Infrared microspectroscopy combined with conventional atomic force microscopy.

This paper reports nanotopography and mid infrared (IR) microspectroscopic imaging coupled within the same atomic force microscope (AFM). The reported advances are enabled by using a bimaterial microcantilever, conventionally used for standard AFM imaging, as a detector of monochromatic IR light. IR light intensity is recorded as thermomechanical bending of the cantilever measured upon illumination with intensity-modulated, narrowband radiation. The cantilever bending is then correlated with the sample's IR absorption. Spatial resolution was characterized by imaging a USAF 1951 optical resolution target made of SU-8 photoresist. The spatial resolution of the AFM topography measurement was a few nanometers as expected, while the spatial resolution of the IR measurement was 24.4 µm using relatively coarse spectral resolution (25-125 cm(-1)). In addition to well-controlled samples demonstrating the spatial and spectral properties of the setup, we used the method to map engineered skin and three-dimensional cell culture samples. This research combines modest IR imaging capabilities with the exceptional topographical imaging of conventional AFM to provide advantages of both in a facile manner.

Label-free characterization of cancer-activated fibroblasts using infrared spectroscopic imaging.

Glandular tumors arising in epithelial cells comprise the majority of solid human cancers. Glands are supported by stroma, which is activated in the proximity of a tumor. Activated stroma is often characterized by the molecular expression of α-smooth muscle actin (α-SMA) within fibroblasts. However, the precise spatial and temporal evolution of chemical changes in fibroblasts upon epithelial tumor signaling is poorly understood. Here we report a label-free method to characterize fibroblast changes by using Fourier transform infrared spectroscopic imaging and comparing spectra with α-SMA expression in primary normal human fibroblasts. We recorded the fibroblast activation process by spectroscopic imaging using increasingly tissue-like conditions: 1), stimulation with the growth factor TGFß1; 2), coculture with MCF-7 human breast cancerous epithelial cells in Transwell coculture; and 3), coculture with MCF-7 in three-dimensional cell culture. Finally, we compared the spectral signatures of stromal transformation with normal and malignant human breast tissue biopsies. The results indicate that this approach reveals temporally complex spectral changes and thus provides a richer assessment than simple molecular imaging based on α-SMA expression. Some changes are conserved across culture conditions and in human tissue, providing a label-free method to monitor stromal transformations.