One specialized type of imaging involves the capture of low intensity fluorescence from animal subjects such as mice. Briefly, fluorescence is a molecular phenomenon in which a substance absorbs light of a particular wavelength and emits light of a longer wavelength. The absorption of light is referred to as the “excitation”, and the emission of longer wave lights as the “emission”. Both organic and inorganic substances can exhibit fluorescent properties.
Fluorescence imaging is performed by illuminating a sample to excite fluorescence molecules in the sample, and then capturing an image of the sample as it fluoresces using a camera. Such imaging applications present particular challenges to the design of a box or chamber in which the sample is contained during imaging. This is especially true in macroscopic applications where the field-of-view is about 1 cm−30 cm in diameter, as compared to microscopic applications where the field-of-view is less than about 1 cm.
Typically, intensified or cooled charge-coupled device (CCD) cameras are used to detect the fluorescence of low intensity light radiating from the sample. These cameras are generally complex, may require specialized cooling, and are typically fixed to a single location on the top of a specimen chamber. A user places a sample at a predetermined position in the specimen chamber within the field of view for the overhead camera.
Due to this static design, one particular challenge to imaging apparatus design is the diverse fluorescent illumination needs required during image capture. Fluorescent image capture, of course, involves the sample being illuminated with an illumination source, while the minute amounts of light emitted from the “excited” sample are detected using a light detector, e.g., a CCD camera. Depending on the application, there are benefits to both epi-illumination (reflection) and trans-illumination for fluorescence imaging. Epi-illumination provides a faster survey of the entire animal, but is subject to higher levels of autofluorescence. Trans-illumination, on the other hand, provides lower levels of autofluorescence and is useful for performing 3D tomographic reconstructions. Therefore, it is desirable to have both epi- and trans-illumination options on a fluorescence imaging system.
It is also desirable to determine the 3D location and concentration of fluorescent probes in tissue. Diffuse tomographic reconstruction algorithms are often used for this purpose. To perform 3D diffuse tomographic reconstructions in fluorescence imaging, however, it is necessary to determine a 3D surface topography of the target specimen. The surface topography is used to define the boundary conditions within the diffuse tomography algorithm. One particularly suitable technique to determine the 3D surface topography of a specimen is through the application of structured light imaging. It would be desirable to provide a single imaging system that is capable of both structured light imaging, to obtain 3D surface topography, and fluorescent imaging, to perform 3D diffuse tomographic reconstructions of a specimen. The present invention describes a dual illumination system that accomplishes these objectives.