Determination of Domain Size and Overlap between Fluorescence Distributions using Fluorescence Microscopy

The lateral distribution of cell-surface proteins and their co-localization in membrane microdomains can be studied by digital imaging microscopy with a resolution of 200-300 nm, as set by Abbe's law [35]. The application of confocal laser scanning microscopy or multiphoton excitation [36] greatly improves image sharpness and contrast by excluding photons arising from out-of-focus planes. This resolution is not sufficient to directly indicate molecular associations; however, it allows the observation of overlap/segregation between different protein clusters or lipid microdomains. Quantitative analysis of the size of clusters/microdomains can be derived from the spatial autocorrelation function of the fluorescence intensity distribution [39]. Using this approach, cluster sizes of predominantly raft-localized proteins IL-2Ra, HLA I and II, as well as GPI-anchored proteins CD48, were determined to be ~600-700 nm, which coincided well with cluster sizes determined from electron microscopic analysis of immunogold-labeled samples. These domain sizes correlated well with the mean barrier-free path measured for the MHC I glycoprotein and its truncation mutants [37], as well as dimensions of confinement zones derived from single particle tracking in which E-cadherin or epidermal growth factor receptor (EGFR) molecules could freely diffuse [38]. Disruption of lipid rafts by cholesterol extraction blurred the boundaries of protein clusters and extended their diameter to ~1000 nm [39].

The extent of overlap between clusters of different membrane proteins and lipid microdomains can be characterized by the cross-correlation coefficient of the pixel intensities of individual fluorescence distributions [11,39,40]. For a pair of images x and y, the cross-correlation coefficient is calculated as:

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Essentials of Human Physiology

Essentials of Human Physiology

This ebook provides an introductory explanation of the workings of the human body, with an effort to draw connections between the body systems and explain their interdependencies. A framework for the book is homeostasis and how the body maintains balance within each system. This is intended as a first introduction to physiology for a college-level course.

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