1 March 2009 Extended-working-distance multiphoton micromanipulation microscope for deep-penetration imaging in live mice and tissue
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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- 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.
© (2009) Society of Photo-Optical Instrumentation Engineers (SPIE)
Milan Makale, Milan Makale, Michele McElroy, Michele McElroy, Peter O'Brien, Peter O'Brien, Robert M. Hoffman, Robert M. Hoffman, Sharon Guo, Sharon Guo, Michael Bouvet, Michael Bouvet, Leo Barnes, Leo Barnes, Elizabeth Ingulli, Elizabeth Ingulli, David Cheresh, David Cheresh, } "Extended-working-distance multiphoton micromanipulation microscope for deep-penetration imaging in live mice and tissue," Journal of Biomedical Optics 14(2), 024032 (1 March 2009). https://doi.org/10.1117/1.3103783 . Submission:

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