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Maple (Acer saccharum) Leaves - High Pressure Freezing and Freeze Substitution for TEM

Application Note for Leica EM HPM100 - Life Science Research

Leaves were immersed in hexadecene and placed under a gentle (0.3 bar) vacuum for 10 minutes to evacuate the internal air spaces. The leaves were then trimmed to fit the carriers and placed in the 200 μm side of a 6 mm Type A specimen carrier. Free space was filled with additional hexadecene after which a 6 mm Type B specimen carrier was placed on top with the flat side down. This assembly was high pressure frozen with the Leica EM HPM100.

The frozen specimens were freeze substituted at –85°C in an Leica EM AFS through a 2 stage process: 3 days in 0.2% glutaraldehyde with 1% tannic acid in acetone, then 2 days in 1% osmium tetroxide with 0.1% uranyl acetate in acetone. After warming to room temperature over 18 hours, they were embedded with Spurr’s resin. Blocks sectioned with a Leica ultramicrotome at 60 nm. Analysis was done with a LaB6 source TEM.

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Large sections of leaves can be easily prepared in 6 mm carriers with the HPM100 if the internal air spaces are filled with hexadecane. The two-step substitution process with tannic acid followed by osmium helps preserve and contrast membranes. The ER, Golgi and tonoplast membranes are particularly visible in this preparation. The chloroplasts were very well preserved with minimal extraction as reflected in the dense stroma especially surrounding the starch grains. This causes the thylakoids to appear in reverse contrast to chemically fixed samples.

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