Micro-analytical Methods

Methods

Examples

SEM image of a germinated fungal spore on a leaf surface EDX spectrum of a precipitation in the soil water Elemental distribution of bean ore

Equipment

Low-Temperature Scanning Electron Microscopy (LTSEM)

General

In the SEM, topographic information is obtained by detecting the emission of secondary electrons. However, this is only a small part of the information to be gained from the specimen due to its interaction with the primary electron beam. This facility's SEM also has the capability of detecting backscattered electrons (BSE) and electro-magnetic radiation in the form of x-rays and normal light near the visible spectrum, i.e. cathodo-luminescence (CL).

LTSEM allows preparation and observation of samples with high water content without altering their moisture content. The technique provides information about the surface structure and allows specimens to be fractured to reveal internal features of tissues. The technique does not affect the degree of hydration in specimens; it can be used to localize water in tissues to determine their degree of hydration. Low-temperature methods have several advantages over conventional methods: immobilization of physiological processes and of elemental translocation within milliseconds and the maintenance of turgescence of cells.

Projects with scientific findings

• In ectomycorrhizal research, frozen-hydrated and frozen, partially freeze-dried samples are investigated with LTSEM. It is possible to prepare surface views of Hartig net only with LTSEM, in combination with complementary or simple low-temperature fracturing. Freezing in liquid nitrogen (LN2) enabled a study of the ontogeny of ectomycorrhizal formation and a study of the host-fungus interface. In order to obtain higher cooling rates, propane was used as a secondary cryogen.
  
• LTSEM combined with image analysis provided accurate and fast measurements of the stomatal aperture in birch leaves. Regression analysis showed a close relationship between the stomatal width and the pore area. The stomatal pores were significantly narrowed by nutrient supply. In addition to the strong influence of nutrient supply, ozone had also narrowed the stomatal pores.
  
• LTSEM of low-temperature fractured lichen thalli enables the localization of free and perturbed water in the symbionts. Collapsed and cavitated drought stressed cells can be easily investigated with this method.
  
• The early development of transplanted vegetative diaspores of epiphytic lichens in an endangered population can be studied by LTSEM.
  

X-ray microanalysis

General

• the relationship of elemental composition to a particular morphological feature
  
• the general distribution of ions and elements in normal plant and animal cells
  
• the localization of ions of known physiological function
  

The SEM has been fitted with a Pioneer energy dispersive x-ray detector (EDX, Noran Instruments). This EDX system is capable of quantitative x-ray spectral analysis as well as qualitative spectral identification. The EDX system is also capable of digitally controlling the SEM for x-ray elemental mapping, and software has been purchased such that areas can be mapped by rastering the specimen stage.

Characteristic x-rays are the emission of electro-magnetic radiation from the specimen due to its interaction with the primary electron beam. The term x-ray is a reference to this radiation's short wavelength and high energy. Not all x-rays are characteristic of the elements present in the specimen, many belong to the background continuum, or "Bremstrahlung" which is the primary contributor to this technique's insensitivity to low elemental concentrations. Characteristic x-rays are generated as a result of a primary electron ionizing an inner electron shell of an atom. When the excited atom relaxes, i.e., an outer electron filling the inner shell, radiation is released which is characteristic of the atom's electron shells' energy levels.

Projects with scientific findings

• EDX of frozen-hydrated plant leaves confirmed the importance of vacuolar K increase with guard cell swelling and thus with active stomatal widening, at constant K values in the subsidiary cells.
  
• With the aid of freeze-fracturing of Picea abies-Hebeloma crustuliniforme ectomycorrhiza for LTSEM, apoplastic tracers such as lanthanum have been detected with EDX in exoplasmic fracture faces of fungal and cortical cells but not in protoplasmic fracture faces supporting the apoplastic pathway.
  
• X-ray microanalysis of cesium in frozen-hydrated, freeze-fractured roots and needles of ectomycorrhizal plants. The technique provided the localization of stable Cs in the various plant tissues and allowed comparisons between cortex and stele, inner and outer cortical cells, or apoplasms and symplasms.
  
• EDX to localize aluminum in the plant tissues. Highest values of Al were found in apoplasts of epidermal and cortical cells of fine roots and to a much lesser extent in cells of stele.