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Sensitive and inexpensive dual-wavelength reflection spectrophotometry using interference filters

Analytical Biochemistry
Publication Date
DOI: 10.1016/0003-2697(82)90377-3


Abstract A mechanical/opto-electronic method for performing reflection spectrophotometry with advantages of at least 50% in size, mobility, and price over previous, monochromator-based instruments is described. The method is applied to the continuous and nondestructive measurement of reduction/oxidation shifts of cerebral cytochrome a, a 3 in vivo but is adaptable to follow kinetically, by dual-wavelength principles, absorption changes of intrinsic or extrinsic optically active molecules from other tissues in vivo or from tissues or samples in vitro by transillumination. Interchangeable glass interference filters select sample and reference wavelengths and provide economy, durability, and high luminous output. Long working distance lenses and flexible fiber optic bundle allow independent selection of illumination and detection angles so that (specular) light reflection is minimized to decrease possible artifact and increase signal-to-noise ratios. Time-sharing modulation of sample and reference illumination is combined with feedback control of the reference signal to compensate for nonwavelength-dependent changes in light scattering. The method is stable and sensitive with slow drift of only 0.12%/h and peak-to-peak noise of 0.20%. Examples of large and small signal changes are provided for cytochrome a, a 3 from rat cerebral cortex.

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