LASER SCANNING CONFOCAL OPTICAL MICROSCOPY

Ribbe A E 1997 Laser scanning confocal microscopy in polymer science Trends Polym. Sc/. 5 333-7 Oliveira M J and Hemsiey D A 1996 Optical microscopy of polymers Sonderb. Prakt. Metallogr. 27 13-22 Nie Sh and Zare R N 1997 Optical detection of single molecules Ann. Rev. Biophys. Biomol. Struct. 26 567-96 Masters B R 1994 Confocal redox imaging of cells Adv. Mol. Cell Biol. 8 1-19... 

More recently Ghiggino and co-workers(32) have applied laser scanning confocal fluorescence lifetime microscopy to the study of polyvinyl alcohol films containing rhodamine B (650 nm emission) and cresyl violet (632 nm emission). Synchronously pumped dye laser excitation and APD detection were used with optical fiber coupling. A schematic diagram of their apparatus is shown in Figure 12.5. 

N. (1985) Three-dimensional microscopy using a confocal laser scanning microscope. Optics Lett. 10, 53-55. 

Carlsson, K., Danielsson, P. E., Lenz, R., Liljeborg, A, Majlof, L., and Aslund, N. (1985) Three-dimensional microscopy using a confocal laser scanning microscope Optics Lett 10,53-55. 

Fig.1 Schematic representing the optical pathway and image information flow of the laser scanning confocal microscopy (LSCM) technique. All principle components of generic LSCM are labeled (Redrawn in part frtnn Carl Zeiss The Confocal Laser Scanning Microscope)...

In the present chapter, we discuss the principles and techniques commonly used for observing biological surface structures, including optical microscopy (light microscopy, laser scanning confocal microscopy), electron microscopy (scanning electron microscopy, transmission electron microscopy), and scanning probe microscopy. We describe and contrast the sample preparation of each technique. Quantitative data analysis as well as the limitations of each technique is also addressed. 

Optical imaging with collinear optical coherence and confocal fluorescence microscopy (OCM-CFM). This noninvasive imaging technique combines the use of reflection confocal microscopy in tandem with optical coherence microscopy to characterize scaffolds and tissue constructs. It can probe the interior of scaffolds with the same resolution as conventional laser scanning confocal microscopy but with greater sensitivity (P). 

More advanced (and expensive) microscopy techniques are differential interference contrast microscopy and laser scanning confocal microscopy. The latter can provide precise optical sectioning so that three-dimensional images of stmctures can be recorded. 

Figure 1 shows the spatial resolution of various types of microscopy and scattering methods, including 3D microscopy techniques such as laser scanning confocal microscopy (LSCM), X-ray computerized tomography (X-ray CT), and TEMT. LSCM has been extensively used to investigate the phase-separated bicontinuous structures of polymer blends. It should be noted that X-ray CT is particularly useful for opaque specimens with micrometer-sized stmctures, while transparent specimens are necessary for LSCM. X-ray CT has been also extensively used in medical sdence. In the early 1990s, the spatial resolution of X-ray CT was limited to ca. 100 im. Recently, the resolution has substantially improved and is now comparable with ( 1 pm) or even superior to that of optical microscopy. 

Kapitza H G 1996 Confocal laser scanning microscopy for optical measurement of the microstructure of surfaces and layers Tech. Mess. 63 136-41... 

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