Histopathology forms the gold standard for the diagnosis of breast cancer. Fourier Transform Infrared (FT-IR) spectroscopic imaging has been proposed to be a potentially powerful adjunct to current histopathological techniques. Most studies using FT-IR imaging for breast tissue analysis have been in the transmission or transmission-reflection mode, in which the wavelength and optics limit the data to a relatively coarse spatial resolution (typically, coarser than 5 μm × 5 μm per pixel). This resolution is insufficient to examine many histologic structures. Attenuated Total Reflectance (ATR) FT-IR imaging incorporating a Germanium optic can allow for a four-fold increase in spatial resolution due to the material's high refractive index in the mid-IR. Here, we employ ATR FT-IR imaging towards examining cellular and tissue structures that constitute and important component of breast cancer diagnosis. In particular, we resolve and chemically characterize endothelial cells, myoepithelial cells and terminal ductal lobular units. Further extending the ability of IR imaging to examine sub-cellular structures, we report the extraction of intact chromosomes from a breast cancer cells and their spatially localized analysis as a novel approach to understand changes associated with the molecular structure of DNA in breast cancer.