SCANNING CONFOCAL MICROSCOPY

In a confocal microscope a small aperture is placed in the plane where rays coming from a particular plane in the object come to a point (see Fig. 6.1, the plane is called the confocal plane). A light detector is placed behind the aperture. The aperture selects one point (x, y) in the plane, and also cuts out most of the light coming from other planes in the specimen. If the illumination is focused onto the selected point in the object, then 

Mechanically registering two scanning systems would be difficult, so commercial LCSMs are reflection microscopes, where (as shown in Fig. 6.1) the beam passes through the same scanner twice. The beam is scanned On the specimen, then de-scanned onto a fixed confocal pinhole. Con-focal microscopes may have a transmission mode, but currently this will not be confocal. It may use a separate regular illumination system, or the scanned illumination. In the latter case the 

For metrology, an image is formed by setting the value of each pixel to the value of z at which the image was brightest. This will produce a topographical map of the most reflective surface, whether external or internal. Line profiles can be selected in any direction, or a surface roughness can be calculated from the whole profile. 

The major use of LCSM up to the present has been as a fluorescence microscope in biology [4]. Its use in materials is growing [13] but many synthetic materials are opaque and can withstand vacuum, so their external surfaces can be 

1520 cm peak, so shorter exposures suppress the plasticizer peaks. Degradation is greatest at the film surface, as expected, but the profile can be analyzed numerically to determine diffusion and reaction kinetics [20]. Clearly this method could be extended to give the spectrum and thus the chemistry of every point in a 2-D image, which can come from an internal plane in a transparent material. 

Another confocal scanning method uses a rapidly rotating disk containing many holes in the confocal plane (a Nipkow disk). These holes act as confocal apertures and mechani- 

Although the major use of the CSLM is as a fluorescence microscope in biology [8], it is also 

---

MJ. Schermer, Confocal scanning microscopy in microarray detection, in DNA Microarrays A Practical Approach (M. Schena, ed.), p. 17, Oxford University Press (1999). 

Shuman, H. (1987) Contrast in confocal scanning microscopy with a finite detector. J. Microscopy 149, 67-71. 

Pinto, M.C. and Macias, P. (1995) Determination of intraparticle immobilized enzyme distribution in porous support by confocal scanning microscopy. Biotechnology Techniques, 9(7), 481-486. 

Confocal scanning microscopy 129 Conformations of molecules 43 - 45 anti 43... 

MICROWAVE HEAT-ASSISTED CONFOCAL SCANNING MICROSCOPY... 

K.J. Stine and C.M. Knobler, Fluorescence Microscopy A Tool for Studying the Physical Chemistry of Interfaces, Ultramicroscopy 47 (1992) 23. (Review short introduction to fluorescence and fluorophores basic instrumentation for fluorescence microscopy and extensions to study dynamics and resonance energy transfer, confocal scanning microscopy results obtained with Langmuir monolayers.)... 

Deconvolution microscopy achieves the same Image-sharpening effect as confocal scanning microscopy but through a different process. In this method, Images from consecutive focal planes of the specimen are collected. A separate focal series of images from a test slide of subresolution size (i.e., 0.2 (jim diameter) bead are also collected. Each bead represents a pinpoint of light that becomes an object blurred by the imperfect optics of the microscope. Deconvolution... 

Why are chemical stains required for visualizing cells and tissues with the basic light microscope What advantage does fluorescent microscopy provide In comparison to the chemical dyes used to stain specimens for light microscopy What advantages do confocal scanning microscopy and deconvolution microscopy provide in comparison to conventional fluorescence microscopy ... 

Schmid, M., Thill, A., Purkhold, U., Walcher, M., Bottero, J. Y, Ginestet, P., Nielsen, P. J., Wertz, S. and Wagner, M.(2003). Characterisation of activated sludge floes by confocal scanning microscopy and image analysis. Water Res., 17, 2043-2052. 

Variations of this method include the use of a fluorescent dye such as fluorescein or Rhodamine. Depending on the visualization technique used, the uncertainty between perfect mixing and multi-lamellae flow may still be detrimental to proper quantification of the mixing quality. Nevertheless, the use of confocal scanning microscopy enables one to perform three-dimensional imaging of the flow and distinguish between these conflgurations [17, 34, 38]. 

Among these, some of the most frequently used are attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. X-ray photoelectron spectroscopy (XPS), static secondary ion mass spectrometry (SSIMS), energy dispersive X-ray spectroscopy (EDS), optical microscopy, laser confocal scanning microscopy (LCSM), scanning electron microscopy (SEM), enviromnental scanning electron microscopy (ESEM), transmission electron microscopy (TEM), atomic force microscopy (AFM), contact angle measurement, and some evaluation methods for the biocompatibility of membrane surfaces. 

Using difference contrast light microscopy and laser confocal scanning microscopy, the presence of giant vesicles could clearly be observed in the oleic acid-... 

Confocal scanning microscopy can use non-laser and laser illumination sources, but in practice only the latter provide sufficient brightness. Confocal scanning optical microscopy (CSOM), in which the image is built up by synchronous scanning of the source and the detector units, can operate in transmission, reflection, or fluorescence mode. The lateral resolution of CSOM is of the order of 200 nm, which is a factor 1.4 better than that of the current optical techniques. CSOM images are usually sharper than those of conventional microscopy. The technique is some twenty years old [39]. 

Ploug H, Jorgensen BB (1998) A net-jet flow system for mass transfer and microsensor studies of sinking a egates. Mar Ecol Prog Ser 176 270-290 Qian Z, Stoodley P, Pitt WG (1996) Effect of low-intensity ultrasound upon biofilm structure from confocal scanning microscopy observation. Biomaterials 17 1975-1980... 

---