Confocal optical methods

Instrumentation. In a typical CLSM experiment, a narrow laser beam scans a surface horizontally, i.e. at constant height, in x- and y-directions using piezo-driven mirrors. A small pinhole is located in front of an optical detector at a position conjugate to the focal point in the sample plane. This way, the detector measures the intensity of light reflected from the surface at every scanned position. Light scattered from out-of-focus positions is focused outside the pinhole and thus does not reach the detector. An image of scattered and/or reflected laser light intensity is created 

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A scanning method for each observation may improve the spatial resolution. A LSM with confocal optics and a scanning near-field optical microscope (SNOM) can provide finer spatial resolution with a limit of a few nanometers. The highest... 

Zeolite membranes are commonly characterized by SEM, XRD. TEM, EPMA, SEM-EDX, TEM-EOS, and Nitrogen adsorption are also used to study the morphology, microstructure and composition of zeolite membranes. Usually single gas permeation, mixed gas separation, pervaporation and vapor permeation are performed to evaluate the properties of zeolite membranes. Recently, some novel characterization techniques have been applied. Infrared reflectance measurement was used to characterize membrane thickness [19]. Fluorescence confocal optical microscopy was used to image the grain boundary structure of zeolite membranes [20]. FTIR-ATR method was used to characterize the T-O vibration of zeolite membranes [21],... 

Raman spectroscopy was also reported to be useful for studying the molecular nature of human SC (Williams etal., 1992a). Recently, Raman microspectroscopy is being used to characterize the Upid domains of SC (Percot and Lafleur, 2001). Combined with confocal microscopy, it is useful as a noninvasive in vivo optical method to measure molecular concentration profiles in the skin (Caspers et al., 2001), which allows measuring percutaneous absorption along with detailed information about the molecular composition of the skin with high spatial resolution (Caspers et al., 2003). 

Kino, G. S. (1989). Intermediate optics in Nipkow disk microscopes. In The Handbook of Biological Confocal Microscopy (J. Pawley, ed.), 1st ed., pp 105-126. IMR Press, Madison, WI. Matsumoto, B. (1993). Cell biological applications of confocal microscopy. Methods Cell Biol. 38, 1-380. 

Confocal optical microscopy can be used to take a sequence of randomly chosen images through a bacterial floe. Methodologies for calculation of three-dimensional fractal dimensions have been described for this approach [18-20]. One method determines the fractal dimension of each section 7)f using a two-point correlation function C(r) [20] ... 

Because Si,N ceramics are partially translucent to light and the strength limiting flaws are normally within a shallow depth under fte sur ce, optical methods are effective to detect and characterize these types of subsurface flaws. Argonne National Laboratory (ANL) has developed and utilized several optical methods for nondestructive evaluation (NDE) of subsurface flaws in silicon nitride ceramics. In this study, three optical methods are presented and evaluated (1) laser backscatter, (2) optical coherence tomography (OCT) and, (3) confocal microscopy. The la.ser backscatter is a two-dimensional method while both (XT and confocal are three-dimensional methods. It is demonstrated in the following that subsurface flaws of various types, sizes, and depths can be identified and imaged by these NDE methods. 

Cross-polarization confocal microscopy is a new 3D imaging method developed at ANL [9]. It combines two well-established optical methods, the cross-polarization backscatter detection and the scanning confocal microscopy, and can achieve 3D subsurface imaging with sub-micron spatial resolutions. A schematic diagram of the system is shown in Fig. 7. 

A more direct optical method has been introduced whereby a membrane-permeant, charged dye equilibrates across the membrane of interest. Ideally the dye will equilibrate rapidly and show minimal binding to materials on either side of the membrane (34). Using quantitative confocal fluorescence microscopy, the fluorescence of the dye on either side of the membrane is measured as fluorescence intensity is linearly proportional to fluorophore concentration, the measured intensities can be substituted for the ion concentrations ([M" ] and [M ]o) in equation 1 and the potential obtained (34). This technique is ultimately limited by the spatial resolution of the confocal microscope—it works well for large compartments in whole cells. Recent computational approaches to image analysis have allowed the spatial resolution to be increased to the level where mitochondrial membrane potential can be monitored by such means in individual cells (35). 

Another way to characterize the electrochemical activity of miCToelectrode arrays is to map the electroactive species generated at each electrode by confocal Raman spectroscopy. Indeed, the use of confocal signal detection enables Raman spectroscopic measurements of very small sample volumes (even down to a few om ). Applied to a microelectrode array, it provides a statistical picture of the distribution of active sites on the array (60). As in the case of SECM, these two optical methods are particularly useful to verify if individual diffusion layers do not overlap and if the microelectrodes in the array are diffusely independent, particularly for random microelectrode arrays. 

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