In the 19th century, the microscope was the principal tool of biologists. The ordinary light microscope is designed to work on specimens that absorb light, but most unstained biological specimens do not absorb light appreciably and almost nothing is visible when such a specimen is viewed with the full aperture of the objective illuminated. However, they do show appreciable differences of refractive index, corresponding to variation in the concentration of solids, and the 19th century microscopists understood how to interpret the image when these were made visible by reducing the illuminating aperture and going slightly out of focus. If the objective is brought closer to the specimen (low focus), regions with high refractive index appear dark (positive image), and it became customary to use this setting, as in Fig. l (upper). As a result, the A bands of striated muscle fibres, with refractive index higher than the I bands, came to be known as the dark bands, although they appear bright with high focus.