A commonly used industrial process to manufacture plant-based meat analogues is high-moisture extrusion (HME) (Fig. . In HME, a dough made of plant-protein isolates or concentrates and of 40-60% water is subjected to high temperatures and shear forces within the extruder barrel section . After leaving the screw region of the extruder, the hot protein melt is subjected to shear and temperature gradients in a cooling die, in which anisotropic structures and textures develop . Protein blends, typically originating from soy or pea, contain around 70% -90% protein with the remainder being predominantly polysaccharide fibres originating from plant cell walls . Extrudates typically show a characteristic parabolic structure, which is caused by high temperature and shear treatments in the cooling die (Fig. ). As such, the parabolic flow profile correlates with higher anisotropy, whereas a plug-flow profile correlates with lower anisotropy . Typically, high temperatures and low shear forces are found perpendicularly to the extrusion direction, in the core region of the cooling die, while low temperatures and high shear forces are present near the cooled wall. These temperature and shear gradients cause protein fibrils to be more structured, and thus aligned, near the cooling wall (Fig. ), while more disordered near the core region (Fig. ) . Currently, our knowledge of how HME processing conditions impact the final structure of the extrudate is limited. Only recently it has become apparent that HME extrudates also have a hierarchical, multiscale, anisotropic structure (van der Sman & van der Goot, 2023). Therefore, gaining a better understanding of how this intricate structure develops, both in the extruder barrel and in the cooling die, is seen as key for optimizing process-property relationships. The structure at the nano-to mesoscale is governed by an anisotropic arrangement of sub-micron protein fibrils and micron-scale polysaccharide fibres. The protein fibrils consist of stacked globular proteins that align in shear flow and have a width of 100 -250 nm . Further, the protein fibrils tend to align with each other and form larger bundles with diameters around 10-500 μm . The second biopolymeric phase, e.g., cell wall polysaccharides, is present in a crystalline form, or dispersed throughout the protein matrix . The cooling in the die induces syneresis, whereby sub-mm alternating protein-rich (PR) and water-rich (WR) lamellae are formed . Two underlying mechanisms have been hypothesized: viscoelastic contraction of crosslinked proteins (van der Sman & van der Goot, 2023) and spinodal phase separation . Alongside the effects of heating and cooling, the structure is also influenced by the amount of moisture and pH . Only recently researchers have been able to analyse such complex food systems (Fig. ) using advanced structural analysis techniques. The analysis is often aimed at identifying anisotropy at different length scales ranging from the nanometre scale to the millimetre scale . The anisotropic structural arrangement of submicron protein fibrils and micron-scale polysaccharide fibres was quantified with Small Angle Scattering . At the μm-scale, diffraction-limited confocal laser scanning microscopy (CLSM) enabled qualitative imaging (Q. . Finally, at the mm-scale, nuclear resonance techniques such as Nuclear Magnetic Resonance Imaging (NMR/MRI) (F. L. ) and X-ray Tomography (XRT) have been used to reveal structural anisotropy in plant-based protein extrudates. Yet, these μm-to-mm scale imaging methods have so far been used mostly for qualitative studies , with limited quantification efforts at the mm scale . No methodological framework for the quantitative assessment of structural anisotropy of extrudates at the μm-mm scale has been introduced, which in turn hampers efforts to establish robust process-structure-texture relationships. For generally quantifying 2D images regarding structural anisotropy, a variety of image analysis algorithms have been developed, ranging from generic image analysis workflows , to more complete image analysis pipelines such as Alignment Fourier Transform (AFT) , OrientationJ and Skeletonization . AFT and OrientationJ allow calculating vector maps indicating dominant angles, of, e.g., microtubules or flow patterns in specific regions. Based on the vector field, AFT can also calculate an order parameter quantifying the degree of alignment of vectors within neighbouring regions. AFT and OrientationJ, however, lack the ability to identify more than a single orientation in any area, which can become an issue with very dense or overlapping structures. This kind of dense structure occurs frequently in the protein phase of plant protein extrudates. Furthermore, the statistical significance of a calculated orientation angle, present in a defined area, cannot be determined with AFT. As such, an angle obtained from an area with a low Signal-to-Noise Ratio (SNR) would be weighed identically to an angle obtained from a high-SNR area. Consequently, the vector fields and the order parameter from AFT do not distinguish between foreground and background signals. Building upon concepts like AFT and sliding window analysis, we have developed a method that can extract additional information on structural anisotropy. Our method, termed Rotated Fourier Transform (RFT), enables the extraction of comprehensive angular and anisotropic information by utilising Fourier transforms together with a rotating-window analysis. This provides information on the size of underlying features, the SNR corresponding to each feature orientation, and the spatial distribution of angles. To demonstrate RFT's functionality, we analysed the position-dependent orientation of fibres, within extrudates, caused by the flow in the HME cooling die of an extruder, and spatially mapped the material's structural anisotropy. The images were obtained via CSLM described in this work, or from high-field MRI measurements reported elsewhere , in order to capture anisotropic structural features over the μm-mm scale. The RFT methodology will be demonstrated on MRI and CLSM images of extrudates that were pH-shifted during HME , in order to induce differences in multiscale anisotropic structure. Furthermore, RFT will be applied to quantify the development of anisotropic structure during the passage of the protein melt in the cooling die. For this purpose, CLSM and MRI images were acquired for samples extracted from the cooling die upon an extrudate deadstop. The obtained RFT methodology will be discussed in light of recently formulated hypotheses concerning plant protein structuring by means of HME .