Fluorescence shell: a novel view of sclereid morphology with the Confocal Laser Scanning Microscope

Confocal Laser Scanning Microscopy (CLSM) was used to observe sclereids from stems of Avicennia germinans and from fruits of two species of pear (Pyrus calleryana "Bradford" and P. communis "Red Bartlett"). The images obtained from thick (25 to 100 microm) free-hand sections were, in certain respects, far superior to those obtained by other, more invasive and time-consuming microscopic techniques upon which previous reports of sclereid morphology were based. The cell wall surfaces, including the "internal" surfaces of the branched pit canals and cell lumens, were much accentuated with the techniques we describe, resulting in a "fluorescence shell" image, meaning the cell wall did not stain all the way through but instead only at the inner and outer wall surfaces, including the edges of ramiform pits. By controlling the time of staining with 1% aqueous Safranin O, or by changing the number of optical sections used in extended focus images, it was possible to get either a conventional view of the cell wall structure or a unique, three-dimensional view of the elaborate cell interconnections. Similar fluorescence shell images of sclereids were also obtained using a periodic-Schiff (PAS) staining system, but the stain was not as specific to sclereid cell walls as was the Safranin O stain. Particularly with the use of a narrow range band pass emission filter of 505-530 nm, the Safranin O staining may be more specific to lignin than reported in the literature.