Imaging single living cells with a scanning near-®eld infrared microscope based on a free electron laser M.K. Hong a , A.G. Jeung b , N.V. Dokholyan a , T.I. Smith b , H.A. Schwettman b , P. Huie c , S. Erramilli a, * a Physics Department and the Center for Photonics, Boston University, MA, USA b Hansen Experimental Physics Laboratory, Stanford University, Stanford, USA c Department of Pathology, Stanford University Medical School, Stanford, USA Abstract We report the ®rst sub-wavelength mid-infrared images under water, and describe an application to obtaining images of single living cells in water using the Stanford Free Electron Laser (FEL). Spatial resolution is enhanced at the peak of infrared absorption of water. Images were obtained of single motile ®broblasts with the FEL wavelength tuned to absorption peaks of both protein and lipid molecules. Analysis of the unexpectedly strong absorption due to lipid molecules in motile ®broblasts suggests that the concentration of lipid molecules in lamellopodia is consistent with membrane ¯ow. Ó 1998 Elsevier Science B.V. All rights reserved. PACS: 87.64t; 41.60Cr; 87.64Je; 87.15Mi 1. Introduction Infrared spectroscopy is one of the most sensi- tive techniques for the analysis of biological sys- tems. Many biomolecules like lipids, proteins and nucleic acids have normal modes that are infrared active [1]. These normal modes are so character- istic that the region of the spectrum between 4 and 12 lm is often called the ``®ngerprint'' region (see Fig. 1). A combination of infrared spectroscopy and microscopy provides a new tool for non-de- structive characterization of samples, and for lo- calization of speci®c molecules within a sample [2,3]. The ability to localize speci®c molecules or molecular groups is useful in a wide range of problems in biological physics. The absorption cross-sections associated with many vibrational modes are suciently high (between 10 19 ±10 16 cm 2 ) that it is possible to identify the presence of speci®c molecules at the sub-picogram level with- out having to use ¯uorescent stains or radioactive labels. Recognition of this sensitivity has led to the commercial development of Fourier transform infrared spectrometers (FTIR) attached to infra- red microscopes as a powerful method for micro- analysis in biomedical applications. These Nuclear Instruments and Methods in Physics Research B 144 (1998) 246±255 * Corresponding author. Address: 590 Commonwealth Av- enue, Boston, MA 02215, USA. Tel.: 617 353 1271; fax: 617 353 9393; e-mail: shyam@bu.edu. 0168-583X/98/$ ± see front matter Ó 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 5 8 3 X ( 9 8 ) 0 0 3 1 1 - 5