O-PTIR measurements of cells with a water dipping objective.

Infrared (IR) spectroscopy of biological samples in water environments remains challenging due to water's strong IR absorption in the mid-infrared region and limitations in spatial resolution. Recently, a new optical photothermal infrared (O-PTIR) spectroscopy, which utilizes a laser working in the visible spectrum to probe the sample for IR absorption, has shown great capabilities in the measurements of biological samples, like cells, at sub-micron resolutions. Here, we report the first implementation of a refractive water-dipping objective in a commercially available O-PTIR system. The measurements were carried out in a counter-propagating configuration, with IR illumination being done from below the sample through a calcium fluorite substrate, and visible light being directed through the 60× water-dipping objective from the top. Such a configuration allowed the direct acquisition of IR spectra from fixed cells in water. We present a detailed protocol for system calibration, background acquisition, and spectral measurement, further verified by measurements of U-2 OS cells and avian erythrocytes. Spectra acquired in water exhibit high signal-to-noise ratios and preserve biochemical specificity, with good differentiation between nuclear and cytoplasmic regions of cells. The spectra obtained in the water match the results of experiments done in the air in other configurations of the O-PTIR microscope. Compared to the usually used "sandwich" configurations, measured with reflective Cassegrain air objectives, the water dipping objective offers much higher image quality and easier sample handling. The approach facilitates routine, high-resolution, label-free measurements of cells in a more physiologically relevant environment, with potential applications in experiments with live cells.