Raman Spectrometer System for Remote Measurement of Cellular Temperature on a Microscopic Scale

A simple setup is demonstrated for remote temperature monitoring of water, water-based media, and cells on a microscopic scale. The technique relies on recording changes in the shape of a stretching band of the hydroxyl group in liquid water at 3,100–3,700 cm . Rather than direct measurements in the near-infrared (IR), a simple Raman spectrometer setup is realized. The measured Raman shifts are observed at near optical wavelengths using an inverted microscope with standard objectives in contrast to costly near-IR elements. This allows for simultaneous visible inspection through the same optical path. An inexpensive 671-nm diode pump laser (<100 mW), standard dichroic and lowpass filters, and a commercial 600–1,000 nm spectrometer complete the instrument. Temperature changes of 1 C are readily distinguished over a range consistent with cellular processes (25–45 C) using integration times below 10 s. Greatly improved sensitivity was obtained by an automated two-peak fitting procedure. When combined with an optical camera, the instrument can be used to monitor changes in cell behavior as a function of temperature without the need for invasive probing. The instrument is very simple to realize, inexpensive compared with traditional Raman spectrometers and IR microscopes, and applicable to a wide range of problems in microthermometry of biological systems. In a first application of its kind, the instrument was used to successfully determine the temperature rise of a cluster of H1299 derived human lung cells adhered to polystyrene and immersed in phosphate-buffered saline (PBS) under exposure of RF millimeter wave radiation (60 GHz, 1.3, 2.6, and 5.2 mW/mm). Raman Spectroscopic Measurements The remote measurement of temperature on a microscopic scale has been enabled by the advent of the IR microscope [1]. However, this instrument is extremely costly (e.g., Thermo Scientific Nicolet iN10 IR microscope is currently priced at US$75,000–100,000), and when used for biological measurements, where specimens are often sandwiched between plastic or glass plates or immersed in liquids, the accuracy suffers from differences in emissivity in the various layers that must be penetrated before reaching the sample. It is also difficult to discern the temperature emanating specifically from a cellular or tissue layer immersed in culture medium since water is a very strong IR absorber. Consequently, only the surface temperature has been accurately determined by this type of system. A novel method for remote probing the temperature of liquid water on a Digital Object Identifier 10.1109/MEMB.2009.935468 BY VICTOR PIKOV AND PETER H. SIEGEL