Microwave‐assisted rapid plant sample preparation for transmission electron microscopy

Abstract

SummaryThe preparation of plant leaf material for transmission electron microscopical investigations can be a very timeand labour-consuming task as the reagents infiltrate the samples quite slowly and as usually most steps have to be performed manually. Fixation, buffer washes, dehydration, resin infiltration and polymerization of the resin-infiltrated leaf samples can take several days before the specimen can be cut ultrathin and used for ultrastructural investigations. In this study, we present a microwave-assisted automated sample preparation procedure that reduces preparation time from at least 3 days to about 5 h -with only a few steps that have to be performed manually -until the plant sample can be ultrathin sectioned and observed with the transmission electron microscope. For studying the efficiency of this method we have compared the ultrastructure of different leaf material (Arabidopsis thaliana, Nicotiana tabacum and Picea abies) which was prepared with a conventional, well-established chemical fixation and embedding protocol and a commercially available automated microwave tissue processor. Despite the massive reduction in sample preparation time no negative effects on cutting properties of the blocks, stability of the sections in the electron beam, contrast and ultrastructure of the cells were observed under the transmission electron microscope when samples were prepared with the microwave-assisted protocol. Additionally, no negative effects were detected on the dimensions of fine structures of grana stacks (including membranes, inter-and intrathylakoidal spaces), the nuclear envelope and the plasma membrane as the diameter of these structural components did not differ between leaf samples (of the same species) that were processed with the automated microwave tissue processor or by conventional fixation and embedding at room temperature.

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by TEM

“…Investigations of ultrastructural changes induced by viruses within plants by using transmission electron microscopy (TEM) are often necessary to clearly identify the viral agent. Nevertheless, with conventional methods such investigations can take several days and are not suited for the rapid diagnosis of plant virus diseases [1]. Microwave assisted plant sample preparation can help to drastically reduce sample preparation time for TEM investigations with similar or even better ultrastructural results as observed after sample preparation with conventional methods [1,2].…”

“…Nevertheless, with conventional methods such investigations can take several days and are not suited for the rapid diagnosis of plant virus diseases [1]. Microwave assisted plant sample preparation can help to drastically reduce sample preparation time for TEM investigations with similar or even better ultrastructural results as observed after sample preparation with conventional methods [1,2]. This massive reduction of sample preparation time can be attributed to dielectric heating induced by microwave irradiation which causes a temperature rise inside the whole sample whereas conventional heating starts at the specimen surface.…”

Towards native-state imaging in biological context in the electron microscope

“…Cell monolayers can be fixed in a sub-minute timescale, improving preservation of cytoskeleton and raising the possibility of studying dynamic processes [81]. Plant material can be notoriously difficult to infiltrate due to the thick cell wall, but using microwave technology sample preparation times can be reduced from more than 3 days to just 5 h [105]. However, the use of microwaves in cell biology EM is in its infancy, and further development of protocols and investigation of microwave-induced artifacts is required [102].…”

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

“…With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days 1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation [3][4][5] …”

“…Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations [5][6][7][8] .…”