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Scanning Electron Microscopy (SEM) of Plant Tissues

Kirsten Bomblies, Vipula Shukla and Charles Graham

Cold Spring Harb Protoc 2008; doi: 10.1101/pdb.prot4933

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Protocol

Scanning Electron Microscopy (SEM) of Plant Tissues

Kirsten Bomblies, Vipula Shukla, and Charles Graham

This protocol was adapted from “How to Analyze a Mutant Phenotypically,” Chapter 4, in
Arabidopsis (eds. Weigel and Glazebrook). Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY, USA, 2002.

INTRODUCTION
Scanning electron microscopy (SEM) is used to produce detailed images of surface structures. This arti-
cle details the steps required to prepare a sample for SEM. In brief, the sample is fixed, dehydrated,
subjected to critical-point drying, mounted, and coated with an electrically conducting material such
as gold or palladium.

MATERIALS
CAUTIONS AND RECIPES: Please see Appendices for appropriate handling of materials marked with <!>, and
recipes for reagents marked with <R>.

Reagents
Ethanol (100%, stored over a molecular sieve)
Ethanol series (30%, 50%, 70%, and 95%)
<R>FAA fixative for SEM
<!>Osmium tetroxide (1% OsO4 [v/v] in 25 mM sodium phosphate buffer at pH 7.2)
Store the solution wrapped in aluminum foil at 4°C, where it will keep for at least 1 mo. Discard the solution
when it turns purple.
Sodium phosphate (25 mM at pH 7.2)
Tissue samples

Equipment
Boxes for storing coated samples
<!>CO2 tank
<!>Colloidal silver
Container (for storing samples until ready for mounting)
Critical-point-drying apparatus
Forceps (fine)
Microdissection tools
Pipette
Scanning electron microscope
Scintillation vials (glass)
Specimen baskets
Sputter coater

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Sticky tabs or double-sided mounting tape

Stubs for mounting samples
Vacuum line
Wire mesh
Wrench (Allen)

METHOD
Tissue Preparation

1. After harvesting the tissue of interest, place it directly into FAA fixative for SEM in glass scintillation
vials.
2. Briefly apply a vacuum to remove air bubbles from the tissue; release the vacuum slowly.

3. Incubate the tissue in fixative for 12-24 h at 4°C.

4. Replace the fixative with just enough 1% OsO4 (v/v) to cover the samples.

5. Incubate the tissue overnight or longer (up to a few days) at 4°C. The tissue should turn black
(some tissue types will remain gray).
6. Rinse the tissue samples three times in 25 mM sodium phosphate (pH 7.2).

7. Dehydrate the tissue with an ethanol series (30%, 50%, 70%, 95%, and twice in 100% ethanol)
for at least 15-30 min each at room temperature.
8. Store the tissue in 100% ethanol until ready to proceed with critical-point drying (at least
overnight).

Critical-Point Drying

9. Place the samples into specimen baskets. Place the baskets into a boat (part of the specimen holder
assembly of the critical-point-drying apparatus) filled with 100% ethanol. Cover the baskets with
wire mesh to hold them in place. Incubate the samples overnight.
10. Use a pipette to remove most of the ethanol from the boat, and immediately transfer the boat into
the critical-point-drying apparatus. Tighten the seal with an Allen wrench.
11. Open the first valve on the CO2 tank and then the inlet valve of the drying apparatus.

12. Relieve the back pressure by opening the vent until the CO2 fills the chamber.

13. Close the inlet valve and open the drain at the bottom of the dryer to remove the ethanol.

14. Repeat Steps 12 and 13.

15. Close the vent and let the CO2 sit in the chamber for at least 10 min.

16. Change the CO2 six times as follows:

i. Open the drain and allow the CO2 level to drop all the way.
ii. Open the inlet valve and relieve back pressure by opening the vent, allowing the CO2 level
to rise to the top of the chamber again.
iii. Close the drain, inlet, and vent and allow the CO2 to sit for at least 10 min.

17. To reach the critical point, drain the CO2 halfway. A meniscus should be clearly visible. Close all of
the valves, including the main tank valve.
18. Turn on the hot H2O inlet to the drying apparatus, watching the meniscus and gauges as the tem-
perature and pressure approach the critical point (1150 psi, 32°C). Once the meniscus disappears,
immediately turn off the hot H2O.

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19. Leave samples at the critical point for 5 min.

20. Slowly (over at least 5 min) bleed the system through the vent in a steady flow. Open the vent fur-
ther as the pressure drops.
21. Store the samples in a dry container until ready for mounting.

Mounting and Coating

22. Mount the samples on stubs using sticky tabs (or double-sided mounting tape) and colloidal sil-
ver. For small plant samples, place a droplet of colloid (not too fluid) on the surface of the stub,
and then stick the sample into colloid with fine forceps. Gently support the sample until the col-
loid begins to set. At this point, allow the colloid to dry for ~1 h if further dissection of sample
is desired.
23. Dissect the samples as needed with microdissection tools.
Homemade tools can be fashioned by drawing out glass pipettes or fixing eyelashes to toothpicks.

24. Coat the samples with gold and palladium according to the instructions for the sputter coater.

25. Examine the coated samples using SEM or store them in boxes (to prevent damage and dust accu-
mulation) until ready to view.
If further dissection is required after viewing, the samples must be recoated.

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