Quantitative reflectance microscopy

Fluorescence microscopy is routinely used to study the location and movement of intracellular species. In general, the fluorescence image reflects the location and concentration of the probe, or that amount of probe remaining in a photobleached sample (Figure 1.8, lower left). Consequently, quantitative fluorescence microscopy... 

D. Gingell and I. Todd, Interference reflection microscopy. A quantitative theory for image interpretation and its application to cell-substratum separation measurement, Biophys. J. 26, 507-526 (1979). 

D.C. Prieve and N.A. Frej Total Internal Reflection Microscopy A Quantitative Tool for the Measurement of Colloidal Forces. Langmuir 6, 396 (1990). 

Schindl, M., WaUraff, E., Deubzer, B. et al. (1995). Cell-substrate interactions and locomotion of Dictyostelium wild-type and mutants defective in three cytoskeletal proteins a study using quantitative reflection interference contrast microscopy. Biophys. ]. 68, 1177-1190. 

Total Internal Reflection Microscopy serves, e.g., for obtaining the height distribution function between an - in most cases spherical - object and a surface from quantitative, time-resolved measurements of the intensity of light scattered within the evanescent wave. From the height distribution function one might find the interaction energy between object and surface. 

Pigment particles to be studied by electron microscopy are sometimes incorporated into ultrathin membranes. At a first glance, a quantitative image analysis of such ultrathin layers seems convenient, since it appears to reflect the distribu-... 

As a consequence, researchers from different disciplines of the life sciences ask for efficient and sensitive techniques to characterize protein binding to and release from natural and artificial membranes. Native biological membranes are often substituted by artificial lipid bilayers bearing only a limifed number of components and rendering the experiment more simple, which permits the extraction of real quantitative information from binding experiments. Adsorption and desorption are characterized by rate constants that reflect the interaction potential between the protein and the membrane interface. Rate constants of adsorption and desorption can be quantified by means of sensitive optical techniques such as surface plasmon resonance spectroscopy (SPR), ellipsometry (ELL), reflection interference spectroscopy (RIfS), and total internal reflection fluorescence microscopy (TIRE), as well as acoustic/mechanical devices such as the quartz crystal microbalance (QCM)... 

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