Analysis of receptor clustering on cell surfaces by imaging fluorescent particles

Abstract

Fluorescently labeled low density lipoproteins (LDL) and influenza virus particles were bound to the surface of human fibroblasts and imaged with a cooled slow-scan CCD camera attached to a fluorescence microscope. Particles were also imaged after attachment to polylysine-coated microscope slides. The digital images were analyzed by fitting data points in the region of fluorescent spots by a two-dimensional Gaussian function, thus obtaining a measure of spot intensity with correction for local background. The intensity distributions for particles bound to polylysine slides were mainly accounted for by particle size distributions as determined by electron microscopy. In the case of LDL, the intensity distributions for particles bound to fibroblasts were considerably broadened, indicative of clustering. The on-cell intensity distributions were deconvolved into 1-particle, 2-particle, 3-particle, etc. components using the data obtained with LDL bound to polylysine-coated slides as an empirical measure of the single particle intensity distribution. This procedure yielded a reasonably accurate measure of the proportion of single particles, but large errors were encountered in the proportions of larger cluster sizes. The possibility of studying the dynamics of clustering was investigated by binding LDL to cells at 4 degrees C and observing changes in the intensity distribution with time after warming to 20 degrees C.

Low density lipoprotein and liposome mediated uptake and cytotoxic effect of N4-octadecyl-1-β-D-arabinofuranosylcytosine in Daudi lymphoma cells

“…The apolipoprotein B 100 content of the LDL preparations was measured with a Bio-Rad protein assay (Bio-Rad, Glattbrugg, Switzerland) using albumin as standard. LDL labelling with DiI was performed as described by Morrison et al (1994). DiI concentration was determined fluorometrically as described for DiI-liposomes yielding an average of 100 DiI molecules per LDL.…”

Interaction of HLA‐DR and CD74 at the cell surface of antigen‐presenting cells by single particle image analysis

“…Computational methods used in SPFI have been described previously (26). Images were analyzed using ImageJ: Interactive 3D Surface Plot (U.S. National Institutes of Health, Bethesda, MD, USA).…”

“…In our laboratory, we have developed the technique of single‐particle fluorescence imaging (SPFI); the basic concept of SPFI is to use the high sensitivity of fluorescence to visualize individual receptors in the plasma membrane of living cells. Receptors are selectively labeled with a fluorescent protein‐antibody conjugate, and the particles imaged using a cooled charge‐coupled device (CCD) camera attached to a fluorescence microscope (26–28). Suitable probes are made by attaching a monoclonal antibody (or its Fab fragment) to either a phycobiliprotein or a fluorescent latex microsphere (29).…”

Analyzing protein–protein interactions in cell membranes

“…Third, one must determine the fluorescence intensity per molecule, a number that depends on excitation efficiency and emission detection effectiveness. This has been attempted, (14) and a detailed analysis yields the distribution of the number of molecules per cluster as well as the average number of molecules per cluster and the total number of clusters. However, in practice, when the noise in the signal is significant, the analysis of each individual cluster is not reliable.…”