1028 Biochemical Society Transactions (2003) Volume 31, part 5 Measurements of associations of cell-surface receptors by single-particle fluorescence imaging R.J. Cherry 1 , I.E.G. Morrison, I. Karakikes, R.E. Barber, G. Silkstone and N. Fern ´ andez Department of Biological Sciences, University of Essex, Colchester CO4 3SQ, U.K. Abstract SPFI (single-particle fluorescence imaging) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labelled with small fluorescent particles. The images of particles are diffraction-limited spots that are analysed by fitting with a two-dimensional Gaussian function. The spot intensities depend on whether they arise from one or more particles; this provides the basis for determining self-association of cell-surface receptors. We have used this approach to determine dimerization of MHC class II molecules and its disruption by interface peptides. We have also exploited the positional information obtained from SPFI to detect co-localization of cell-surface molecules. This involves labelling two different molecules with different coloured fluorophores and determining their positions separately by dual wavelength imaging. The images are analysed to quantify the overlap of the particle images and hence determine the extent of co-localization of the labelled molecules. The technique provides quantification of the extent of co-localization and can detect whether co-localized molecules occur singly or in clusters. We have obtained preliminary data for co-localization of lipopolysaccharide and CD14 on intact cells. We also show that HLA-DR (human leukocyte antigen-DR) and CD74 are partially co-localized and that interaction between these molecules involves the peptide-binding groove of HLA-DR. Introduction Signalling and other functions of cell-surface receptors often involve transient or long-lived associations with other membrane-bound molecules. Self-association, particularly dimerization, of receptors is also often essential to function. The existence of molecular associations may be deduced from applying biochemical and molecular biological techniques while more direct evidence of associations in intact cells is often obtained from FRET (fluorescence resonance energy transfer) measurements. There has of late been much interest in techniques for detecting events at the level of single molecules. Such an approach can provide much more detailed information than that obtained from more traditional methods. In our labora- tory, we have developed applications of SPFI (single-particle fluorescence imaging) to study molecular associations. The basic concept of SPFI is to use the high sensitivity of fluorescence to visualize individual receptors in the plasma membranes of living cells. Receptors are selectively labelled with a small fluorescent particle and the particles imaged using a cooled CCD camera attached to a fluorescence microscope [1–3]. Suitable probes are made by attaching a monoclonal antibody (or its Fab fragment) to either a phycobiliprotein or a fluorescent latex microsphere [4]. Phycobiliproteins are particularly advantageous because of their small size [PhyE (R-phycoerythrin) is 11 nm × 8 nm] and minimal Key words: CD74, CD14, co-localization, lipopolysaccharide, MHC class II, phycobiliprotein. Abbreviations used: FRET, fluorescence resonance energy transfer; SPFI, single-particle fluorescence imaging; CLIP, class II invariant chain peptide; LPS, lipopolysaccharide; PhyE, R- phycoerythrin; APhyC, allophycocyanin; HLA, human leukocyte antigen. 1 To whom correspondence should be addressed (e-mail cherr@essex.ac.uk). non-specific binding. The images of the small particles used for SPFI are diffraction patterns that approximate to a two- dimensional Gaussian function. By fitting the intensity distribution in an individual fluorescent spot to a two- dimensional Gaussian function, the intensity of the spot and its position can be determined. Determination of receptor self-association by SPFI Analysis of spot intensities obtained in SPFI experiments provides a method for detecting self-association of receptors in living cells. The principle of the method is that monomers, dimers, trimers . . . of a receptor will bind 1, 2, 3 . . . particles provided the probe is added at saturating concentrations. The spot intensities thus have intensities that are multiples of the intensity of a single particle. A histogram of spot intensities will in principle exhibit peaks corresponding to different oligomeric states of the receptor (see Figure 1). In practice the peaks are considerably broadened even if, as is the case with phycobiliproteins, all particles have the same number of fluorophores. This is because the low-intensity images are inherently noisy, also exposure times that optimize signal-to-noise cause some random photobleaching of the probe. Consequently, histograms of spot intensities have to be deconvolved to determine the contributions from 1, 2, 3 . . . particles. To aid this analysis, the intensities of single particles are normally determined by imaging the probe bound to poly-L-lysine-coated slides. Spots that have intensities corresponding to two or more particles occur when the particles are too close to be resolved C  2003 Biochemical Society