Scanning confocal optical microscopy

Fig. 5. Scanning confocal optical microscopy for single-molecule detection. Sample scanning configuration. The fiber exit and the active area of the SPAD serve as confocal pinholes. S sample, O high-NA microscope objective, DM dichroic mirror, L1,L2 lenses, F Filters.

Cork T and Kino G S 1996 Confocal Scanning Optical Microscopy and Related Imaging Systems (New York Academic) Gu Min 1996 Principles of Three Dimensional Imaging In Confocal Microscopes (Singapore World Scientific)... 

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... 

For samples thicker than the depth of field, the images are blurred by out-of-focus fluorescence. Corrections using a computer are possible, but other techniques are generally preferred such as confocal microscopy and two-photon excitation microscopy. It is possible to overcome the optical diffraction limit in near-field scanning optical microscopy (NSOM). 

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. 

A number of methods are available for the characterization and examination of SAMs as well as for the observation of the reactions with the immobilized biomolecules. Only some of these methods are mentioned briefly here. These include surface plasmon resonance (SPR) [46], quartz crystal microbalance (QCM) [47,48], ellipsometry [12,49], contact angle measurement [50], infrared spectroscopy (FT-IR) [51,52], Raman spectroscopy [53], scanning tunneling microscopy (STM) [54], atomic force microscopy (AFM) [55,56], sum frequency spectroscopy. X-ray photoelectron spectroscopy (XPS) [57, 58], surface acoustic wave and acoustic plate mode devices, confocal imaging and optical microscopy, low-angle X-ray reflectometry, electrochemical methods [59] and Raster electron microscopy [60]. 

Sherar, M. D., Noss, M. B., and Foster, F. S. (1987). Ultrasound backscatter microscopy images the internal structure of living tumour spheroids. Nature 330,493-5. [174] Shimada, H. (1987). Propagation of multi-mode ultrasonic pulses in non-destructive material evaluation. In Ultrasonic spectroscopy and its application to Materials science (ed. Y. Wada), pp. 50-6. Ministry of Education, Science and Culture, Japan. [148] Shotton, D. M. (1989). Confocal scanning optical microscopy and its applications for biological specimens. J. Cell. Sci. 94,175-206. [177,200]... 

Shotton, D. M. (1989) Confocal scanning optical microscopy and its applications for biological specimens J Cell Sci 94, 175-206... 

Three major advancements in resolution have occurred since Hookes s discovery of the optical microscope in 1665 [46]. In 1873, Ernst Abbe established fundamental criteria for the resolution limit in optical microscopy [47], which did not exceed the range of a couple of 100 nanometers even after the introduction of the confocal optical microscope [43,48]. The invention of the transmission electron microscope by Ernst Ruska in 1933 extended the resolution of microscopes to the nanometer scale [49]. Finally, scanning tunnelling microscopy introduced, by Binnig and Rohrer in 1981, made a breakthrough when atomic... 

Keywords Single-molecule detection single emitters confocal microscopy scanning near-field optical microscopy. 

Refs. [i] Minsky M (1988) Scanning 10 128 [ii] Wilson T, Sheppard CJR (1984) Scanning optical microscopy Academic Press, London [Hi] Corle TG, Kino GS (1996) Confocal scanning optical microscopy and related techniques. Academic Press, New York [iv] Delhaye M, Barbil-lat J, Aubard J, Bridoux M, Da Silva E (1996) Instrumentation. In Rur-rell G, Corset J (eds) Raman microscopy developments and applications. Academic Press, San Diego [v] Ren B, Lin XF, Jiang YX, Cao PG, Xie Y, Huang QJ, Tian ZQ (2003) Appl Spectrosc 57 419... 

Confocal SERS microscopy combined with subsequent local scanning electron microscopy (SEM) is able to deliver valuable optical and chemical information on the location of SERS active spots. The method as to how to retrieve in the electron micrograph the active spots seen in the confocal images is explained. The spots were analyzed with respect to band position and spectral intensity. 

Fig. 8.8 Optical images (at an active zone I) of isolated silver clusters electrodeposited onto ITO by means of the double-pulse method ( i = —1,550 mV 2 = —VOO mV, 25 s) [37] (a) Scanning confocal microscopy image (topography mode), (b) Raman/fluorescence image of the same sample area, (c) SEM image corresponding to (a) and (b)...

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