Monitoring cell cycle distributions in MCF-7 cells using near-field photothermal microspectroscopy

¹ Lancaster University

^* To whom correspondence should be addressed. E-mail: f.martin{at}lancaster.ac.uk.

Submitted on October 4, 2004
Revised on January 5, 2005
Accepted on 8 February 2005

	Abstract

Microspectroscopic techniques such as Fourier transform infrared (FTIR) have played an important role in "fingerprinting" the biochemical composition of cellular components. Based on structure and function, complex biomolecules absorb energy in the mid-IR ( = 2-20 µm) yielding characteristic vibrational IR spectra. However, optical detection FTIR microspectroscopy may not be suitable for IR-absorbing sample materials. Photothermal microspectroscopy (PTMS) permits the direct measurement of heat generated as a result of sample material absorbing radiation. This approach generates true absorption spectra and is implemented by interfacing a scanning probe microscope and an FTIR spectrometer. Detection is performed using a near-field ultra-miniaturised temperature sensor. Employing PTMS, IR spectra of MCF-7 cells were examined in spectral regions (900-2000 cm-1) corresponding to proteins, DNA, RNA, glycoproteins, carbohydrates, lipids and levels of protein phosphorylation. As a cell passes through the cell cycle, its nuclear material de-condenses and condenses and this has led to ambiguity as to whether the intensity of such spectral regions may be associated with the G1-, S- or G2-phases of the cell cycle. Cultured cells were tracked over a time-course known to correspond to marked alterations in cell-cycle distributions, as determined using flow cytometry. Experiments were carried out in the absence or presence of lindane, a pesticide known to induce G1-arrest in MCF-7 cells. Significant (P <0.05) elevations in spectral intensities were associated with exponentially growing cell populations, predominantly in S-phase or G2-phase, compared to more quiescent populations predominantly in G1-phase. Increases in the absorption band at 970 cm-1, associated with elevated protein phosphorylation, were observed in vibrational spectra of exponentially growing cell populations compared to those exhibiting a slowing in their growth kinetics. These results seem to suggest that intra-cellular bulk changes, associated with transit through the cell cycle, can be tracked using PTMS.

Key Words: Cell cycle, Flow cytometry, G1-arrest, Lindane, MCF-7 cells, Photothermal microspectroscopy (PTMS)

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