Spectral band optimization for multispectral fluorescence imaging

Multispectral imaging has the potential to improve sensitivity and specificity in biomedical imaging through simultaneous acquisition of both morphological (spatial) and chemical (spectral) information. Performing multispectral imaging in real time with spectrally resolved detector arrays (SRDAs), for example in endoscopy or intraoperative imaging, requires a direct trade off between spatial and spectral resolution. We sought to quantitatively assess the impact of spectral band selection on contrast agent detection in fluorescence endoscopic imaging. As a proof of concept, we measured the ‘ground truth’ spectra from a dilution series of a single near-infrared fluorescent contrast agent using a spectrometer incorporated into the detection path of our endoscope. We then modeled the influence of an SRDA on these spectra and calculated the theoretical endmembers associated with reflectance and fluorescence signals from the pure contrast agent. To test the accuracy of our model, we incorporated into the same endoscope an off-the-shelf SRDA with a 3x3 filter deposition pattern of 9 spectral bands. After spectral unmixing using the modeled endmembers, the amplitude of the fluorescence recorded with the SRDA compared favorably with the amplitude of fluorescence derived from the ‘ground truth’ spectra recorded with the spectrometer. In the future, this approach could be used to minimize the number of spectral bands required in a given imaging system and hence maximize the spatial resolution of the multispectral camera.