Spectral resolved imaging of biological samples

The spectral resolved imaging technique has a typical characteristic that it allows simultaneous recording of real-color RGB image representation and corresponding spectra. In this paper, we present a nonlinear spectral resolved imaging approach based on two-photon excited autofluorescence and second harmonic generation of biological tissues to elucidate microstructure and spectroscopic features of unstained rabbit arterial wall. Coupled to the image-guided spectral analysis method, we use a series of recorded nonlinear spectral resolved images excited by a broad range of laser wavelengths (730-910nm) to identify five components in unstained rabbit arterial wall including in NADH, elastin, flavin, porphyrin derivatives and collagen. Using the combination of a nonlinear spectral resolved imaging approach and two-channel synchronized detection, we obtained high contrast 2-13 and 3-D images of collagen and elastic fiber in the arterial wall. Our results demonstrate that simultaneously linking a nonlinear spectral resolved imaging approach with the image-guided spectral analysis method and multi-channel synchronized detection technique is a very useful method because it can in tandem acquire the structural modifications of different tissue components and quantitatively record the change of corresponding emission signal. This method has the potential to provide more comprehensive diagnostic information for determining tissue pathology which associated with mechanical properties of aorta wall and pharmacodynamical studies of vessels.