KEYWORDS: Raman spectroscopy, Tomography, Optical tomography, Imaging systems, Signal detection, 3D image processing, 3D image reconstruction, Projection systems
Volumetric imaging enables rapid, quantitative and global measurements of cells, tissues or organisms to obtain their biomolecular information and has become a powerful tool for studying cellular metabolism, brain function and developmental biology. Optical projection tomography (OPT) plays an important role in whole-body imaging of cells, organs, embryos and organisms because it enables three-dimensional (3D) imaging with high spatial and temporal resolution of samples at the millimeter level. However, the OPT technique relies on fluorescent labels for chemical targeting, which can perturb the biological function of living system. As a label-free molecular imaging technique, widefield Raman imaging enables high-resolution analysis of large field-of-view samples. Its combination with projection tomographic strategy enables high-resolution 3D imaging of large-scale samples in a label-free manner. However, this technique was failure to determine the tissue microstructure and specific spatial distribution. Here, we proposed a concept of new label free volumetric imaging, dual-modality of optical-Raman projection tomography. In this concept, Raman projection tomography was assigned to achieve volumetric imaging of chemical composition and distribution in a 3D volume, and the OPT was used to obtain structural information of the 3D volume with micron-level spatial resolution. We further homebuilt a dual-modality imaging system for optical-Raman projection tomography and the feasibility of the system was validated by imaging polystyrene microspheres and dimethyl sulfoxide. Finally, we demonstrated the application potential by a series of bio-sample experiments.
Raman spectroscopic imaging can provide three-dimensional data set of samples, including two-dimensional spatial image and one-dimensional Raman spectral data. Currently, three strategies can be used to achieve Raman spectroscopic imaging, including point scanning, line scanning, and wide-field illumination. Point scanning method provides the best resolution but has low imaging speed. On the contrary, wide-field illumination can image fast but provides lower spatial resolution. To integrate the advantages of two methods, a new strategy for large-field Raman spectroscopic imaging was proposed, which uses the frequency modulation based spatially encoded light as the excitation. In this method, millions of single beams simultaneously illuminate on the sample to act as the wide-field illumination. Each beam illuminates on different positions of the sample, whose intensity are modulated with different frequencies. Thus, each excitation beam has its own modulation frequency and the excited Raman signal will carry the modulation information. At the detection end, a single point detector was used to collect the time series Raman signals carrying the unique modulation information. Using the sparse reconstruction based on demodulation strategy, the Raman image can be recovered effectively. The feasibility of the method was verified with numerical simulations. The results showed that it is feasible to conduct Raman spectroscopic imaging with high-resolution and high speed under the illumination of frequency modulation based spatially encoded light and the detection of single-point detector.
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