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J Neurophysiol 91: 1908-1912, 2004. First published December 10, 2003; doi:10.1152/jn.01007.2003
0022-3077/04 $5.00
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Innovative Methodology

In Vivo Multiphoton Microscopy of Deep Brain Tissue

Michael J. Levene, Daniel A. Dombeck, Karl A. Kasischke, Raymond P. Molloy and Watt W. Webb

School of Applied and Engineering Physics, Cornell University, Ithaca, New York 14853

Submitted 20 October 2003; accepted in final form 1 December 2003

Although fluorescence microscopy has proven to be one of the most powerful tools in biology, its application to the intact animal has been limited to imaging several hundred micrometers below the surface. The rest of the animal has eluded investigation at the microscopic level without excising tissue or performing extensive surgery. However, the ability to image with subcellular resolution in the intact animal enables a contextual setting that may be critical for understanding proper function. Clinical applications such as disease diagnosis and optical biopsy may benefit from minimally invasive in vivo approaches. Gradient index (GRIN) lenses with needle-like dimensions can transfer high-quality images many centimeters from the object plane. Here, we show that multiphoton microscopy through GRIN lenses enables minimally invasive, subcellular resolution several millimeters in the anesthetized, intact animal, and we present in vivo images of cortical layer V and hippocampus in the anesthetized Thy1-YFP line H mouse. Microangiographies from deep capillaries and blood vessels containing fluorescein-dextran and quantum dot-labeled serum in wild-type mouse brain are also demonstrated.

Address for reprint requests and other correspondence: W. W. Webb, Clark Hall, Rm. 223, Cornell Univ., Ithaca, NY 14853 (E-mail: www2{at}cornell.edu).




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