Microscopy sample preparation

Key words Confocal microscopy, Sample preparation, DAPI, FRAP, FRET, 2-Photon imaging... 

Overview. Microscopy Techniques Light Microscopy Sample Preparation for Light Microscopy X-Ray Microscopy. Sample Handling Comminution of Samples. Sampling Theory Practice. Sulfur. X-Ray Fluorescence and Emission Energy Dispersive X-Ray Fluorescence. 

In this chapter, online size classification techniques for both diameter and length of gas phase nanofibers are reviewed. In addition, unipolar diffusion charging theories for fibers are discussed. Based on the findings of this review, an approach to online size characterization of carbon nanotubes (and nanofibers) is developed and experimental results are presented. Because of the importance of TEM analysis for size measurement confirmation and for structure and compositional analysis, a brief discussion of microscopy sample preparation and analysis is also presented. 

Electron Beam Techniques. One of the most powerful tools in VLSI technology is the scanning electron microscope (sem) (see Microscopy). A sem is typically used in three modes secondary electron detection, back-scattered electron detection, and x-ray fluorescence (xrf). AH three techniques can be used for nondestmctive analysis of a VLSI wafer, where the sample does not have to be destroyed for sample preparation or by analysis, if the sem is equipped to accept large wafer-sized samples and the electron beam is used at low (ca 1 keV) energy to preserve the functional integrity of the circuitry. Samples that do not diffuse the charge produced by the electron beam, such as insulators, require special sample preparation. 

Also a good interface resolution is obtained with transmission electron microscopy (TEM), where, however, a dedicated sample preparation and treatment are necessary to achieve nanometer resolution and suitable contrast [64]. Thus the... 

Scanning electron microscopy and replication techniques provide information concerning the outer surfaces of the sample only. Accurate electron microprobe analyses require smooth surfaces. To use these techniques profitably, it is therefore necessary to incorporate these requirements into the experimental design, since the interfaces of interest are often below the external particle boundary. To investigate the zones of interest, two general approaches to sample preparation have been used. 

The authors wish to acknowledge the work of Paul McCarthy in scanning electron microscopy, Michael Saculla in x-ray radiography, and Steven Buckley and Chuck Chen in sample preparation and modulus measurement. This work was performed under the auspices of the U.S. Department of Energy by the Lawrence Livermore National Laboratory under Contract No. W-7405-ENG-48. 

MgO-supported model Mo—Pd catalysts have been prepared from the bimetallic cluster [Mo2Pd2 /z3-CO)2(/r-CO)4(PPh3)2() -C2H )2 (Fig. 70) and monometallic precursors. Each supported sample was treated in H2 at various temperatures to form metallic palladium, and characterized by chemisorption of H2, CO, and O2, transmission electron microscopy, TPD of adsorbed CO, and EXAFS. The data showed that the presence of molybdenum in the bimetallic precursor helped to maintain the palladium in a highly dispersed form. In contrast, the sample prepared from the monometallie precursors was characterized by larger palladium particles and by weaker Mo—Pd interactions. ... 

Scattering experiments can be performed to help determine the size and shape of the vesicles without the need for the extensive sample preparation required for electron microscopy and AFM. Dynamic (DLS) and static light scattering (SLS) are widely used to determine the size and possible shape of vesicle systems [40,42,48,49,51,... 

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. 

Sample Preparation for Transmission Electron Microscopy Analysis... 

Most of the sample preparations and contact angle measurements were made by A. D. Karnas. K. W. Littlepage and G. G. Engerholm did the ESCA spectroscopy. J. Burns did the electron microscopy on the block copolymers. I thank D. F. Hager and G. B. Butler for helpful discussions. 

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. 

Figure 8.3 Positive ion LD TOF mass spectrum of blood from a P. vivax infected human patient (only asexual parasites have been observed by microscopy estimated parasitemia approximately 72 parasites/pl). Protocol C is used for sample preparation estimated number of parasites deposited per well is approximately 90. A commercial TOF system is used laser wavelength 337 nm. All one hundred single laser shot spectra, obtained from hnear scanning of an individual well, are averaged (no data smoothing). The characteristic fingerprint ions of detected heme are denoted.

