Label-free live brain imaging and targeted patching with third-harmonic generation microscopy
2011
Witte, Stefan | Negrean, Adrian | Lodder, Johannes C. | de Kock, Christiaan P.J. | Testa Silva, Guilherme | Mansvelder, Huibert D. | Louise Groot, Marie
The ability to visualize neurons inside living brain tissue is a fundamental requirement in neuroscience and neurosurgery. Especially the development of a noninvasive probe of brain morphology with micrometer-scale resolution is highly desirable, as it would provide a noninvasive approach to optical biopsies in diagnostic medicine. Two-photon laser-scanning microscopy (2PLSM) is a powerful tool in this regard, and has become the standard for minimally invasive high-resolution imaging of living biological samples. However, while 2PLSM-based optical methods provide sufficient resolution, they have been hampered by the requirement for fluorescent dyes to provide image contrast. Here we demonstrate high-contrast imaging of live brain tissue at cellular resolution, without the need for fluorescent probes, using optical third-harmonic generation (THG). We exploit the specific geometry and lipid content of brain tissue at the cellular level to achieve partial phase matching of THG, providing an alternative contrast mechanism to fluorescence. We find that THG brain imaging allows rapid, noninvasive label-free imaging of neurons, white-matter structures, and blood vessels simultaneously. Furthermore, we exploit THG-based imaging to guide micropipettes towards designated neurons inside live tissue. This work is a major step towards label-free microscopic live brain imaging, and opens up possibilities for the development of laser-guided microsurgery techniques in the living brain.
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