We demonstrate that the surface topography of a sample can be reconstructed from electron backscatter diffraction (EBSD) patterns collected with a commercial EBSD system. This technique combines the location of the maximum background intensity with a correction from Monte Carlo simulations to determine the local surface normals at each point in an EBSD scan. A surface height map is then reconstructed from the local surface normals. In this study, a Ni sample was machined with a femtosecond laser, which causes the formation of a laser-induced periodic surface structure (LIPSS). The topography of the LIPSS was analyzed using atomic force microscopy (AFM) and reconstructions from EBSD patterns collected at 5 and 20 kV. The LIPSS consisted of a combination of low frequency waviness due to curtaining and high frequency ridges. The morphology of the reconstructed low frequency waviness and high frequency ridges matched the AFM data. The reconstruction technique does not require any modification to existing EBSD systems and so can be particularly useful for measuring topography and its evolution during in situ experiments.

A brief description is given of the events surrounding the development of biological X-ray microanalysis during the last 30 years, with particular emphasis on the contribution made by research workers in Cambridge, UK. There then follows a broad review of some applications of biological X-ray microanalysis. A more detailed consideration is given to the main thrust of current procedures and applications that are, for convenience, considered as four different kinds of samples. Thin frozen dried sections which are analyzed at ambient temperatures in a transmission electron microscope (TEM); semithin frozen dried sections which are analyzed at low temperature in a scanning transmission electron microscope (STEM); thick frozen hydrated sections which are analyzed at low temperature in a scanning electron microscope (SEM), and bulk samples which are analyzed at low temperature in the same type of instrument. A brief outline is given of the advantages and disadvantages of performing low-voltage, low-temperature X-ray microanalysis on frozen hydrated bulk biological material. The article concludes with a consideration of alternative approaches to in situ analysis using either high-energy beams or visible and near-visible photons.

Experimental work in a number of research groups over the past five years have demonstrated, unequivocally, that low voltage i.e. 5kV and below, quantitative x-ray microanalysis is a valid analytical technique which can be applied to a wide range of specimens. The advantages of this approach are now generally well known and include improved image contrast, diminished bulk charging of the sample and for bulk specimens, a reduction in the size of the beam-sample interactive volume from which the x-rays are generated, and as a consequence, a considerable improvement in the microanalytical spatial resolution. In addition, low voltages ensure a sensitive surface analysis procedure which provides true topographic contrast and that in the case of light element analysis, quantitation is simplified because the atomic number (Z), absorption (A) and fluorescence (F) effects are minimised. All these advantages are improved further if one uses a field emission gun as the primary electron source.

Water is the most abundant and most important molecule in the hydrosphere, outer lithosphere and the biosphere of our planet. It is also the most abundant and energetically the least expensive building block of living material, forms an integral parts of natural inorganic matrices such as soil and is a constituent of many synthetic organic materials such as paints and polymers. Paradoxically, water does not exist naturally, in the pure state. Water, when converted to the solid state, can provide the perfect matrix in which we may observe the structure and study the in situ chemistry of hydrated samples. We will consider the nature of this solid matrix, and its constituent components in a range of sample, and show how it may be formed, manipulated, examined and analysed. In the short amount of time and space available, one can do little more than highlight the main features of the subject.

The matrix of bio-organic samples is composed primarily of carbon, hydrogen, oxygen and nitrogen. Dispersed within this matrix there is a wide spectrum of other elements whose local concentration can be measured in situ by x-ray spectroscopy. The analysis continues to present a challenge because most samples are highly hydrated, beam sensitive and of low density. In light of these restrictions there is only one sensible approach to the use of an analytical procedure which depends on the intereaction of high energy beam electrons with a labile specimen. Samples must be prepared by the least invasive preparative procedure and irradiated with the minimum amount of energy in order to obtain the maximun amount of chemical information. Low temperature sample preparation and low voltage electron beam instruments are central to these processes. By way of example, a brief description will be given of an on-going biological investigation which makes full use of these processes.

An earlier investigation had shown that it is possible to perform quantitative energy dispersive x-ray microanalysis at primary beam voltages as low as 5kV on bulk biological material maintained in either the fully frozen hydrated or the dried state. This present study extends the earlier work and provides data which shows that the same type of analysis can be achieved at voltages as low as 3kV on organic samples composed primarily of hydrogen, carbon and oxygen which contain varying amounts of bromine, sulphur and chlorine. Such materials are equally beam sensitive as fully frozen hydrated biological samples and range in density from 1.1 to 2.2kg.m3. The samples fall into three main classes. 1. Chlorinated and/or sulphonated organic polymers. 2. A brominated organic sample. 3. Three experimental brominated plastics kindly supplied by Ms. B.S.Chuah, Melville Laboratory for Polymer Synthesis, Cambridge University. The remaining samples were purchased from Aldrich Chemicals at the highest purity available. The atomic weight percent and density was calculated for each of the eight specimens using the Electron Flight Simulation™ software programme.

Low-voltage energy-dispersive X-ray microanalysis has a number of distinct advantages for measuring the concentration of light elements (Z = 11−20) in a largely organic matrix. Between 10 and 4 kV, there is a 50-fold decrease in the size of the incident beam-specimen interaction volume which enables discrete subcel-lular compartments to be analyzed. Experiments with mineral and organic samples of known chemical composition show that two of the most widely used analytical algorithms are capable of providing quantitative data at accelerating voltages as low as 5 kV. This technique is used at 5 kV to analyze frozen hydrated fracture faces of leaves of the tea plant which are known to contain aluminium. Higher levels of aluminium are found in the cell walls and are associated with increased levels of silicon and magnesium. The advantages and disadvantages of the experimental approach are discussed in relation to analysis carried out at higher voltages.

Although high resolution (2nm), low voltage (lkV), SEM of bio-organic materials can now be performed more or less routinely using instruments fitted with a field emission source, virtually no low voltage x-ray microanalysis has been carried out on this type of specimen. Boyes and Nockolds showed that quantitative microanalytical information could be obtained from polished inorganic samples at a spatial resolution of l00nm at 5kV and Johnson et al obtained similar type of data at a spatial resolution of 150nm at 3kV. High spatial resolution (l0nm) microanalysis can be achieved in frozen dried or chemically compromised sections of biological material examined at high voltage in the TEM but frozen hydrated chemically unfixed sections are damaged. The other approach is to use the SEM with frozen hydrated, chemically uncompromised samples, usually at about 10-15kV, in order to obtain sufficient signal from the elements of interest which typically lie in the range Na (Z=l 1) to Ca (Z=20).