Label-free mid-infrared dichroism-sensitive photoacoustic microscopy for assessing engineered heart tissue

Structural alignment in fibrous tissues, such as myocardium and tendons, is a key biomarker for physiological integrity and pathological remodeling. However, conventional imaging relies on staining and indirect quantification, limiting its objectivity and throughput. For example, in histochemical or confocal fluorescence microscopy (CFM) images, fiber structural alignment is non-physically estimated via 2D gradients. Here, we present mid-infrared dichroism-sensitive photoacoustic microscopy (MIR-DS-PAM)—a label-free, dual-contrast imaging system that combines molecular specificity with polarization sensitivity for histostructural analysis. MIR-DS-PAM employs a mid-infrared (MIR) light source with a center wavelength of 6.0 μm to selectively image protein-rich extracellular matrix (ECM). In addition, a polarization-control module enables quantifying the degree and angle of linear dichroism (DoLD and AoLD, respectively), which reflect the density and alignment of fibrous structures. In particular, the resultant vector length of AoLD indicates the uniformity of alignment to interpret tissue integrity. We applied MIR-DS-PAM to engineered heart tissues (EHTs) and demonstrated its ability to assess ECM development during maturation and to detect structural disorganization in fibrotic models. Compared to CFM, MIR-DS-PAM offers consistent, label-free evaluation of tissue anisotropy. In fibrosis, MIRDS- PAM captured distinct patterns between the phenotypes, associated with disease progression and mechanical dysfunction. We further extended MIR-DS-PAM to assembled EHTs to analyze spatial heterogeneity, linking microstructural disruption with functional impairment. Consequently, this analytical platform demonstrates its capability for quantitative and label-free imaging of tissue organization, offering new opportunities for functional histopathology, regenerative medicine, and fibrosis remodeling research.