In situ microscopic techniques

Recently a whole new family of microscopic techniques has been invented which enable the in situ imaging of solid surfaces using local probes. By local we mean that resolution can approach atomic dimensions, implying that probe size and accuracy of controlling its movement over the surface limit the resolution. The probe is usually located at atomic distances from the sample and is not influenced by the medium. 

Not only can experiments be performed in situ, but also the time evolution of surface topography can be monitored. Accurate scanning of the surface is accomplished by means of piezoelectric drives—the size of a piezoelectric crystal changes linearly with the applied potential difference. Drives are applied in x, y and z directions as shown schematically in Fig. 12.14. Generally, successive scans are applied in the x direction incrementing y between scans, so that a series of x-z profiles are recorded. These can be converted by computer software into gray-scale or coloured images if desired. 

From a practical point of view, adequate vibration-free experimental conditions must be assured given the resolution in question. Vibration-free tables are available, but in many cases suspending a concrete block on which the working part of the instrument is located from the ceiling with elastic cords is perfectly adequate. 

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Spectroscopic techniques can be carried out in situ (low-energy photon, etc.) and ex situ or in vacuo (high-energy photon and electron techniques). Ex situ microscopic techniques have been employed for many years to examine surfaces, and are now widely used tools. However, in situ microscopic techniques with resolution approaching the atomic scale... 

Although the emphasis of this chapter is on isolated cells in culture, responses at the cellular level can be assessed in intact tissue after exposure of the whole animal. Observations of cellular responses are most often made with in situ detection techniques and microscopic observation, such as immunohistochemisty (Chapter 7) and nucleic acid hybridization (Chapter 2). Preparations used for these in situ techniques are generally tissue that has been fixed after toxicant treatment, then embedded and sliced thinly enough (-5 pm) to enable observation by microscopy, usually... 

Optimization of internal engine combustion in respect of fuel efficiency and pollutant minimization requires detailed insight in the microscopic processes in which complex chemical kinetics is coupled with transport phenomena. Due to the development of various pulsed high power laser sources, experimental possibilities have expanded quite dramatically in recent years. Laser spectroscopic techniques allow nonintrusive measurements with high temporal, spectral and spatial resolution. New in situ detection techniques with high sensitivity allow the measurement of multidimensional temperature and species distributions required for the validation of reactive flow modeling calculations. The validated models are then used to And optimal conditions for the various combustion parameters in order to reduce pollutant formation and fuel consumption. 

For in-situ experiments most commonly used microscopic and spectroscopic techniques are environmental transmission electron microscopy (E-TEM) [207-209], In-situ vibrational spectroscopic [210-212], ambient pressure X-ray photoelectron spectroscopy [206,210,213], X-ray absorption spectroscopy [213,214-217], and Raman spectroscopy [218]. Making use of this in-situ experiments, the solar fuel generation processes will get a new dimension to the state-of-the-art beliefs. Moreover, the catalysts structure, coverage and composition also change with time, the combination of ultrafast of in-situ spectroscopic techniques reveal the structure and catalytic activity relationships (See Table 7) [217]. 

Recently, the structure of the solid/liquid interface has been studied with a wide range of in-situ structural techniques. In particular, scanned probe microscopes [1-5] and synchrotron-based methods [6-9] have yielded a wealth of structural information. The ultimate goal of this work is an understanding of the structure and reactivity of the electrode surface at the atomic level. One of the most extensively studied processes is metal underpotential deposition (UPD) [10], which involves the formation of one or more metal monolayers at a potential positive of the reversible Nemst potential for bulk deposition. 

The importance of low pressures has already been stressed as a criterion for surface science studies. However, it is also a limitation because real-world phenomena do not occur in a controlled vacuum. Instead, they occur at atmospheric pressures or higher, often at elevated temperatures, and in conditions of humidity or even contamination. Hence, a major tlmist in surface science has been to modify existmg techniques and equipment to pemiit detailed surface analysis under conditions that are less than ideal. The scamiing tunnelling microscope (STM) is a recent addition to the surface science arsenal and has the capability of providing atomic-scale infomiation at ambient pressures and elevated temperatures. Incredible insight into the nature of surface reactions has been achieved by means of the STM and other in situ teclmiques. 

The main technique employed for in situ electrochemical studies on the nanometer scale is the Scanning Tunneling Microscope (STM), invented in 1982 by Binnig and Rohrer [62] and combined a little later with a potentiostat to allow electrochemical experiments [63]. The principle of its operation is remarkably simple, a typical simplified circuit being shown in Figure 6.2-2. 

TUNEL is the name given to the in-situ DNA endlabelling technique which serves as a marker of apoptotic cells. This method is based on the specific binding of terminal de-oxy nucleotidyl transferases to the 3-hydroxy ends of DNA. The technique is normally used for examination under the light microscope but can also be adapted for examination under the electron microscope. 

Mechanical and chemical methods for qualitative and quantitative measurement of polymer structure, properties, and their respective processes during interrelation with their environment on a microscopic scale exist. Bosch et al. [83] briefly discuss these techniques and point out that most conventional techniques are destructive because they require sampling, may lack accuracy, and are generally not suited for in situ testing. However, the process of polymerization, that is, the creation of a rigid structure from the initial viscous fluid, is associated with changes in the microenvironment on a molecular scale and can be observed with free-volume probes [83, 84]. 

Hence, for modern FRET and FLIM techniques in Molecular Biology and Biochemistry it is important to keep the enthusiasm for the in situ technique, yielding unprecedented rich information on molecular states in live cells, and to keep the advantages of easy labeling techniques, modern microscopes and automated data processing. However, we need to educate the new generations of FRET scientists in the theoretical background of the technique, how it should be done correctly, and what the sources of errors are. Only then it will be clear that FRET-(FLIM) is a very direct, robust, extremely sensitive, and reliable technique. 

Measuring FRET by fluorescence lifetime imaging microscopy (FRET-FLIM) offers the ability to see beyond the resolution of the optical system ( 10-100 times that of modern far field microscopes [5]). FRET efficiency can be used as a proxy for molecular distance, thereby allowing the easy detection and somewhat more challenging quantification of molecular interactions. Although many types of assay exist, FRET-FLIM is a highly suitable technique that is capable of in situ measurements of molecular interactions and conformation in living and fixed cells. 

The Scanning Tunneling Microscope has demonstrated unique capabilities for the examination of electrode topography, the vibrational spectroscopic imaging of surface adsorbed species, and the high resolution electrochemical modification of conductive surfaces. Here we discuss recent progress in electrochemical STM. Included are a comparison of STM with other ex situ and in situ surface analytic techniques, a discussion of relevant STM design considerations, and a semi-quantitative examination of faradaic current contributions for STM at solution-covered surfaces. Applications of STM to the ex situ and in situ study of electrode surfaces are presented. 

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