In: Phyton 58, Fasc. 2 (2018): S. 103-108 DOI: 10.12905/0380.phyton58(2)-2018-0103 Published online on 16th November 2018

Published online on 16th November 2018

Rapid sample preparation of Nicotiana tabacum leaves for scanning and transmission electron microscopy

Bernd Zechmann & Maria Müller

Key words: Beam deceleration, microwave irradiation, Nicotiana tabacum, SEM, TEM.

Summary
Zechmann B. & Müller M. 2018. Rapid sample preparation of Nicotiana tabacum leaves for scanning and transmis­sion electron microscopy. – Phyton (Horn, Austria) 58 (2): 103–108, with 3 figures and 1 table.

Conventional preparation of biological samples for scanning and transmission electron microscopy (SEM, TEM) takes several days and is very labor intensive. The main reasons are that substances used during preparation infiltrate the specimen slowly and that many steps are performed manually. Microwave irradiation combined with automated sample preparation can drastically reduce labor and sample preparation time by automatically exchanging solutions used during sample preparation and by increasing the internal temperature of the specimen, which accelerates diffusion and reactiv­ity of reagents. The aim of this study was to develop microwave-assisted sample preparation of Nicotiana tabacum leaves for both SEM and TEM investigations.

Samples prepared conventionally at room temperature and with microwave irradiation showed similar ultrastruc­ture when investigated with SEM and TEM. The surface structures of leaves including stomata cells and trichomes were well preserved when investigated in the SEM and no differences were observed between samples prepared convention­ally and with the help of microwave irradiation. Additionally, no differences regarding sample stability and behavior under the beam were observed. Similar results were found for TEM investigations where the ultrastructure of leaf cells did not differ between cells prepared with the support of microwave irradiation and cells prepared conventionally. Ad­ditionally, microwave irradiation did not negatively affect the cutting properties of the blocks, stability of sections in the electron beam, and contrast.

It can be concluded that microwave-assisted, automated sample preparation is well suited for the rapid preparation of plant leaves for SEM and TEM investigations. The main advantages of this procedure over conventional sample prepa­ration at room temperature are (i) the massive reduction of sample preparation time, (ii) faster reproducibility of results, and (iii) reduction of the amount of manual labor necessary for SEM and TEM sample preparation.