells inside live, externalized mouse lymph nodes. Effective imaging depths range between 100 and 500 μm. This compares favorably with the 100- to 300 μm micron depth attained by many 2-P systems, especially descanned 2-P instruments, and 40 μm-deep imaging with confocal microscopes. The greater depth penetration is attributable to the use of high-NA long-working-distance water-dipping lenses incorporated into a nondescanned instrument with carefully configured laser beam introduction and image-acquisition optics. Thus the new system not only has improved imaging capabilities, but allows micromanipulation and maintenance of tissues and organs.

Paper Details

Date Published: 1 March 2009
PDF: 10 pages
J. Biomed. Opt. 14(2) 024032 doi: 10.1117/1.3103783
Published in: Journal of Biomedical Optics Volume 14, Issue 2
Show Author Affiliations
Milan Makale, Univ. of California, San Diego (United States)
Michele McElroy
Peter O'Brien, New York Medical College (United States)
Robert M. Hoffman, AntiCancer, Inc. (United States)
Sharon Guo, Univ. of California, San Diego (United States)
Michael Bouvet, Univ. of California, San Diego (United States)
Leo Barnes, Univ. of California, San Diego (United States)
Elizabeth Ingulli, Univ. of California, San Diego (United States)
David Cheresh, Univ. of California, San Diego (United States)


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