Research Papers

Extended-working-distance multiphoton micromanipulation microscope for deep-penetration imaging in live mice and tissue

[+] Author Affiliations
Milan Makale, Michele McElroy

University of California, San Diego, Moores Cancer Center, 3855 Health Sciences Drive, La Jolla, California 92093

Peter O’Brien

New York Medical College, Department of Cell Biology & Anatomy, Valhalla, New York 10595

Robert M. Hoffman

University of California, San Diego, Department of Surgery, 200 West Arbor Drive, San Diego, California 92103 and AntiCancer Inc., 7917 Ostrow Street, San Diego, California 92111

Sharon Guo

University of California, San Diego, Department of Bioengineering, 9500 Gilman Drive, 0412, La Jolla, California 92093-0412

Michael Bouvet

University of California, San Diego, Moores Cancer Center, 3855 Health Sciences Drive, La Jolla, California 92093 and University of California, San Diego, Department of Surgery, 200 West Arbor Drive, San Diego, California 92103

Leo Barnes, Elizabeth Ingulli, David Cheresh

University of California, San Diego, Moores Cancer Center, 3855 Health Sciences Drive, La Jolla, California 92093

J. Biomed. Opt. 14(2), 024032 (April 28, 2009). doi:10.1117/1.3103783
History: Received July 17, 2008; Revised January 29, 2009; Accepted February 10, 2009; Published April 28, 2009
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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-to3.5-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 750to800μm, 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 500μm. This compares favorably with the 100-to300μm 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.

Figures in this Article
© 2009 Society of Photo-Optical Instrumentation Engineers

Citation

Milan Makale ; Michele McElroy ; Peter O’Brien ; Robert M. Hoffman ; Sharon Guo, et al.
"Extended-working-distance multiphoton micromanipulation microscope for deep-penetration imaging in live mice and tissue", J. Biomed. Opt. 14(2), 024032 (April 28, 2009). ; http://dx.doi.org/10.1117/1.3103783


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