Quick and Easy Cryo-Fixation with Aluminium Pockets
Tips & Tricks for a Preservation Method of Delicate Botanical Objects for EDX
- Fig. 1: A: Trap of Utricularia australis (diameter = 4 mm) with prey content. B: Typical X-shaped glands (length: 60 µm) at the inner epidermis of the traps of Utricularia sp.
- Fig. 2: A: cone shaped aluminium pockets. B: sealed aluminium pocket. C: Styrofoam container with LN2 showing plunged traps in Styrofoam cup and precooled pockets ready to be filled.
Carnivorous plants build traps to catch prey organisms like little insects to supplement nutrition. Digestive substances in the traps degrade the animal tissue so that nutrients can be taken up by glands. The delicate, bladder like suction-traps of aquatic Utricularia are a few mm in size (fig. 1A), mostly two cell layers thick and equipped with various sensitive glands (fig. 1B). They are responsible for catching prey by performing hydrostatic underpressure and uptake of degraded prey tissue . The glandular structures of traps of carnivorous plants are particularly interesting in their ion composition. They are physiologically very active and change their ion content in the cytoplasm to produce and secrete digestive enzymes [2,3] or they activate endocytosis mechanisms for uptake of prey derived nutrients . The aim of this project was to measure the elemental composition in glands and epidermal tissue by EDX (Energy dispersive X-ray spectroscopy).
For measuring with EDX, traps of the plants have to be completely dry, like for conventional Scanning Electron Microscopy (SEM). Furthermore, elemental translocation due to the use of fixation chemicals or artefacts by slow cell death must be avoided completely. Therefore, we applied an innovative combination of rapid physical fixation in liquid nitrogen followed by conventional freeze drying.
Materials and Methods
Aquatic species of Utricularia australis R. Br. (Lentibulariaceae) were collected at the natural site in a pond in “Schwarzes Moos” (N 48° 52.345’, E 14° 58.743’, 510 m) in Lower Austria. The main shoots were carefully transported to the laboratory.
Cryo-Fixation and Freeze Drying
Selected living traps with a size of 2-4 mm were microscopically separated in two groups: with and without prey content. Traps (10 – 20) were blotted with filter paper and directly transferred to specially prepared aluminium pockets (fig. 2A). These cone shaped pockets were made by conventional aluminium foil with a thickness of 30 µm by wrapping over 1.5 ml Eppendorf tubes.
The foil was perforated several times with a needle to guarantee rapid gas exchange. The pockets were sealed by folding of the open end (fig. 2B) and then directly placed into the precooled freeze-drying (FD) device (Leica EM MED020).
Alternatively, traps were directly plunged into liquid nitrogen (LN2) in styrofoam cups. The floating traps were poured into a bigger styrofoam container (fig. 2C) to transfer them gently into the aluminium pockets. Finally, the sealed pockets were transferred directly into the FD device without thawing.
The FD device was either cooled manually or with an attached automated Norhof LN2 pumping system for cooling. The process started at high vacuum of 10-5 mbar and a temperature of about -140°C. The sublimation process was performed during the following 24 h until room temperature was established. Duration and temperature had to be adjusted individually for each sample, particularly for sensitive plant material .
The dry traps were mounted on aluminium stubs using double-sticky carbon conductive tape. Some of the traps were gently fractured with a Teflon tip to investigate the inner parts, carbon coated (Leica EM MED020) and analysed in a SEM with attached EDX sensor.
Element analysis of traps and their glands was performed in a SEM Phillips XL20 with Genesis software and in a JSM-JEOL IT300 with TEAM software 4.3 and attached EDX-microanalysis system (EDAX-Ametek). Measurements were obtained at a working distance of 11 mm and at 20 kV acceleration voltage, the SDD-detector (Silicium-Drift-Detector) reached ~20.000 counts per second (cts/sec) at 30% dead time as stable measurement parameters.
Results and Conclusion
Preservation of elemental content without dislocation is a challenge in delicate plant tissues like gland cells and epidermis. Thus, a physical fixation was essential. Other fixation or drying methods would translocate elements to other parts of the sample or even extract them . Cryo-fixation via conventional plunge freezing was impractical due to the size of the propane container and samples were easily lost in the freezing vessel. On the other hand, traps were too big for a high pressure freezer. Consequently, we prepared the traps very efficiently in LN2 followed by FD. Furthermore, we distinguished between two physiologically different groups (traps with and without prey) and therefore prepared a minimum of 10-20 traps per pocket at once. The little pockets met all these prerequisites. A high cooling rate was realized via the good heat conductance of the thin aluminium foil; rapid gas exchange was accomplished by the needle sized holes.
