Label-free mid-infrared dichroism-sensitive photoacoustic microscopy for histostructural analysis of engineered heart tissues.

Many biological tissues, such as cardiac muscle, tendons, and the cornea, exhibit highly organized microstructural alignment that is critical for mechanical and physiological functions. Disruptions in this structural organization are commonly associated with pathological conditions such as fibrosis, infarction, and cancer. However, conventional histological imaging techniques rely on immunofluorescence or histochemical staining, and they evaluate tissue alignment via non-physical 2D gradient-based calculation, which is labor-intensive, antibody-dependent, and prone to variability. Here, we demonstrate label-free mid-infrared dichroism-sensitive photoacoustic microscopy (MIR-DS-PAM), an analytical imaging system for cardiac tissue assessments. By combining molecular specificity with polarization sensitivity, this method selectively visualizes protein-rich engineered heart tissue (EHT) and quantifies the extracellular matrix (ECM) alignment without any labeling. The extracted dichroism-sensitive parameters, such as the degree of dichroism and the orientation angle, enable histostructural evaluation of tissue integrity and reveal diagnostic cues in fibrotic EHT. This technique offers a label-free analytical tool for fibrosis research and tissue engineering applications.