For infrared microspectroscopy, samples such as cells and biological tissues are often supported on or between infrared-transparent window materials such as barium fluoride (BaF2), potassium bromide (KBr), and zinc selenide (ZnSe). These materials have refractive indices that are frequency dependent, so the focal point that one sees with visible light through the infrared microscope is different than the infrared focal point where the spectrum is taken. In other words, a sample that appears focused with visible light is really out of focus to the infrared light, so that the sample Harea chosen for data collection is really larger than it visibly Happears. The smaller the aperture, the more significant is this focal Hpoint ?error?. Since synchrotron infrared microspectroscopy of Hbiological samples involves apertures of less than 20 ?m, we find that Ha focus shift correction must be applied in order to obtain spectra Hwith high spatial resolution. Using the frequency-dependence of the Hrefractive indices for BaF2, KBr, and ZnSe, we have calculated the Hexpected focus shifts for these materials as a function of infrared Hfrequency. Experimentally, we have taken infrared spectra on these Hmaterials with a range of focus-shifts from the visible focal point Hand we find good agreement with our calculated values. For windows Hwith a 2 mm thickness, we find differences of ~ 20, 25, and 60 ?m Hbetween the visible and infrared focal points of BaF2, KBr, and ZnSe, Hrespectively. Therefore, after visibly focusing a sample that is Hmounted on or between the above-mentioned materials, the sample must Hbe ?defocused? by translating the sample stage so that the sample is Hfocused for collecting the infrared spectrum.
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