Linear unmixing protocol for hyperspectral image fusion analysis applied to a case study of vegetal tissues

Hyperspectral imaging (HSI) is a useful non-invasive technique that offers spatial and chemical information of samples. Often, different HSI techniques are used to obtain complementary information from the sample by combining different image modalities (Image Fusion). However, issues related to the different spatial resolution, sample orientation or area scanned among platforms need to be properly addressed. Unmixing methods are helpful to analyze and interpret the information of HSI related to each of the components contributing to the signal. Among those, Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) offers very suitable features for image fusion, since it can easily cope with multiset structures formed by blocks of images coming from different samples and platforms and allows the use of optional and diverse constraints to adapt to the specific features of each HSI employed. In this work, a case study based on the investigation of cross-sections from rice leaves by Raman, synchrotron infrared and fluorescence imaging techniques is presented. HSI of these three different techniques are fused for the first time in a single data structure and analyzed by MCR-ALS. This example is challenging in nature and is particularly suitable to describe clearly the necessary steps required to perform unmixing in an image fusion context. Although this protocol is presented and applied to a study of vegetal tissues, it can be generally used in many other samples and combinations of imaging platforms.