The FluoroDichroSpectroPhotometer: Multi-function Instrumentation for Biophysical Spectroscopy in the Ultraviolet, Visible and Near Infrare

Abstract

The decade-wide region of the electromagnetic spectrum from wavelengths of roughly 150 nm in the far ultraviolet (UV) to 1.5 μ in the near infrared (NIR) is particularly important for biophysical spectroscopy because water is relatively transparent, while most other molecules important in bio-molecular systems absorb in some part of this "water window". In addition to spectrophotometers, which measure the absorption spectrum of a sample, the most widely used instruments are fluorometers, which measure the light emitted after absorption of a photon, and dichrometers, which measure the difference between the absorption or emission of two polarizations, either circular or linear. I will describe the rationales for, and the design and construction of a laboratory instrument capable of measuring fluorescence as well as circular dichroism (CD) and magnetic CD (MCD). A second instrument of the same general type was optimized for CD in the far and vacuum UV by the use of UV radiation from a synchrotron light source. Other developments have included the simultaneous measurement of CD and the absorption spectrum of the sample, fluorescence detected CD/MCD, and the measurement of fluorescence polarization anisotropy using the components normally associated with a dichrometer. Since its introduction in 1969, essentially all dichrometers have used photoelastic modulators (PEM) to periodically modulate the polarization of a monochromatic photon beam, which makes possible the detection of very small differences in the absorption of different polarization components of the same wavelength – to about one part in a million. I will also discuss the programming of PEMs as a function of wavelength to achieve their proper operation for the measurement of CD/MCD, linear dichroism (LD), and fluorescence polarization anisotropy, and the limits of certain approximations made in the derivation of the mathematical descriptions of the operations of dichrometers.