Figure 1: Schematic illustration of qVRI imaging process, from data collection and spectral unmixing to 3D reconstruction and quantification.
From: Quantitative volumetric Raman imaging of three dimensional cell cultures
The confocal microscope provides control over x × y × z dimensions of the sample position for 3D imaging. Each imaging plane is described by a hyperspectral dataset. Hyperspectral datasets are 3D datasets with x × y (number of pixels in a single imaging plane) spatial dimensions and w (wavenumbers) spectral dimension. Each voxel in 3D is associated with a single Raman spectrum. Combining hyperspectral datasets from multiple imaging planes creates a volumetric hyperspectral dataset with z × x spatial dimensions and z being equal to the sum of yn imaging planes. For spectral unmixing analysis the volumetric hyperspectral dataset is unfolded to form a matrix D=M × w with M=z × x. D is unmixed using N number of ‘pure’ components (e.g., here N=4) into two matrices C and ST. C contains the relative abundance values of the pure components in each voxel in an M × N matrix with every column associated to one component. ST is an N × w matrix containing a ‘pure’ component spectrum in every row. Each column of C contains all the spatial information needed to reconstruct every components’ 3D architecture by refolding it to the original x × y × z dimensions. Each voxel contains the concentration profile of the reconstructed component. The number of voxels within an isosurface at a chosen threshold can be used as a metric for quantification, comparing experimental conditions (e.g., comparing Cell A to Cell B).
