Label-free live brain imaging and targeted patching with third-harmonic generation microscopy

Abstract

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.

Quantitative comparison of 3D third harmonic generation and fluorescence microscopy images

“…Third harmonic generation (THG) microscopy [1][2][3][4] shows great potential for real-time pathology of brain tumor tissue [5,6]. THG microscopy is also establishing itself as an important tool for studying intact tissue.…”

“…THG microscopy is also establishing itself as an important tool for studying intact tissue. It has been successfully applied to image unstained samples such as insect embryos, plant seeds and intact mammalian tissue [3], epithelial tissues [7][8][9], zebrafish embryos [10], zebrafish nervous system [4], and mouse brain [5]. THG is a nonlinear optical process that depends on the third-order susceptibility c (3) of the tissue and the phase-matching conditions.…”

“…THG is a nonlinear optical process that depends on the third-order susceptibility c (3) of the tissue and the phase-matching conditions. Three incident photons are converted into one photon with triple energy and one third of the wavelength [1][2][3][4][5][6].…”

“…Like other multi-photon techniques [10][11][12][13][14][15][16][17][18], THG not only enables the recording of label-free images of unfixed 3D volumes of tissue [1][2][3][4][5][6][7][8][9][10] free from spatial distortion artifacts inherent to histopathology, but also potentially allows feedback on the nature of the tissue, i. e. whether it is healthy or tumorous, to the surgeon during surgery, as the relative speed of the imaging modalities approaches 'real' time, and no preparation steps of the tissue are required [5,6,[19][20][21][22][23]. THG was shown recently to yield label-free images of ex-vivo human tumor tissue of histopathological quality, in real-time [6].…”

“…The active contour models [33][34][35][36][37][38][39][40][41][42][43] became popular for automatic cell/nuclei segmentation at the beginning of this century when the classical CV model [33] was proposed. THG images, with Raman and other nonlinear microscopy images, differ from labeled-fluorescence images in their complexity, inherent to their high information density [5,6,20,21,24,25,[44][45][46][47]62]. A limited number of segmentation methods has been explored on THG images of several tissue types.…”