Localization of Chlorophyll in Spinach Chloroplast Lamellae by Fluorescence Microscopy

Chlorophyll in green plants is thought to reside in the internal membranes or lamellae of chloroplasts (1). These membranes are observable by electron microscopy and can be roughly grouped, on the basis of size and arrangement , into grana lamellae and stroma lamellae which exist as closed, flattened sacs, or thylakoids (2). Al though the chlorophyll molecule is not yet demonstrable by electron microscopy, it is readily detected by fluorescence microscopy (3-5). The deep red fluorescence of chlorophyll, observed by Sir David Brewster in 1833, was used to advantage in 1924 by Francis E. Lloyd (6) who found the chloroplasts of higher plants to be the source of the fluorescence, and again in 1962 by Spencer and Wildman (7) who further restricted the red fluorescence to the chloroplast grana. The technique described here permits observation of the same specimen by fluorescence and electron microscopy, yielding precise information on the distribution of chlorophyll in the membranes through a direct comparison of ultrastructure and fluorescence.