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 3100 to 3700 cm-1. Rather than direct measurements in the near-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.
iiiiFig.1 Block diagram of the Diaphot microscope setup and optical paths.
iiiiThis spectral signature of water, although very distinct, falls well into the near-IR region making direct observation through a typical optical system very difficult. However, by employing well-established Raman spectroscopy techniques, it is possible to shift the measured spectrum to the near-visible region of 800-900 nm, which permits the use of standard microscope objectives and filters while avoiding the background bio-fluorescence of the visible spectral region.
iiiiThe setup which was used to excite and record the spectrum, is based on a Nikon DiaphotTM inverted microscope with standard 4, 10 and 20X objectives and TCC-5.0ICE cooled CCD camera.
¡¡¡¡Fig2. CCD picture through eyepiece showing co-aligned 671nm laser light spot and top white light illumination spot on one mm diameter circle drawn on a piece of ground glass positioned at the objective focus.
¡¡¡¡Fig3. Microphotograph of H1299 cells adhered to the OptiCell membrane, taken through the CCD camera port with a 20X objective. The black circle (1 mm in diameter) is marked on the membrane for subsequent localization of the cells for time-lapse imaging.