Multispectral Imaging

Background
Developmental studies and disease modeling in the zebrafish increasingly depend on in vivo detection of fluorescent signals. This approach is extremely flexible, but in the visible emission range (500 to 700 nm) in-vivo imaging is hampered by bright tissue autofluorescence which is close to the fluorescent signal of green fluorescent protein, limiting achievable signal-to-noise ratios.
Multispectral imaging can overcome these limitations. It offers the ability to capture images at multiple wavelengths using a liquid crystal tunable filter, combined with algorithms to discern different signals based on their unique emission spectra.

Instrumentation
The Nuance™ multispectral imaging system (CRI. Inc.) uses a liquid crystal tunable filter (LCTF) optically coupled to a sensitive scientific-grade, cooled megapixel CCD. With no moving parts, vibration, image shift or noise, a series of images at different wavelengths are taken. The imaging unit can be combined with a regular camera lens for macroscopic images of adult fish or mounted on the C-mount of a fluorescence microscope (see Fig.1).

Physics
The liquid crystal tunable filter acquires a set of images over a range of emission wavelengths. In this way, the emission spectrum for each pixel in the image is obtained.

The schematic shows two different emission spectra obtained for two different points in a zebrafish. The green spectrum indicates the signal obtained from green fluorescent protein in a target organ, the black spectrum depicts the background tissue autofluorescence. Note the overlap of autofluorescence and GFP spectra (Fig.2).

Figure 3 shows an example of what we obtained using multispectral imaging. The zebrafish thymus undergoes involution, just like its mammalian counterpart. With increasing age of the fish it therefore becomes more and more difficult to visualize a Rag-2-GFP transgenic thymus . And this results in an almost undetectable fluorescent signal in one-year old Rag-2-GFP transgenic fish (Figure 3, left panel), where the eye is indicated with a yellow arrow. However, after spectral unmixing, we now get a clearly defined fluorescent signal, that even gives an impression of texture and 3D orientation (Figure 3, right panel).