Ultrafast Third Harmonic Micro-spectroscopy Reveals a Two-Photon Resonance in Human Hemoglobin

The recently developed technique of ultrafast third harmonic generation (THG) micro-spectroscopy is discussed. The approach is easily adapted to a standard laser scanning microscope and allows for two and three photon resonances to be identified in non-fluorescent unlabeled samples. This work provides nonlinear microscopists with a tool for further understanding the contrast and damage mechanisms they will encounter under nonlinear excitation. Here, we use THG micro-spectroscopy to investigate the nonlinear optical properties of hemoglobin over the spectral range of 770 -1000 nm with 100 fs long nJ energy laser pulses. We demonstrate the ability of this approach to distinguish different ligand binding states in physiological solutions of human hemoglobin.