Our group has an active NSF-Major Research Instrumentation – Instrument Development (MRI-ID) grant to develop a nonlinear optical microscope capable of performing detailed polarization analysis of biological systems with high spatio-temporal resolution. The emergence of nonlinear optical imaging as a powerful tool for selective detection of native structures in living systems (e.g., collagen) has opened up new opportunities for label-free biological imaging. Randomly oriented assemblies result in complete destructive interference, producing negligible background. However, the intrinsic orientation-selectivity of second harmonic generation (SHG) enables sensitive detection of highly specialized classes of materials. Furthermore, both the intensity and polarization-dependence of the resulting signals depend sensitively on local structure and orientation. The primary goals of the imaging project are to develop experimental and computational methods to efficiently mine the nascent information contained within the polarization-dependence.

            An added advantage of the instrument under development is the availability of multi-modal imaging. The same beam used to generate SHG can also perform two-photon excited fluorescence (TPEF). Consequently, a single instrument allows simultaneous imaging of two photon fluorescence, brightfield (scattering), and SHG in epi (back through the excitation objective) and transmission. The ability to image up to 8 channels simultaneously with our custom-built detection electronic package providee unique abilities in sample imaging.  Further improvements to the instrument to enable full polarization ellipsometry, resonant Z-scanning, and novel beam-scanning methods for signal to noise enhancement are currently underway.

Second order non-linear optical imaging of chiral crystals (SONICC) is demonstrated as a sensitive and selective detection method for protein crystallization. This figure contains representative results comparing TPEF and a comparison of imaging techniques for GFP crystals; bright field (a), epi-detected TPEF (b), epi-detected SHG (c), transmitted SHG (d), and a line scan on a log scale for TPEF (green) and SHG (blue) (e). Scale bar: 50 µm.

Wampler, R.D.; Kissick, D.J.; Dehen, C. J., Gualtieri, E.J.; Grey, J.L.; Wang, H.; Thompson, D.H.; Cheng, J.; Simpson, G.J., Selective detection of protein crystals by second harmonic microscopy. Journal of American Chemical Society 2008,  130, 43, 167.

SONICC was recently highlighted in C&EN

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