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Chemically Resolved Imaging of Biological Cells and Thin Films by Infrared Scanning Near-Field Optical Microscopy

Antonio Cricenti ^*, Renato Generosi ^*, Marco Luce ^*, Paolo Perfetti ^*, Giorgio Margaritondo , David Talley , Jas S. Sanghera , Ishwar D. Aggarwal , Norman H. Tolk , Agostina Congiu-Castellano ^¶, Mark A. Rizzo ^|| and David W. Piston ^||

^* Istituto di Stuttura della Materia, Rome, Italy; Institut de Physique Appliquée, Ecole Polytecnique Fédérale, Lausanne, Switzerland; Optical Sciences Division, U.S. Naval Research Laboratory, Washington, District of Columbia; Department of Physics and Astronomy, Vanderbilt University, Nashville, Tennessee; ^¶ Department of Physics, Università di Roma "La Sapienza," Rome, Italy; and ^|| Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee

Correspondence: Address reprint requests to David W. Piston, 702 Light Hall, Vanderbilt University, Nashville, TN 37232-0615. Tel.: 615-322-7030; Fax: 615-322-7236; E-mail: dave.piston{at}vanderbilt.edu.

The infrared (IR) absorption of a biological system can potentially report on fundamentally important microchemical properties. For example, molecular IR profiles are known to change during increases in metabolic flux, protein phosphorylation, or proteolytic cleavage. However, practical implementation of intracellular IR imaging has been problematic because the diffraction limit of conventional infrared microscopy results in low spatial resolution. We have overcome this limitation by using an IR spectroscopic version of scanning near-field optical microscopy (SNOM), in conjunction with a tunable free-electron laser source. The results presented here clearly reveal different chemical constituents in thin films and biological cells. The space distribution of specific chemical species was obtained by taking SNOM images at IR wavelengths () corresponding to stretch absorption bands of common biochemical bonds, such as the amide bond. In our SNOM implementation, this chemical sensitivity is combined with a lateral resolution of 0.1 µm (/70), well below the diffraction limit of standard infrared microscopy. The potential applications of this approach touch virtually every aspect of the life sciences and medical research, as well as problems in materials science, chemistry, physics, and environmental research.