Microscopy techniques selection

Peng, L., Stephens, B.J., Bonin, K. et al. 2007b. A combined atomic force/fluorescence microscopy technique to select aptamers in a single cycle from a small pool of random oligonucleotides. Microsc Res Tech 70(4) 372-381. 

As previously discussed, electron, light, and confocal microscopy techniques may be used to visualize the position of electron-dense precipitates, radioactive substances, and fluorescent probes, respectively, in the sample tissue. However, none of these techniques possess the capability both to visualize and to selectively measure the flux of a molecule across the skin. SECM, however, permits the measurement and subsequent imaging of the local flux of an electroactive species across biological membranes. Scott et al. [3] used SECM to investigate the effect of pretreatment of the penetration enhancer sodium dodecyl sulfate (SDS), on the ion transport rate and transport pathways of Fe(CN) across hairless mouse skin. Increasing the time of SDS exposure from 10 min to 30 min increased the overall (porous and nonporous) transport of Fe(CN) by 17-fold. More specifically, the SDS-induced increase in Fe(CN)g transport was found to be associated with nonporous (i.e., intercellular) transport routes, while transport via porous routes was significantly reduced. The fraction of Fe(CN)g transport through pores, as measured by... 

It should be noted that most of the microstructural data to be presented rely on different characterization techniques using electron microscopy in combination with other scanning probe microscopy techniques, e.g. scanning tunneling microscopy (STM) and atomic force microscopy (AFM). The details of specimen preparation and the analysis techniques of electron microscopy are described in other chapters of this book and are therefore not further covered in this chapter. The references provided in the chapter are merely a selection, since the field of research is vivid and the number of publications is large. The goal is to show the salient features which can be further explored by use of the referenced literature. 

The morphology of these nanostructures strongly depends on the technique selected for the preparation and on the operating conditions. Figures reports, as an example, some of the morphologies that may be achieved for tin and zinc oxide. The most important features of these nanowires for chemical sensors are their high surface to volume ratio and their single crystalline nature confirmed by transmission microscopy. 

Diffraction Techniques. Diffraction techniques can readily reveal the crystalline structure of bulk diamond or graphite. However, in many cases, a material may be a complex mixture of diamond, graphite, and amorphous constituents on a size scale that makes them difficult to resolve even with electron microscopy and selected area diffraction (SAD). Consequently, the results of these diffraction techniques have to be interpr ed cautiously. 

The steps involved in the problem solving protocol are outlined in Table 7.1. They are rather simple and do not take much time to consider and such a protocol can save time in the long run. The protocol involves steps typical of scientific inquiry collect all the currently known facts, determine the nature of the problem, state the objective of the study, obtain the correct specimen, be sure to have experimental controls, look at the sample with the naked eye and then with a stereo microscope. These provide an aid to selection of the specific microscopy techniques and preparation methods needed to begin to address the objectives. The result should be that clearly defined analyses are conducted. 

The first step in the selection of a microscopy technique is to know the size of the polymer structures to be characterized. In fundamental... 

Once the objective of the experiment is known and the specimens selected for study, the next major step is the selection of the microscopy techniques and the specimen preparation methods required to image the polymer structures of interest (Table 7.2). If lamellar crystals must be... 

Fig. 7.1 Problem solving flow chart. Questions to consider when selecting a microscopy technique.

Just as for any other fluorescence microscopy technique, the choice of the fluorescent probe is of significant importance for PCS. Drops in the autocorrelations curves can occur as a result of photophysical and photochemical processes. In particular, the contribution of saturation effects and triplet blinking has been investigated [97, 98] and the rates of intersystem crossing and triplet decay as well as the excitation cross section of fluorophores could be determined [12]. In addition, antibunching is determined by the photophysics of the fluorophore. Therefore, the choice of appropriate dyes is essential to obtain meaningful results. Apart from that, the fluorescent probe should also serve as a selective label to... 

Once the objective of the experiment is known and the specimens selected for study, the next major step is the selection of the microscopy techniques and the specimen preparation methods required to image the polymer structures of interest (Table 6.2). If lamellar crystals must be evaluated, for instance, there is no point in considering most optical techniques as they will only provide an overview of these structures. Comparisons are made in this section regarding the various techniques, in both the text and tables, as an aid in this selection process. Observations of... 

The specific microscopy techniques and appropriate preparation methods required to solve structural problems must be selected now that the techniques and the problem solving protocol (Table 6.1) have been considered. The advantages and limitations of these techniques have already been considered but questions still remain. When should optical techniques be utilized in solving polymer structural problems How can experiments be conducted so that it is clear if further study is required Are there any... 

The most important issues in the solution of structural problems are image interpretation and development of structure-property relations. Imaging techniques and preparative methods must be chosen that provide images of the needed structures by the most efficient experiments. Several major principles have been emphasized for imaging of structures. First, the problem solving protocol (Table 6.1) should be considered prior to developing an experimental plan. As part of this protocol the important properties of the material to be studied should be determined and the overall objective of the study developed. The size of the polymer structures required should be determined (Tables 6.2 and 6.3) as an aid to the selection of the appropriate microscopy techniques. Specimen preparation methods should be selected after considering the nature of the specimen itself, the types of structures to be determined and the potential artifacts. If a specimen can be examined directly, that is preferred over a less direct specimen preparation method, especially... 

It is impossible to comprehensively discuss all non-vibrational in situ techniques with a potential application to oxidation catalysts within this chapter. Therefore, we have selected only those methods for a more detailed presentation which have seen a widespread application so far and/or offer unique opportunities for understanding the functioning of real catalysts. For more specific in situ methods, such as the microscopy techniques mentioned above, Mossbauer spectroscopy which is restricted to the viewing of elements only, or thermo-analytical studies using an oscillating microbalance reactor,the reader is referred to the respective reviews. 

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