Techniques for Obtaining Absorption Spectra on Intact Biological Samples

Absorption spectra can be obtained on a wide range of biological samples with little or no sample preparation by using a high sensitivity, low-noise spectrophotometer with the sample in close juxtaposition with the photocathode. An instrument designed for such measurements is described, and possible applications are discussed. The spectrophotometer is a single-beam recording unit using a double-prism monochromator, 100-watt tungsten source, end-window multiplier-type phototube and an X-Y recorder. The phototube is operated at a constant anode current and a logarithmic voltmeter measures the dynode voltage, providing a photometer which is linear with density change over an opticaldensity range of 8. The noise level for samples of low density is equivalent to an optical-density change of 0.002 with a response time of 1 second for full scale pen travel. Any part or all of the wavelength region from 200 to 1200 mμ may be scanned with a wide choice of scanning speeds. Provision is included for electrical correction of system response to give a flat baseline characteristic for a selected region of the spectrum. This permits measurements at high sensitivity on samples with high scatter loss. Versatile sample mounting arrangements permit measurement of a wide range of materials. Liquids, powders, and homogenates are measured in sample cells of appropriate size. Tissue slices and solid samples are mounted on a metal plate with an aperture in the center for the transmitted light to reach the phototube.