Confocal microscopy and multi-photon fluorescent microscopy provide unsuppressed capabilities of optical sectioning. Therefore, volumetric data sets can be obtained by sequentially scanning successive levels in a specimen. The quality of this sequential imaging method requires a stable specimen over the period of data acquisition. We have re-designed our previous specimen chamber (Cheng et al., Multi-dimensional Microscopy. Springer, 339-380, 1993) and fabricated a new chamber which allows significantly better specimen holding capabilities to handle maize pollen grains and small embryos.

The specimen chamber consists of two aluminum blocks (upper cover and holder body; Figure 1). The opening of the cover plate is tapered to allow for easy exchange of different objective lenses using a revolving nosepiece. The opening of the holder body allows for the possibility of viewing the specimen in conventional wide-field mode and also enables the scanning of samples in transmitting mode. Specimens are held between a cover glass and a flexible plastic film (e.g. Mylar or acetic cellulose film). An O-ring is installed to provide a clamping force for the cover slip and supporting film and also prevents mounting medium leakage. The flexible plastic film conforms to the curvature of the specimen, resulting in a firm holding force on the specimen.

The holding force experienced by the specimen depends on the stiffness of the supporting film. Therefore, it is important to select a suitable supporting film for a specific sample. Figure 2 and Figure 3 show the effect of two different supporting films for holding maize anthers. Figure 2 is an x-z scan of an anther held by a sheet of acetic cellulose (dotted line, 70µm in thickness) and Figure 3 shows a x-z scan of an anther held by a sheet of Mylar film (dotted line, 10µm, SyranWrap®). The solid line on both figures indicates the surface of cover glass. Note the acetic cellulose film was too stiff and caused a noticeable tissue deformation. The specimen chamber described above is suitable for holding small maize tissue for confocal and multiphoton fluorescent microscopy.
 

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