There is a long history of standard H. & E. staining for pathological diagnostic samples, where the organism is fixed with formaldehyde. However, one should not be neglect that the pathological diagnostic sample has an artificial, secondary protein structure, differing from the structure of just frozen‐sectioned organism. We observed quantitatively the cholesterol distribution in the atheroma of a frozen‐section of autopsied aorta by imaging, for the first time, using the Fourier transform infrared microscopy (FT‐IRM) technique. We detected the cholesterol distribution and found that the protein structure changed to a &bgr;‐sheet conformation, from an &agr;‐helix conformation on the inside edge of the calcified area. © 2004 American Institute of Physics