Attenuated total reflectance Fourier-transform infrared spectroscopic imaging for breast histopathology

Abstract

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.

Using Fourier transform IR spectroscopy to analyze biological materials

“…Diffraction-limited resolution with ATR-FTIR imaging can also be advantageous as it allows analysis of live cells in aqueous systems 21,48 . In addition, the spatial resolution of the image can be increased by incorporating optics with a high refractive index 21,34 .…”

“…The spatial resolution of non-aperture-based techniques is determined by the optics chosen, and it has been shown that a germanium optic is preferential, although ZnSe and diamond crystals can also be used 34 . Although transmission and transflection imaging have been widely implemented in biological tissues, imaging in ATR mode is a versatile option, because little sample preparation is required owing to minimal sample-thickness restrictions, which thus means that it has been implemented in biological fields such as pharmacology and subcellular interrogation 59,78,79 .…”

High-definition Fourier Transform Infrared (FT-IR) Spectroscopic Imaging of Human Tissue Sections towards Improving Pathology

“…There are a number of modifications that can be made to the imaging system including imaging in ATR mode 10,24,26 and using nanoscale thermal approaches 52,53 to allow for high resolution IR imaging. The main limitation with high resolution IR imaging is that the tissues must be carefully prepared and thin enough for IR to pass through (typically 4 μm thickness).…”

“…, with many very exciting studies showing its applications [19][20][21][22][23][24][25] . In addition, it was recently demonstrated that the increased spatial resolution associated with ATR imaging can allow for the visualization and classification of endothelial and myoepithelial cells in breast tissue which form a key component of breast cancer diagnosis 26 . While ATR imaging is very useful, this technique requires the SIL to make contact with the tissue to form FT-IR images; therefore, its use is somewhat limited for tissue pathology where large regions of tissues must be quickly imaged.…”

High-definition Fourier transform infrared spectroscopic imaging of breast tissue

“…3d that used the same NA = 0.62 objectives, but with a total magnification of 7.3x, resulting in 5.5 μm image pixels that make the basement membrane and distinction between myoepithelial and luminal epithelial cells impossible. These cells are of key histopathological interest since the destruction of the basement membrane and the disappearance of the myoepithelium are markers for invasive breast cancer 10 . …”