"Third-harmonic generation (THG) imaging: 3D contrast without absorption" M. Müller, J. Squier, and G. J. Brakenhoff. Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing IV, eds. D. Cabib, C. J. Cogswell, J. -A. Conchello, J. M. Lerner, and T. Wilson. Proceedings of SPIE–The International Society for Optical Engineering, 3605. Bellingham, WA: SPIE, 1999
 

Description:

Imaging biological samples often involves telling apart one wet thing from another. A relatively new way of gaining the needed contrast is to exploit the nonlinear optical features of the sample itself by using a process in which a high-power laser beam can, when it is brought to a tight focus in certain media, generate subsidiary light waves at twice the original frequency (second harmonic), three times the frequency (third harmonic), and so on. If the detector is sensitive to just the third harmonic radiation, say, then by scanning the laser focus across the face of the sample, an image can be built up with a spatial resolution as small as the focal size. This scheme was used to produce the first 3-dimensional third-harmonic image of a living system.

Above is the third harmonic generation image of a spiral algae formation. The image is rendered using a series of cross sectional images produced by third harmonic generation at interfaces within the specimen. The excitation wavelength was 1.2 microns, and the detected wavelength was 400 nm. the laser pulse duration was 100 fs. Each cross sectional image was acquired in 1.6 seconds. The specimen in the image measures approximately 100 microns long by 50 microns wide.

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