Abstract
This paper reports the first use of a novel completely optically based photothermal method (O-PTIR) for obtaining infrared spectra of both fixed and living cells using a quantum cascade laser (QCL) and optical parametric oscillator (OPO) laser as excitation sources, thus enabling all biologically relevant vibrations to be analyzed at submicron spatial resolution. In addition, infrared data acquisition is combined with concomitant Raman spectra from exactly the same excitation location, meaning the full vibrational profile of the cell can be obtained. The pancreatic cancer cell line MIA PaCa-2 and the breast cancer cell line MDA-MB-231 are used as model cells to demonstrate the capabilities of the new instrumentation. These combined modalities can be used to analyze subcellular structures in both fixed and, more importantly, live cells under aqueous conditions. We show that the protein secondary structure and lipid-rich bodies can be identified on the submicron scale.
Highlights
This paper reports the first use of a novel completely optically based photothermal method (O-PTIR) for obtaining infrared spectra of both fixed and living cells using a quantum cascade laser (QCL) and optical parametric oscillator (OPO) laser as excitation sources, enabling all biologically relevant vibrations to be analyzed at submicron spatial resolution
We present data obtained using both the QCL and a tunable optical parametric oscillator (OPO) laser that for the first time enables the higher wavenumber region (3600− 2700 cm−1) containing the O−H, N−H, and C−H stretching vibrations to be accessed by O-PTIR
A recent study on polystyrene beads has shown that the O-PTIR signal is broadly related to the amount of material being sampled,[67] but for a complex biological cell, it is possible that deviations from the Beer−Lambert law could occur
Summary
This paper reports the first use of a novel completely optically based photothermal method (O-PTIR) for obtaining infrared spectra of both fixed and living cells using a quantum cascade laser (QCL) and optical parametric oscillator (OPO) laser as excitation sources, enabling all biologically relevant vibrations to be analyzed at submicron spatial resolution. The pancreatic cancer cell line MIA PaCa-2 and the breast cancer cell line MDA-MB-231 are used as model cells to demonstrate the capabilities of the new instrumentation These combined modalities can be used to analyze subcellular structures in both fixed and, more importantly, live cells under aqueous conditions. The vast majority of infrared single-cell analyses to date have reported the use of chemically or cryogenically fixed, dried cells These preservation methods allow the biochemistry to be captured at a specific moment in time and have enabled the elucidation of significant spatial resolution = 0.61 λ/NA (1)
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