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A single human oesophageal adenocarcinoma cell (OE33) has been imaged using aperture infrared scanning near-field optical microscopy (IR-SNOM) in transmission and reflection and also by Fourier-transform infrared (FTIR) microspectroscopy in transmission only. This work presents the first images obtained in both transmission and reflection of the same specimen using the aperture IR-SNOM technique. The results have been used to compare the two SNOM modes and also the two techniques, which have complementary capabilities. The SNOM technique necessitates a very stable source and a careful choice of wavelengths, since it is too slow to yield images at the thousands of wavelengths obtained with FTIR. However the SNOM technique is not diffraction limited and with careful fabrication of tips can yield images with high spatial resolution. There is no significant correlation between the SNOM images obtained in transmission and reflection and the correlations between images obtained at different wavelengths vary with the different imaging modes. These results are attributed to the strong dependence of the evanescent wave on both the wavelength and the distance between the tip and the source of the signal within the sample. While both transmission and reflection SNOM images show some correlation with topography this is not a dominant effect. These results indicate that with suitable calibration a combination of reflection and transmission aperture IR-SNOM measurements has the potential to reveal information on the depth distribution of the chemical structure of a specimen.

Cervical cancer remains a major cause of morbidity and mortality among women, especially in the developing world. Increased synthesis of proteins, lipids and nucleic acids is a pre-condition for the rapid proliferation of cancer cells. We show that scanning near-field optical microscopy, in combination with an infrared free electron laser (SNOM-IR-FEL), is able to distinguish between normal and squamous lowgrade and high-grade dyskaryosis, and between normal and mixed squamous/glandular pre-invasive and adenocarcinoma cervical lesions, at designated wavelengths associated with DNA, Amide I/II and lipids. These findings evidence the promise of the SNOM-IR-FEL technique in obtaining chemical information relevant to the detection of cervical cell abnormalities and cancer diagnosis at spatial resolutions below the diffraction limit (≥0.2 μm). We compare these results with analyses following attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy; although this latter approach has been demonstrated to detect underlying cervical atypia missed by conventional cytology, it is limited by a spatial resolution of ~3 μm to 30 μm due to the optical diffraction limit.Cervical cancer is associated with the persistent infection of high-risk types of human papillomavirus (HPV), together with other socioeconomic co-factors 1 . Screening involves cytological and histological classification of cervical cells. In the UK, cytological examination of cervical squamous cells is classified as normal, borderline or mild dyskaryosis, moderate or severe dyskaryosis and invasive cervical cancer (ICC). Histology is defined as normal, cervical intra-epithelial neoplasia (CIN): CIN1, CIN2, CIN3, or invasive cervical cancer. For atypical cells found in the glandular cells of the cervix, the pre-invasive lesion of adenocarcinoma is defined by changes termed cervical glandular intraepithelial neoplasia (CGIN), and sub-classified as low-grade cervical glandular intra-epithelial neoplasia (LGCGIN) and high-grade cervical glandular intra-epithelial neoplasia (HGCGIN). Squamous and glandular lesions may co-exist together and are defined by the level of CIN together with either LGCGIN or HGCGIN. Conventional screening is flawed as it is dependent on the subjective visual inspection of cytology; this often results in mis-diagnoses when grading samples 2 .

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The development of more accurate and sensitive diagnostic techniques is a key factor in efforts to improve cancer survival rates. The technique of infrared aperture fibre scanning near-field optical microscopy (IR-SNOM), together with radiation from the infrared free-electron laser (IR-FEL) on ALICE at Daresbury Laboratory (UK), has been used to obtain IR images of a cryptlike feature and the surrounding tissue; the tissue was taken from a patient with oesophageal adenocarcinoma and with a history of Barrett's oesophagus. We have shown that the DNA signal is enhanced relative to other contributions in the region of the crypt, and the glycoprotein signal shows a less pronounced increase in the region of the crypt. The Amide II signal is found to be anti-correlated with the DNA and glycoprotein profiles. The absorbance of the Amide II signal is found to differ for three different types of cancer tissue. High-resolution IR images of the crypt reveal additional structure that would not be resolved in diffraction-limited techniques.