High-Speed, Label-Free Second Harmonic Generation Holographic Microscopy of Biological Specimens

We apply holography to second harmonic generation (SHG) microscopy to obtain three-dimensional images of biological specimens without the use of external labeling. Nonlinear scanning microscopy techniques like SHG, has proven to be a powerful technique for imaging biological samples such as collagen, myosin, and muscle fibrils, but it lacks in acquisition speed for video rate 3D imaging. In nonlinear holography however, full 3D images can be numerically reconstructed from one hologram, the recorded intensity interference pattern between the endogenous SHG and coherent reference fields. The femtosecond Yb:KGW laser oscillator used in these experiments appears in biologically compatible window at a center wavelength near 1 mu m. The limited absorption helps prevent damage to our samples, even for prolonged exposures. Holograms were recorded with exposure times as short as 1 ms using a fast mechanical shutter. Extrapolating the trend measured above this limit, we anticipate a 10 dB SNR at 40 mu s integration time. Such high 3D image frame rates would yield a great deal of information of chemical waves, neurological networks, and other biological systems.