Background:Atrial fibrosis creates a vulnerable tissue for atrial fibrillation (AF), but the spatial disarray of collagen fibers underlying atrial fibrosis is not fully elucidated. Objective:This study hypothesizes that harmonics optical microscopy can illuminate the spatial mal-alignment of collagen fibers in AF via a layer-by-layer approach. Patients and Methods:Atrial tissues taken from patients who underwent open-heart surgery were examined by harmonics optical microscopy. Using the two-dimensional Fourier transformation method, a spectral-energy description of image texture was constituted and its entropy was used to quantify the mal-alignment of collagen fibers. The amount of collagen fiber was derived from its area ratio to total atrial tissue in each image. Serum C-terminal pro-collagen pro-peptide (CICP), pro-matrix metalloproteinase-1 (pro-MMP-1), and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) were also evaluated. Results:46 patients were evaluated, including 20 with normal sinus rhythm and 26 with AF. The entropy of spectral-energy distribution of collagen alignment was significantly higher in AF than that in sinus rhythm (3.97±0.33 vs. 2.80±0.18, p<0.005). This difference was more significant in the permanent AF group. The amount of collagen was also significantly higher in AF patients (0.39±0.13 vs. 0.18±0.06, p<0.005) but serum markers of cardiac fibrosis were not significantly different between the two groups. Conclusions:Harmonics optical microscopy can quantify the spatial mal-alignment of collagen fibers in AF. The entropy of spectral-energy distribution of collagen alignment is a potential tool for research in atrial remodeling.