Imaging Protease Activity

Systematic efforts are under way to develop novel technologies that would allow molecular sensing in intact organisms in vivo. Using near-infrared fluorescent molecular beacons and inversion techniques that take into account the diffuse nature of photon propagation in tissue, we were able to obtain three-dimensional in vivo images of a protease in orthopic gliomas.

Setupipa

Figure 1.
The experimental imager shown in Figure1. consisted of a 675 nm laser diode (Fig 1a) and a beam splitter (1b) that divided light to a reference channel (1c) and an optical switch (1d). 24 source fibers (1e) and 36 fiber bundles (g) were employed to illuminate and collect light respectively from the optical imaging bore (f). The fiber bundles and reference fiber were arranged on a grid (1h) and imaged with a CCD camera (1j) using appropriate filters (1i). The insert surrounded by the dotted line describes the typical measurements (F, I, B) and formation of the composite field (C) used to produce quantified FMT images.

This system was used to three dimensionally image enzyme-activatable fluorochromes, within animal bodies. It was found that fluorescence activity can be detected with high positional accuracy in deep tissues, that molecular specificities of different beacons towards enzymes can be resolved and that tomography of beacon activation is linearly related to enzyme concentration. The tomographic imaging method offers a range of new capabilities for studying biological function; for example, identifying molecular-expression patterns by multispectral imaging or continuously monitoring the efficacy of therapeutic drugs.

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Figure 2.
In vivo FMT of cathepsin B expression levels in 9L gliosarcomas stereotactically implanted into unilateral brain hemispheres of nude mice. Fig 2a and 2b, Axial and sagittal MR slices of an animal implanted with a tumor, which is shown in green after gadolinium enhancement. c, d and f, Consecutive FMT slices obtained from top to bottom from the volume of interest shown on b by thin white horizontal lines. e, Superposition of the MR axial slice passing through the tumor a onto the corresponding FMT slice c after appropriately translating the MR image to the actual dimensions of the FMT image. g and h, Axial brain section through the 9L tumor imaged with white light and with monochromatic light at the excitation wavelength (675 nm), respectively, and i, fluorescence image of the same axial brain section demonstrating a marked fluorescent probe activation, congruent with the tumor position identified by gadolinium-enhanced MRI and FMT

For details see Ntziachristos et al, Nature Medicine. 8 (7): 757-760 (2003)