The aim of this project is to extend optical microscopy into the deep ultraviolet (DUV) wavelength range, and characterize DUV endogenous signals obtained from unlabeled biological tissue. We define DUV wavelengths as ranging from 230 nm to 350nm. This range is rarely investigated in microscopy because DUV light does not transmit through ordinary glass, and hence cannot be observed with standard microscope optics. Our basic hypothesis is that DUV optical microscopy can reveal clinically relevant information on cells and tissue structures that is normally unobservable. Our proposed technique will be based on the use of a nonlinear microscope to produce DUV three-photon excited autofluorescence (3PEF) and third-harmonic generation (3HG). These signals are intrinsic and do not require sample labeling. We will exploit the advantages of nonlinear microscopy over standard (linear) microscopy for accessing the DUV spectral window, and will develop a novel light collection technique specifically designed for detection in the backward direction, thereby allowing DUV imaging in thick tissues. In addition, we will integrate our nonlinear microscope with a custom designed spectrograph to allow both high-resolution imaging and spectral analysis. As far as we know, DUV imaging (as defined here) in a thick tissue with a nonlinear microscope has never been performed before. Our goal in this project is to demonstrate sub-cellular resolution DUV imaging in unlabelled brain and cancer tissue, and to demonstrate the unambiguous identification of molecular species in the DUV spectral range using recently developed techniques of spectral decomposition. Ultimately, we hope to confirm our hypothesis that intrinsic optical signals in the DUV spectral window, which has largely been overlooked so far, can provide useful and clinically relevant information.
| Lillis, Kyle P; Eng, Alfred; White, John A et al. (2008) Two-photon imaging of spatially extended neuronal network dynamics with high temporal resolution. J Neurosci Methods 172:178-84 |
| Leray, A; Lillis, K; Mertz, J (2008) Enhanced background rejection in thick tissue with differential-aberration two-photon microscopy. Biophys J 94:1449-58 |
