INFRARED MICROSPECTROSCOPY IN BIOLOGICAL RESEARCH

At present, infrared microspectrophotometry can be defined as the study of infrared absorption intensities for samples smaller than the mechanical slit of the spectrometer. This definition arises from the fact that the smallest sample that can be studied by a spectrophotometer without auxiliary optics is that which just covers the entrance or exit slit of the monochromator. Ordinarily the slit' will be no shorter than 10 mm., and may vary from 0.01 mm. to 1 mm. in width, depending on the particular region of the spectrum under consideration. If an appropriate sample thickness is about 0.025 mm., then, with no occlusion of the radiation beam incident on the slit, the minimum sample volume would be 2.5 x ~ m . ~ , or a quarter of a milligram for substances of unit density. This is, therefore, the maximum sample size for microspectrophotometry, or the minimum for macrospectrophotometry, according to the present definition. For various reasons this minimum is not reached in actual practice, but the limit may be set in this arbitrary way for the present discussion. The most elementary microilluminator consists2 of a pair of infrared-transmitting lenses, one producing a reduced image of the source in the sampling space, and the other restoring the radiation to its former path with an image of the source on the entrance slit. In the original work with this device, the reduction in the minimum sample size was about 3 times, and the final minimum sample size was about 0.75 X 6 mm., since the slit was rather larger than 1 X 10 mm. Application of the arrangement to any spectrometer may reduce the sample size by a similar factor, and for the case under consideration would give a minimum of 0.33 X 3.3 mm., or 2.5 X lop6 cm.3 One drawback of such a system arises from the errors of the refracting elements involved, especially the chromatic aberrations. The wave-length range usually investigated covers the wide interval from 2 . 5 ~ to 1 5 ~ , a factor of 6 i n wave length compared to the factor of 2 at most required to cover the visible spectrum. Thus, chromatic aberration may be considerable, especially since not many transparent materials are available for the construction of compound lenses for the infrared. The fairly large aperture required of such a system also causes the aberrations other than chromatic to be considerable for simple lenses. The solution to many of the optical problemsconnected with microilluminator design has come with the development of totally reflecting mic ro -~p t i c s .~ -~ I n fact, the first report of infrared microspectroscopy as such6 came shortly after the description of the Schwarzschild-type totally reflecting microscope objectives For smaller samples a microilluminator of some sort must be used.