ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTIntensity fluctuation spectroscopy of laser light scattered by solutions of spherical viruses. R17, Q.beta., BSV, PM2, and T7. I. Light-scattering techniquePeter N. Pusey, Dennis E. Koppel, Dale E. Schaefer, Rafael D. Camerini-Otero, and Seymour H. KoenigCite this: Biochemistry 1974, 13, 5, 952–960Publication Date (Print):February 1, 1974Publication History Published online1 May 2002Published inissue 1 February 1974https://pubs.acs.org/doi/10.1021/bi00702a020https://doi.org/10.1021/bi00702a020research-articleACS PublicationsRequest reuse permissionsArticle Views367Altmetric-Citations141LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
During spermiogenesis and epididymal transit, proteins on the sperm surface become localized to specific domains. In at least one case (PH-20), the protein is initially inserted throughout the membrane and subsequently becomes restricted to a domain by some mechanism that has not yet been determined. Other proteins could become localized through localized insertion. The sperm surface is a dynamic structure that is altered even after the spermatozoon leaves the male. In the female reproductive tract the spermatozoa undergo capacitation and the acrosome reaction that enables them to fertilize the egg. Both of these processes are accompanied by alterations in protein localization: the PT-1 protein migrates during capacitation, and the PH-20 protein migrates after the acrosome reaction. In addition, an upregulation of the surface expression of PH-20 occurs during the acrosome reaction. This additional PH-20 is incorporated into the plasma membrane by the irreversible fusion of the acrosomal membrane with the plasma membrane. The acrosomal membrane contains PH-20 protein that has been stored there since the formation of the acrosome at the spermatid stage of spermiogenesis. Proteins that are freely diffusing must be maintained in a domain by a mechanism that does not involve immobilization or slowing of protein diffusion. We have suggested that barriers to membrane protein diffusion exist at the equatorial region, the posterior ring, and the annulus and that they are responsible for maintaining a localized distribution of at least some of the surface proteins. The migration of surface proteins could result from an alteration of these barriers, a change in the protein structure so that it can pass through the barrier, or active transport across the barrier. These observed changes in surface expression (localization and the level of expression) may be acting to control surface function post-testicularly.
The photocount autocorrelation of non-Gaussian scattered laser light is discussed as a tool for the study of both very dilute or highly correlated scattering systems. A scaled photocount correlation technique is introduced and shown to produce a function directly proportional to the correlation function of the integrated scattered intensity, independent of the field statistics. This technique overcomes the serious problems of interpretation that can arise when the commonly used clipped correlation technique is applied to non-Gaussian fields. The scaled correlation technique is demonstrated experimentally with a simple scattering system, a small number of particles under uniform translational motion.
SUMMARY The method of cumulants has been applied to digital video fluorescence microscopy. The method is used to reconstruct the distribution of fluorescent molecules before the initiation of fluorescence photobleaching, and to characterize heterogeneous photobleaching by imaging one or more of the cumulants of the bleaching decay rate. Using the pipelined pixel processor of the image analysis system for the bulk of the calculations, rather than the general‐purpose host‐computer CPU, the video kinetics imaging can be performed in near real‐time. The method is applied to chick embryo myotubes labelled with fluorescein‐conjugated α‐bungarotoxin. The pre‐bleach fluorescence distribution is derived, and the image of fluorescein fluorescence is separated from glutaraldehyde‐induced autofluorescence on the basis of the spatially resolved average photobleaching decay rate.
The lateral mobility of fluorescein-labeled membrane glycoproteins was measured in whole unlysed erythrocytes and erythrocyte ghosts by the technique of “fluorescence redistribution after fusion.” Measurements were made on polyethylene glycol-fused cell pairs in which only one member of the couplet was initially fluorescently labeled. Diffusion coefficients were estimated from the rate of fluorescence redistribution determined from successive scans with a focused laser beam across individual fused pairs. This technique allows for the analysis of diffusion within cell membranes without the possible damaging photochemical events caused by photobleaching. It was found that lateral mobility of erythrocyte proteins can be increased by the addition of polyphosphates (i.e., ATP and 2,3-diphosphoglycerate) and decreased by the addition of organic polyamines (i.e., neomycin and spermine). This control is exerted by these molecules only when they contact the cytoplasmic side of the membrane and is not dependent upon high-energy phosphates. Microviscosity experiments employing diphenylhexatriene demonstrated no changes in membrane lipid state as a function of these reagents. Our results, in conjunction with data on the physical interactions of cytoskeletal proteins, suggest that the diffusion effector molecules alter the lateral mobility of erythrocyte membrane proteins through modifications of interactions in the shell, which is composed of spectrin, actin, and component 4.1.
