Microscopy detection techniques

SPM encompasses a group of surface-detection techniques that include AFM and scanning tunneling microscopy (STM) that allow the topographic profiling of surfaces. SPM techniques investigate only the outermost few atomic layers of the surface with nanometer resolutions and at times atomic-level resolution. 

For a particular biomedical application of CRS microscopy, the best choice whether to use CARS or SRS detection depends on the optimal balance between the pros and cons of each technique regarding its detection sensitivity, image acquisition time, and interpretability of image contrast and spectrum. In the following, we provide a critical discussion of the advantages and disadvantages of both complementary detection techniques ... 

The technique of immunohistochemistry is very similar to fluorescence microscopy. This technique differs only in the method of detection or localization of the antibody and can be performed with a conventional light microscope. As with the ELISA and Western blot, the antibody used in this experiment is covalently conjugated to an enzyme, such as horseradish peroxidase. This enzyme is then incubated with a substrate that is converted to an insoluble colored product that will precipitate or deposit at the site of enzyme activity. The distribution and location of the colored product is readily detected with an ordinary light microscope. 

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... 

As consequence of implementation of parallelization in microfluidic cell culture chips, detection of biologically relevant cellular parameters imposes further requirements on the development of the applied detection techniques. Using available motorized microscope stages, time-lapse fluorescence microscopy is a widely applied technique in monitoring cellular responses. Alternatively, fluorescent plate readers facilitate real-time monitoring in highly parallelized systems (readouts for 1,536 well microtiter plate format). 

One interesting new Add in the area of optical spectroscopy is near-field scanning optical microscopy, a technique that allows for the imaging of surfaces down to sub-micron resolution and for the detection and characterization of single molecules [80,81] When applied to the study of surfaces, this approach is capable of identifying individual adsorbates, as in the case of oxazine molecules dispersed on a pol mier film, illustrated in figure Bl. 22,11 [82]. Absorption and emission spectra of individual molecules can be obtained with this technique as well, and time-dependent measurements can be used to follow the d5mamics of surface processes. 

A complementary single molecule detection technique is FCS [110,111] (fig. 3.4). Here, confocal microscopy is used to record minute fluorescence fluctuations in a very small... 

On the other hand, spectroscopic detection methods, such as laser-induced fluorescence, UV/Vis absorption, chemiluminescence, and thermal lens microscopy, have been used for on-chip detection. Among these methods, the fluorescence detection method has been most widely used because of its high sensitivity and low detection limits for biologically relevant species. However, the fluorescence detection technique has some disadvantages. Many chemical... 

Nanofluidic systems are also ideally suitable for certain single-molecule detection techniques such as total internal reflection fluorescence (TIRF) microscopy. TIRF utilizes evanescent waves, which are generated by total internal reflection of a laser beam, to excite the fluorescence signal, and since evanescent waves decay exponentially, the molecule of interest must locate to the close proximity of the interface of the glass and liquid. Nanofluidic systems confine molecules of interest in nanochannels, which is well in the evanescent field of TIRF microscopy. 

This technique is also known as scanning acoustic tomography. This is a powerful tool for spotting delaminations, cracks, and other anomalies non-destructively. Not only does acoustic microscopy detect the failures but it can also provide the specific location of the problem. SAM high-resolution images and advanced diagnostic tools are used to ... 

TLC remains one of the most widely used techniques for a simple and rapid qualitative separation. The combination of TLC with spectroscopic detection techniques, such as FTIR or nuclear magnetic resonance (NMR), is a very attractive approach to analyze polymer additives. Infrared microscopy is a powerful technique that combines the imaging capabUities of optical microscopy with the chemical analysis abilities of infrared spectroscopy. FTIR microscopy allows obtaining of infrared spectra from microsized samples. Offline TLC-FTIR microscopy was used to analyze a variety of commercial antioxidants and light stabilizers. Transferring operation and identification procedure by FTIR takes about 20 min. However, the main drawbacks of TLC-FTIR are that TLC is a time-consuming technique and usually needs solvent mixtures, which makes TLC environmentally unsound, analytes must be transferred for FTIR analysis, and TLC-FTIR cannot be used for quantifying purposes. 

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