Three‐photon‐resonance‐enhanced third‐harmonic generation for label‐free deep‐brain imaging: In search of a chemical contrast

Within the past decade, nonlinear Raman microscopy has earned a well‐deserved status of a gold‐standard technology for chemically selective imaging. Even though second‐ and third‐harmonic microscopy is much less demanding on a laser source and multifrequency beam arrangement, it is increasingly falling behind nonlinear Raman scattering as a method of bioimaging because it offers no mechanism whereby imaging could be made chemically specific. Here, we show, however, that such a mechanism does exist, helping harmonic‐generation microscopy overcome its no‐chemical‐specificity handicap. We demonstrate that, with the laser wavelength tuned to a three‐photon resonance with the Soret band of hemoglobin, third‐harmonic generation provides a chemically specific method for a high‐contrast imaging of red blood cells in a broad class of biological systems, including live brain. Moreover, third‐harmonic generation imaging can be conveniently combined with second‐harmonic microscopy on a compact laser platform, providing, as our experiments on rat brain show, a powerful resource for three‐dimensional, cell‐specific label‐free deep‐brain imaging.