Infrared spectroscopic characteristics of cell cycle and cell death probed by synchrotron-based FTIR spectromicroscopy Hoi-Ying N. Holman a , Michael C. Martin b , Eleanor A. Blakely c , Kathy Bjornstad c , and Wayne R. McKinney b a Center for Environmental Biotechnology, b Advanced Light Source, c Life Sciences Division, Ernest Orlando Lawrence Berkeley National Laboratory, University of California, Berkeley, California 94720, USA INTRODUCTION Conventional Fourier Transform Infrared (FTIR) spectroscopy and spectromicroscopy has been widely used as a diagnostic tool for characterizing the composition and structure of cellular components within intact tissues. 1-5 However, the spatial resolution of traditional FTIR spectromicroscopy is limited to ~75 μm with sufficient signal-to-noise at reasonably short data collection times. 6, 7 Synchrotron radiation-based FTIR spectromicroscopy (SR-FTIR), on the contrary, provides several hundred times higher brightness at a nearly diffraction-limited spatial resolution of 10 μm or better (depending on wavelength), and is therefore a sensitive analytical technique capable of providing molecular information at a significantly finer spatial resolution on biological specimens. 6-11 With this 10 μm or smaller spot size, SR-FTIR is ideally suited for the non-destructive, in situ study of processes that are taking place in individual cells. In this study we use SR-FTIR spectromicroscopy to investigate the spectral changes that occur in individual living human lung cells as a function of cell cycle and cell death. EXPERIMENTAL DETAILS The normal human fetal lung fibroblast IMR-90 P4 (passage 4) cell line was used for this study. They were cultured and grown to confluence on a plastic petri dish, as shown in the photograph in Figure 1(a). Cells were rinsed twice in PBS, scraped in 5mls media, mixed, and pipetted onto dried cleaned gold- coated glass slide pieces in 100 μl aliquots (2x10 4 cells) which were spread out with the pipette tip to insure single cells could be visualized. A photomicrograph of cells ready for infrared analysis is shown in Figure 1(b). Fluorescence-activated cell sorting (FACS) 14-16 was used to determine the distribution of cells in each phase of the cell cycle. The results of the FACS analysis showed 82.2% G1 phase, 9.3% S phase, and 8.5% G2/M phase. No cells of abnormal DNA content were observed. Spectromicroscopic measurements were made at beamline 1.4.3 at the Advanced Light Source. 8, 17-19 The cells on gold coated glass were inserted into a small chilled chamber with a thin IR-transparent ZnSe window to maintain a more constant humidity, and prolong cell viability. This chamber was placed on the motorized microscope stage. Most of the liquid medium remaining on the slide surface was removed by carefully holding the slide at an angle against a piece of dry sterile gauze. a b Figure 1. (a) IMR-90 cells grown to confluence, and (b) cells placed on gold- coated slides ready for SR-FTIR analysis.