Fluorescence optical microscopy

Optical fluorescence microscopy is a powerful and sensitive method for obtaining information about the orientation of luminescent dye molecules in small crystals. In Figure 1.10, we show unpolarized and linearly polarized fluorescence of two perpendicularly lying zeolite L crystals loaded with DSC. 

Normanno, D., van Zanten, X, and Garcia-Parajo, M. (2010) in Nanoscopy and Multidimensional Optical Fluorescence Microscopy (ed. A. Diaspro), CRC Press, Boca Raton, FL, pp. 18-1-18-28. 

Journal of Applied Polymer Science 69, No.4, 25th July 1998, p.645-55 MAPPING PHASES OF POLY(VINYL ALCOHOL) AND POLY(VINYL ACETATE) BLENDS BY FTIR MICROSPECTROSCOPY AND OPTICAL FLUORESCENCE MICROSCOPY Dibbem-Brunelli D Atvars T D Z Joekes I Barbosa V C Campinas,Universidade Estadual... 

Figure 9.5 Logical operation of image of cracks sorted and aligned and image of parallel lines. Reprinted from Cement and Concrete Research, Vol 36, Litorowicz, A., identification and quantification of cracks in concrete by optical fluorescent microscopy, p. 8., Copyright (2006), with permission from Elsevier.

Litorowicz, A. (2006) Identification and quantification of cracks in concrete by optical fluorescent microscopy. Cement and Concrete Research, 36 1508-15. 

Brunelli, D.D., Atvars, T.D.Z., Joekes, I., and Barbosa, V.C. (1998) Mapping phases of poly(vinyl alcohol) and poly(vinyl acetate) blends by FTIR microspectroscopy and optical fluorescence microscopy. 

Optical fluorescence microscopy was employed to study the phase morphology of PVOH and PVAc blends [78]. Fluorescein (green) and anthracene (blue) were added to provide the contrast as fluorescein preferred the PVOH domains and anthracene concentrated in the PVAc domains. 

A wide variety of measurements can now be made on single molecules, including electrical (e.g. scanning tunnelling microscopy), magnetic (e.g. spin resonance), force (e.g. atomic force microscopy), optical (e.g. near-field and far-field fluorescence microscopies) and hybrid teclmiques. This contribution addresses only Arose teclmiques tliat are at least partially optical. Single-particle electrical and force measurements are discussed in tire sections on scanning probe microscopies (B1.19) and surface forces apparatus (B1.20). 

Osborne M A, Balasubramanian S, Furey W S and Klenerman D 1998 Optically biased diffusion of single molecules studied by confocal fluorescence microscopy J. Chem. Phys. B 102 3160-7... 

Neil, M. A. A., Squire, A., Juskaitis, R., Bastiaens, P. I. H. and Wilson, T. (2000). Wide-field optically sectioning fluorescence microscopy with laser illumination. J. Microsc. 197, 1-4. 

Various solutions have been proposed for the reduction or elimination of autofluorescence. One way is to chemically suppress the autofluorescence signal with some reagents such as sodium borohydride, glycine or toluidine blue. However, in many cases, these approaches are either infeasible or ineffective, and none of them fully eliminates the problem. The second way is to use spectral unmixing algorithms subtracting the background fluorescence. This is only possible if you have at your disposal complicated, expensive confocal optics with sophisticated automated software (http //www.cri-inc.com/applications/fluorescence-microscopy.asp). 

Fluorescent pH indicators offer much better sensitivity than the classical dyes such as phenolphthalein, thymol blue, etc., based on color change. They are thus widely used in analytical chemistry, bioanalytical chemistry, cellular biology (for measuring intracellular pH), medicine (for monitoring pH and pCC>2 in blood pCC>2 is determined via the bicarbonate couple). Fluorescence microscopy can provide spatial information on pH. Moreover, remote sensing of pH is possible by means of fiber optic chemical sensors. 

Finally, in Chapter 11 some advanced techniques are briefly described fluorescence up-conversion, fluorescence microscopy (confocal excitation, two-photon excitation, near-field optics, fluorescence lifetime imaging), fluorescence correlation spectroscopy, and single-molecule fluorescence spectroscopy. 

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