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APRIL 2003

Coregistration method images exogenous tissue

By: Charles Bankhead

CONTEXT: Multiphoton microscopy studies have typically relied on two-photon excited fluorescence (TPEF) to image biological systems. As researchers gained experience with TPEF, they learned that second-harmonic generation (SHG) from structural proteins could be exploited as another potentially useful contrast mechanism. But until recently, they were unable to coregister TPEF/SHG, so the utility of the combined imaging strategy for in vivo thick-tissue applications was limited.

RESULTS: By combining a form of multiphoton microscopy and SHG, Aikaterini Zoumi, Ph.D., and colleagues at the Beckman Laser Institute at the University of California, Irvine, imaged endogenous signals unique to different types of tissue, a capability that has potentially major implications for monitoring disease processes and response to therapy.

The UC Irvine team found that coregistration can be easily achieved by using simple backscattering geometry. The capability was demonstrated across a broad range of excitation wavelengths (Proc Natl Acad Sci, USA 2002;99:11014-11019).

An initial investigation concluded that the TPEF/SHG technique could be used to determine the origin of image-forming signal from various tissue constituents within a 3D tissue model. Specifically, the researchers could distinguish between signals emitted by cells and the signal emitted by the surrounding extracellular matrix.

"Using different excitation wavelengths, we can manipulate signals so they become spectally distinct," Zoumi said.

IMAGE: A fibroblast cell and surrounding collagen were obtained from the same site of an organotypic raft tissue model (a type of ex vivo culture that closely resembles a cervix). Images were acquired from tissue at depth of 67 microns for an excitation wavelength of 840 nm, using different emission filters. A 440/40-nm band-pass emission filter was used to collect the SHG from the collagen in the raft. A 520/40-nm band-pass emission filter was used to collect the TPEF from the fibroblast in the raft before the images were coregistered. (Provided by A. Zoumi)

IMPLICATIONS: The ability to distinguish signal from cells versus extracellular matrix has potentially major applications for the evaluation of cancer. Other possible applications include signal analysis of the cornea, arterial tissue, and protein-protein interactions.