Fourier transform infrared spectroscopy (FTIR) microscopic imaging technology combines infrared ray spectroscopy with microscopy, so that not only spectral information but also space distribution of sample can be provided. However, because severe peak overlapping exists in the FTIR microscopic image of complex samples, distribution information of the target chemical compound cannot be displayed directly. To overcome the drawbacks, a method of principal component analysis-2nd derivative spectral imaging, combining factor analysis and the spectral unmixing technology is developed. Image analysis of distribution of cholesterol in rabbit arteries with FTIR microscopic imaging technology verifies the feasibility and effectiveness of the proposed method. The experimental results show that the proposed method is an effective method to improve the spectral resolution and find the useful information hidden in overlapping spectral peak.