The preservation state of glands and epidermis were superb; tissue and glands only show minor shrinkage. Both preparation methods were comparable showing no effect in EDX measurements. Huge changes in elemental composition were revealed on the cellular level depending on the trap content.
EDX is a semiquantitative method to measure physiologically important elements in e.g. the cell wall or in the cytoplasm. Interestingly, it was only rarely used in plant tissue so far. A few reports contain the uptake of heavy metals in bryophytes , silification in epidermis cells of sugarcane leaf  or the cation exchange capacity in the plant cell wall of Picea abies . Preparation methods for EDX were diverse from simply air drying  or freeze fixation with propane/isopentane  or with Freon-22  followed by FD. Our novel experimental design combines a good preservation for large amounts of small botanical objects without elemental dislocation.
The cryo-fixation of sensitive plant tissue is a challenge, particularly in elemental analyses. Handmade aluminium pockets provided a quick and easy way to handle several mm-sized objects at a time. For investigations with EDX, the preservation was equally fine for plunge-frozen and directly freeze-dried samples.
 Simon Poppinga, Carmen Weisskopf, Anna Sophia Westermeier, Tom Masselter and Thomas Speck: Fastest predators in the plant kingdom: functional morphology and biomechanics of suction traps found in the largest genus of carnivorous plants, AoB PLANTS 8 : plv140 (2016) http://doi.org/10.1093/aobpla/plv140
 B. E. Juniper, R. J. Robins, D. M. Joel: The Carnivorous Plants., Academic Press Limited, 353 pp. (1989)
 Marianne Koller-Peroutka, Wolfram Adlassnig, Thomas Lendl, Kornelija Pranjic and Irene Lichtscheidl: Functional biology of carnivorous plants. In: Jamie A. Texeira da Silva (ed): Floriculture, Ornamental and Plant Biotechnology. Advances and Topical Issues, Vol. V, Global Sciences Books Isleworth, 266-286 (2008)
 Wolfram Adlassnig, Marianne Koller-Peroutka, Sonja Bauer, Edith Koshkin, Thomas Lendl and Irene K. Lichtscheidl: Endocytotic uptake of nutrients in carnivorous plants, The Plant Journal 71, 303-313 (2012) http://doi.org/10.1111/j.1365-313X.2012.04997.x
 Patrick Echlin: Handbook of Sample Preparation for Scanning Electron Microscopy and X-Ray Microanalysis 2009, Springer, 332 p., http://doi.org/10.1007/978-0-387-85731-2
 Hans John Seymour Heslop-Harrison: Energy Dispersive X-Ray Analysis. In: Hans-Ferdinand Linskens and John F. Jackson (eds): Physical Methods in Plant Science. Modern Methods of Plant Analysis 11, 244-277.
 Stefan Sassmann, Marieluise Weidinger, Wolfram Adlassnig, Florian Hofhansl, Barbara Bock and Ingeborg Lang: Zinc and copper uptake in Physcomitrella patens: Limitations and effects on growth and morphology, Environmental an Experimental Botany 118: 12-20 (2015) http://doi.org/10.1016/j.envexpbot.2015.05.003
 William S. Sakai and W.G. Sanford: A developmental study of silification in the abaxial epidermal cells of sugarcane leaf blades using scanning electronmicroscopy and energy dispersive X-ray analysis, American Journal of Botany 71:1315-1322 (1984)
 Eberhard Fritz : Measurement of cation exchange capacity (CEC) of plant cell walls by X-ray microanalysis (EDX) in the transmission electron Microscope , Microscopy and Analysis 13: 233-244 (2007) http://doi.org/10.1017/S1431927607070420
Barbara Hefel1, Marianne Koller-Peroutka1, Marieluise Weidinger1, Stefan Sassmann2, Wolfram Adlassnig1, Ingeborg Lang1
1 University of Vienna, Core Facility Cell Imaging and Ultrastructure Research, Vienna, Austria
2 University of Exeter, College of Life and Environmental Sciences, Biosciences, Exeter, United Kingdom
Dr. Ingeborg Lang
University of Vienna
Core Facility Cell Imaging and Ultrastructure Research