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We use photothermal induced resonance (PTIR) imaging and spectroscopy, in resonant and non-resonant mode, to study the cytoplasmic membrane and surface of intact cells. Non-resonant PTIR images apparently provide rich details of the cell surface. However, we show that non-resonant image contrast does not arise from the infrared absorption of surface molecules and is instead dominated by the mechanics of tip-sample contact. In contrast, spectra and images of the cellular surface can be selectively obtained by tuning the pulsing structure of the laser to restrict thermal wave penetration to the surface layer. Resonant PTIR images reveal surface structures and domains that range in size from about 20 nm to 1 μm and are associated with the cytoplasmic membrane and its proximity. Resonant PTIR spectra of the cell surface are qualitatively comparable to far-field IR spectra and provide the first selective measurement of the IR absorption spectrum of the cellular membrane of an intact cell. In resonant PTIR images, signal intensity, and therefore contrast, can be ascribed to a variety of factors, including mechanical, thermodynamic and spectroscopic properties of the cellular surface. While PTIR images are difficult to interpret in terms of spectroscopic absorption, they are easy to collect and provide unique contrast mechanisms without any exogenous labelling. As such they provide a new paradigm in cellular imaging and membrane biology and can be used to address a range of critical questions, from the nature of membrane lipid domains to the mechanism of pathogen infection of a host cell.

Photoacoustic spectroscopy (PAS) measures the photon absorption spectrum of a sample through detection of the acoustic wave generated by the photothermal effect as one modulates the intensity of the incident radiation at each wavelength. We have recently demonstrated the implementation of PAS in a microscopy configuration with mid-infrared radiation (microPAS). In the present work, we describe the performance of microPAS using synchrotron radiation (SR) in diffraction-limited spectromicroscopy and imaging experiments. Spectra were obtained for polystyrene beads, polypropylene fibres, and single fibres of human hair. SR produced microPAS spectra of much higher intensity as compared with those obtained using conventional mid- and near-infrared sources. For hair samples, the penetration depth of mid-infrared light, even with bright SR, is significantly shorter than the probed sample thickness at very low modulation frequencies resulting in saturated PAS spectra. In contrast, microPAS spectra of polymer beads were in general of much better quality than those obtained with conventional sources. We also demonstrated the capability to collect line profiles and line spectra at diffraction limited spatial resolution. The microPAS spectra of beads appear free from appreciable bandshape distortions arising from the real part of the refractive index of the sample. This observation confirms microPAS as an absorption-only technique and establishes it as a valuable new tool in the microspectroscopic analysis of particulates and of samples with a complex topography.

Over the years Fourier-Transform Infrared (FTIR) spectroscopy has been widely employed in the structural and functional characterization of biomolecules. The introduction of infrared (IR) microscopes and of synchrotron light sources has created expectations that FTIR could become a generally viable technique to study both structure and reactivity in vivo, inside single cells, by performing measurements that up to a few years ago were the preserve of in vitro experiments on purified macromolecules. In this review we present the state-of-the-art in the application of FTIR spectromicroscopy as a technique for the study of structure and dynamics in single cells, we discuss the performance requirements for this application and review developments in sample handling methods.

We have tested an approach to identify putative cancer stem cells that involves measurement of the infrared absorption spectrum of individual cells in an aqueous environment, and their subsequent classification using multivariate data analysis techniques. Two primary esophageal cell lines were characterized: the immortalized normal esophageal epithelial cell line, Het-1A, and the esophageal adenocarcinoma cell line, OE33. In addition, we also evaluated spheroids, reflecting stem-like cell populations, which were derived from each parent cell line when grown in serum-free media. As differences in cell size appeared to be a strong discriminating factor, a correction needs to be performed to allow a reliable classification based on infrared absorption spectra. We demonstrated that stem-like cells derived from Het-1A could easily be discriminated on the basis of absorbance differences in the 1000-1200 cm(-1) spectral interval, whereas this was not possible for OE33. Furthermore, we found that changes due to aging of OE33 cells in culture dominated the infrared absorption spectra and somewhat limited the potential of this approach to identify stem-like cell populations using this in vitro model system.

We demonstrate the capability of synchrotron-based Fourier-Transform Infrared spectromicroscopy to detect metabolite formation in single, living cells of the unicellular algae Chlamydomonas reinhardtii. We show that the high brightness of the source provides a sufficient signal-to-noise ratio to detect small molecular species accumulating in a spot about 15 microm in size. Time resolved measurements are carried out on cells grown heterotrophically under low-light conditions to study the evolution of products of anaerobic metabolism. The formation of small molecular species, including ethanol and at least one carbonyl containing compound, can be detected with a time resolution of the order of one minute.

The objective of this exploratory study was to evaluate the feasibility of using Fourier-Transform Infrared (FTIR) spectromicroscopy to characterize formalin-fixed, paraffin-embedded human esophageal tissues. Matched histologically normal esophageal squamous epithelium (NS), premalignant Barrett esophagus (BE), and primary esophageal adenocarcinoma (EADC) tissues, each defined according to strict clinicopathologic criteria, were obtained from patients who underwent esophageal resection. Using confocal IR microscopy, measurements in the mid-IR spectral region were carried out in transflection configuration, scanning regions of interest in 15 microm steps. A multidimensional dataset reporting the spectroscopic properties at each sampled point were analyzed by performing a hierarchical cluster analysis on the second derivative of spectral traces. Normal esophageal epithelia were characterized by a few well defined regions, mostly of large size (tens of contiguous pixels), which correlated with tissue histology, specifically the basal cell layer. BE tissues had characteristic regions localized to gland crypts, ranging in size from one pixel to a few tens of pixels, which displayed IR spectra with defined absorption features characteristic of glycoproteins. The incorporation of synchrotron light to improve the resolution of individual cells in BE tissues has demonstrated that these glycoproteins are associated with goblet cells, the characteristic cell type defining BE. Whereas the highly fragmented regions identified in EADC likely reflect tumor heterogeneity, FTIR mapping would appear to be a potentially useful technique to identify premalignant BE tissues. The technical feasibility of using FTIR to characterize formalin-fixed, paraffin-embedded human esophageal tissues demonstrates the potential of this technique to study archival human BE tissue specimens via automated screening techniques.