Biology Reference
In-Depth Information
FIGURE 15.3
Diagram of a microscope slide before mounting the top cover glass to demonstrate the
position of the spacers. Gaps in the vacuum grease allow for the displacement of some liquid
mounting media while positioning the cover glass.
damaging the seedling and altering its physiology. The distance between the slide
and the coverslip is an important factor to consider and the width of the plant organ
being observed dictates this distance. Cover glass spacers can be used to avoid squ-
ishing the plant while gently mounting the cover glass ( Fig. 15.3 ). Spacers are made
by gluing together multiple coverslips to achieve the proper distance between the
slide and cover glass. We have found that the ideal spacer for 4- to 5-day-old
light-grown Col-0 seedlings is two #2 coverslips glued together, for etiolated hypo-
cotyls is a single #1 cover glass, and for root tips is a #0 slip. The coverslip is secured
to the slide with vacuum grease to prevent evaporation of the mounting media during
imaging, to maintain the space between the slide and cover glass, and to hold the
cover glass in place when using an inverted microscope.
Before being moved to slides, seedlings are typically grown on sterile half-
strength Murishage and Skoog (0.5
MS) agar plates under the appropriate environ-
mental conditions as determined by the experimental setup. To minimize osmotic
disruptions, seedlings should be mounted in 0.5
MS liquid media on the slide.
Tap water should never be used as it can contain herbicides and fertilizers from
agricultural runoff that vary seasonally. Seedlings should be allowed to acclimate
on slides for at least 15 min before imaging. The media do not need to be exchanged
during experiments lasting less than 2 h, and new media should be perfused onto the
slide during longer-term experiments (see Buschmann, Sambade, Pesquet, Calder, &
Lloyd, 2010; Shaw, 2006 for more information about perfusion chambers).
15.1.3 Imaging
Seedlings are thick specimens that produce many autofluorescent compounds that
contribute extensive out-of-focus fluorescent information that substantially degrades
image quality. Many plant biologists use confocal microscopes to capture thin
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