Volumetric tomography of fluorescent proteins through small animals in vivo

Abstract

Volumetric detection and accurate quantification of fluorescent proteins in entire animals would greatly enhance our ability to monitor biological processes in vivo. Here we present a quantitative tomographic technique for visualization of superficial and deep-seated (>2-3 mm) fluorescent protein activity in vivo. We demonstrate noninvasive imaging of lung tumor progression in a murine model, as well as imaging of gene delivery using a herpes virus vector. This technology can significantly improve imaging capacity over the current state of the art and should find wide in vivo imaging applications in drug discovery, immunology, and cancer research.fluorescence ͉ whole-body imaging ͉ imaging gene expression ͉ gene transfer ͉ multispectral imaging F luorescent proteins (FP) have become essential reporter molecules for the elucidation of the function of proteins within cells, the biodistribution of immune and stem cells, and evaluation of drug candidates in vivo (1, 2). Confocal and multiphoton microscopy have emerged as powerful methods for imaging superficial locations down to a few hundred micrometers and in limited field of views (typically 1-2 mm) (3). Alternative technologies to date use macroscopic epiillumination imaging using sensitive color cameras that obtain ''photographs'' of fluorescence activity emanating from up to a few millimeters under the skin surface of animals (4). Such images are often biologically informative, but they are surfaceweighted, i.e., they preferentially image superficial over deepseated activity because the intensity of the fluorescence signal recorded drops with a strong nonlinear dependence as a function of depth. In addition, photographic imaging yields a single projection that cannot resolve depth; signal quantification is complicated with varying tissue optical properties, for example because of angiogenesis; and superficial fluorescence signals or skin autofluorescence can reduce or even shield deep-seated contrast.To improve on imaging of FPs in whole animals, it is important to develop methods that account for the nonlinear propagation effects of photons into tissues and resolve depth. Such technology has been recently reported for tomographic imaging of extrinsically administered fluorescence probes in the nearinfrared (NIR) (5, 6). However, the FPs currently available absorb in the visible (Ͻ600 nm) and many also emit in the visible (7). The translation of tomographic methods for operation in the 450-600 nm spectral region comes with some unique challenges. These challenges arise because tissue offers significantly higher photon attenuation in the visible necessitating different instrumentation than implementations that have appeared so far for imaging in the NIR. Similarly, adept theoretical models are required to model the corresponding change of photon propagation patterns in highly absorbing media (8). We have recently developed a system and method for tomographic imaging in the visible (9) achieving 400-m resolution at 6-mm depth in homogenous slabs of 1.2-cm thickn...

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