Microscopy sample coating

For scanning electron microscopy of electrically insulating materials, the surface of the specimen may be electrically isolated when bombarded with electrons. This leads to charge buildup on the specimens that makes imaging or other analysis difficult. To address this issue, special sample coating steps are often required and have been discussed in Section 9.2.3. 

Scanning Electron Microscopy. Samples of unweathered and weathered untreated and formaldehyde-treated wools were mounted or specimen stubbs using conducting silver paint and coated with two thin layers of silver. Scanning electron micrographs of the samples were prepared and examined for changes In the fiber surface (Fig. 1). 

The thickness of the deposited layers was measured by SEM microscopy and profilometry [39]. For both methods silicon platelets were used as substrates. For SEM microscopy, the coated Si platelets were broken and such a cross-section can be obtained. Fig. 14A shows the SEM cross-section of a broken Si sample coated with carbon for 30 min. The silicon substrate is represented by the bright white part on the left of the image, while the gray shadow part represents photo-deposited carbon layer. The dependence of the carbon layer thickness on deposition time measured by SEM and by profilometry is presented in Fig. 14B. Data of both methods show a nearly linear increase of the thickness with the deposition time. 

More recently, the introduction of environmental scanning microscopy provided an important alternative which avoided drying artifacts, particularly in the foulant layer. Environmental scanning electron microscopy (ESEM) can operate in either high-vacuum mode (dried sample) or low-vacuum mode (hydrated conditions). Fully saturated water vapour conditions as high as 7kPa (SOTorr) and the elimination of any sample coating allow sample analysis without dehydration [42, 49]. 

This technique can be applied to samples prepared for study by scanning electron microscopy (SEM). When subject to impact by electrons, atoms emit characteristic X-ray line spectra, which are almost completely independent of the physical or chemical state of the specimen (Reed, 1973). To analyse samples, they are prepared as required for SEM, that is they are mounted on an appropriate holder, sputter coated to provide an electrically conductive surface, generally using gold, and then examined under high vacuum. The electron beam is focussed to impinge upon a selected spot on the surface of the specimen and the resulting X-ray spectrum is analysed. 

Indeed, a bDNA assay for diagnosis of African trypanosomiasis was developed and compared with buffy coat microscopy for detection of T brucei in human blood samples (Harris etal., 1996). Two repetitive DNA sequences found only in the T. brucei complex, a 177-bp satellite repeat and the ribosomal mobile element, were selected as targets in the bDNA assay. The assay used the standard bDNA components capture probes, target probes, amplifier molecules, and alkaline phosphatase-labeled probes. Various blood fractions and sample preparation methods were examined. Ultimately, buffy coat samples resulted in the highest sensitivity. Although typanosomes do not infect leukocytes, they cosediment with them. 

The limit of detection of the assay was estimated to be 200 parasites/ml of blood. The detection limit is well within the range of sensitivity needed to diagnosis trypanosomiasis, as the parasitemia may vary from 5000 to 1,500,000 parasites/ml (Vickerman, 1974). The bDNA assay was compared with buffy coat microscopy for detection of T brucei in 56 blood samples (36 buffy coat positive and 20 buffy coat negative by microscopy). There was complete concordance between the results of the two tests in terms of identifying specimens as positive of negative. However, the numbers of parasites observed by microscopy were lower overall than those calculated with the bDNA assay. The authors suggested that the excess of leukocytes in the buffy coat could interfere with the microscopic detection of typanosomes, resulting in lower apparent parasitemia than the true value. 

Valuable results with Tof-SIMS imaging have been obtained e.g., with paper samples. The distribution of papermaking chemicals on the surface of coated and uncoated papers is very important for its further treatment, such as printing. Figure 16 [70] is an example of use of the technique for chemical microscopy analysis of paper surfaces. 

Procedure Germination conditions were 25 1°C under continuous fluorescent light of 25 mE m 2 sec-1. Seed germination was monitored by observing the seeds directly in the Petri dishes with a stereomicroscope. They were considered germinated, when the radicle had protruded through the seed coat. Seeds sampled at different times after the beginning of imbibition were used for microscopy studies. 

Samples for infra-red absorption measurement were introduced between two rubbed nylon coated calcium fluoride substrates spaced 10 pm apart. To insure proper parallel alignment, samples were cooled at 0.02° C/minute from the isotropic to the Smectic C phase. The alignment was then checked using polarizing microscopy. Polarized IR spectra (32 scans per spectrum) were obtained using an FTIR spectrometer (IFS-66 Bruker, Pillerica, MA) equipped with a wire grid polarizer at a resolution of 2 cm"1. 

Figure 4.11 Scanning electron microscopy images of bare and coated magnesium chips. Clear morphological differences are seen between the coated and uncoated samples. (Reproduced from ref. 13, with permission.)...

The basic approach with the axoneme-based analysis is to combine tubulin and axonema fractions and then to quench the reaction with glutaraldehyde. Samples of sufficient axoneme concentration are then added directly onto Formvar-coated sample grids for staining and electron microscopy. In some cases where the axoneme count is too low, samples may be sedimented onto grids by the method of Gould and Borisy (1977). With the methodology perfected by Borisy and Bergen (1982) samples can be taken as frequently as every 20 seconds, and the... 

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