In the first LDMS-based detection of malaria in human subjects (unpublished), lOOpl P. falciparum or P. v/vax-infected blood samples, grouped into three different parasitemia ranges—low (10-150 parasites/pl), mid (2 x 103 parasites/pl), and high (25 x 103-60 x 103 parasites/pl)—have been examined using both sample preparation protocols. Parasitemia levels in these samples were previously determined independently for each sample by optical microscopy examination of blood smears. The LDMS data clearly indicate that... 

The goal of our investigations was to characterise the morphology of the sample, and to determine the size and location of the PTFE and silicone oil phases by different methods [46,47], For phase characterization using Raman microscopy, no special sample preparation was necessary. For FTIR imaging, microtomed sections (5 pm in thickness) had to be prepared by cutting the sample with a diamond knife at — 80°C ("cryo-microtomy") to prevent smearing and to obtain flat surfaces. 

Infrared spectroscopy/microscopy certainly is the primary method of choice when organic substances have to be identified. Sample preparation usually is simple for identification purposes, but will be an issue for imaging experiments, and spatial resolution may then well be only in the range of a few micrometers, depending on the used experimental approach (transmission, ATR). 

Raman microscopy provides a spatial resolution slightly better than IR, and no sample preparation is necessary in many cases. It has advantages with special types of substances (e.g., systems containing conjugated double bonds, oriented systems, amorphous and crystalline carbon, oxides). SNOM techniques (with spatial resolution below 1 pm) have been more popular with Raman than with IR, so far, but as yet are not routinely practiced. 

A comprehensive review of compositional and failure analysis of polymers, which includes many further examples of analysis of contaminants, inclusions, chemical attack, degradation, etc., was published in 2000 [2], It includes details on methodologies, sampling, and sample preparation, and microscopy, infrared spectroscopy, and thermal analysis techniques. 

The strategy that is followed for mapping a phase diagram depends on the specific problem considered. In general there are two complementary approaches that must be used. In the first, samples prepared at a particular temperature-pressure condition and subsequently quenched from these conditions are analyzed, typically by optical or electron microscopy and X-ray diffraction. This approach does not... 

Retention of a protein or protein activity after 105,000y, 1 hr Chromatography on gel filtration columns with large pore sizes Electron microscopy—however, sample preparation may partially reconstitute membranes Decrease in solution turbidity, which may be detected by a diminution in light scattering or an enhancement in light transmission Diffusion of membrane lipids as assayed by nuclear magnetic resonance and electron spin resonance... 

Transmission electron microscopy (TEM) is a powerful and mature microstructural characterization technique. The principles and applications of TEM have been described in many books [16 20]. The image formation in TEM is similar to that in optical microscopy, but the resolution of TEM is far superior to that of an optical microscope due to the enormous differences in the wavelengths of the sources used in these two microscopes. Today, most TEMs can be routinely operated at a resolution better than 0.2 nm, which provides the desired microstructural information about ultrathin layers and their interfaces in OLEDs. Electron beams can be focused to nanometer size, so nanochemical analysis of materials can be performed [21]. These unique abilities to provide structural and chemical information down to atomic-nanometer dimensions make it an indispensable technique in OLED development. However, TEM specimens need to be very thin to make them transparent to electrons. This is one of the most formidable obstacles in using TEM in this field. Current versions of OLEDs are composed of hard glass substrates, soft organic materials, and metal layers. Conventional TEM sample preparation techniques are no longer suitable for these samples [22-24], Recently, these difficulties have been overcome by using the advanced dual beam (DB) microscopy technique, which will be discussed later. 

The OLED is composed of hard and soft layers so that the conventional cross-sectional TEM sample preparation techniques cannot be applied. Figure 10.3 is a first DB microscopy-prepared TEM image of an OLED in cross-sectional view [37], The glass substrate, ITO, organic layers, and A1 cathode are indicated in the image. The microstructure and interfaces of all these layers can be well studied now. The nanometer-sized spots in organic layers are indium-rich particles. We believe the combination of DB microscopy and TEM will greatly advance the OLED research and development in the near future. 

Electron microscopy does have some limitations. For example, this technique usually requires special sample preparations. Caution also needs to be exercised to minimize ary electron beam-induced... 

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