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

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 microm, 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 microm. This compares favorably with the 100- to 300 microm micron depth attained by many 2-P systems, especially descanned 2-P instruments, and 40-microm-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.