Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging.

The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on alpha-galactosylceramide (alphaGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from approximately 25 to approximately 160 nm, with the smaller domains corresponding to a single CD1d molecule.

Improving the contrast of topographical AFM images by a simple averaging filter.

New image-processing methods were applied to atomic force microscopy images in order to visualize small details on the surface of virus particles and living cells. Polynomial line flattening and plane fitting of topographical images were performed as first step of the image processing. In a second step, a sliding window approach was used for low-pass filtering and data smoothing. The size of the filtering window was adjusted to the size of the small details of interest. Subtraction of the smoothed data from the original data resulted in images with enhanced contrast. Topographical features which are usually not visible can be easily discerned in the processed images. The method developed in this study rendered possible the detection of small patterns on viral particles as well as thin cytoskeleton fibers of living cells. It is shown that the sliding window approach gives better results than Fourier-filtering. Our method can be generally applied to increase the contrast of topographical images, especially when small features are to be highlighted on relatively high objects.

Recognition force microscopy/spectroscopy of ion channels: applications to the skeletal muscle Ca2+ release channel (RYR1).

The skeletal muscle Ca2+ release channel (ryanodine receptor 1, RYR1) plays an important role in the excitation-contraction coupling process. We purified ryanodine receptor type 1 from rabbit white muscle and adsorbed it to mica sheets with the cytoplasmic side facing up. Single receptors of uniformly distributed size and shape of 10-12 nm height and 40-50 nm width, and occasionally some aggregates were seen in contact mode AFM images. These immobilized RYR1 were specifically recognized by rabbit anti-RYR1 (antibody#8) with at least 30% efficiency, as measured by an enzyme immunoassay with goat-anti-rabbit. Single specific antibody-antigen recognition events were detected with AFM tips to which an antibody#8 was tethered. In linear scans, the occurrence of antibody-antigen binding showed significant lateral dependence, which allowed for the localization of binding sites with nm resolution. Variation of the loading rate in force spectroscopy experiments revealed a logarithmic dependence of the unbinding forces, ranging from 42 to 73 pN. From this dependence, a bond width of the binding pocket of L = 0.2 nm and a kinetic off-rate of koff = 12.7s(-1) was determined.

Anomalous fluorescence enhancement of Cy3 and cy3.5 versus anomalous fluorescence loss of Cy5 and Cy7 upon covalent linking to IgG and noncovalent binding to avidin.

This study provides a critical examination of protein labeling with Cy3, Cy5, and other Cy dyes. Two alternate situations were tested. (i) Antibodies were covalently labeled with Cy dye succinimidyl ester at various fluorophore/protein ratios and the fluorescence of the labeled antibodies was compared to that of free Cy dye. (ii) Fluorescent biotin derivatives were synthesized by derivatizing ethylenediamine with one biotin and one Cy3 (or Cy5) residue. The fluorescence properties of these biotin-Cy dye conjugates were examined at all ligand/(strept)avidin ratios (0

Properties of lipid microdomains in a muscle cell membrane visualized by single molecule microscopy.

The lateral motion of single fluorescence labeled lipid molecules was imaged in native cell membranes on a millisecond time scale and with positional accuracy of approximately 50 nm, using 'single dye tracing'. This first application of single molecule microscopy to living cells rendered possible the direct observation of lipid-specific membrane domains. These domains were sensed by a lipid probe with saturated acyl chains as small areas in a liquid-ordered phase: the probe showed confined but fast diffusion, with high partitioning (approximately 100-fold) and long residence time (approximately 13 s). The analogous probe with mono-unsaturated chains diffused predominantly unconfined within the membrane. With approximately 15 saturated probes per domain, the locations, sizes, shapes and motions of individual domains became clearly visible. Domains had a size of 0.7 micrometer (0.2-2 micrometer), covering approximately 13% of total membrane area. Both the liquid-ordered phase characteristics and the sizes of domains match properties of membrane fractions described as detergent-resistant membranes (DRMs), strongly suggesting that the domains seen are the in vivo correlate of DRMs and thus may be identified as lipid rafts.

Preparation of thiol-reactive Cy5 derivatives from commercial Cy5 succinimidyl ester.

The present study offers reliable protocols for the preparation of new thiol-reactive Cy5 derivatives which are urgently needed for single molecule fluorescence microscopy. In a systematic approach, two alternate strategies were found for the extension of commercial amine-reactive Cy5 with thiol-reactive end groups. In the two-step method, Cy5 succinimidyl ester was first reacted with ethylenediamine under conditions which gave approximately 99% asymmetric "Cy5-amine" and only approximately 1% symmetric product with two Cy5 residues. Subsequently, "Cy5-amine" was derivatized with commercial heterobifunctional cross-linkers to introduce thiol-reactive end groups (maleimide or pyridyldithio). Alternatively, commercial Cy5 succinimidyl ester was reacted with a primary amine (MTSEA, methanethiosulfonylethylamine, or PDEA, pyridyldithioethylamine) or a secondary amine (PEM, piperazinylethylmaleimide) to give the corresponding thiol-reactive derivatives in a single step. Results were good for MTSEA, moderate for PEM, and poor for PDEA. An additional drawback of the one-step method was the need for rigorous removal of unreacted Cy5 succinimidyl ester, which would label lysine residues on probe molecules. It is concluded that, except for the Cy5-MTSEA conjugate, the two-step method is much more general, reliable, and easier to follow by the typical biophysicist, biologist, etc., for whose benefit, these procedures are being published. All thiol-reactive Cy5 derivatives showed similar absorption and fluorescence properties as Cy5 succinimidyl ester, and fluorescence was fully retained after binding to thiols on proteins. The kinetics of protein labeling was also examined in order to get an idea of proper labeling conditions.

Accurate measurement of avidin and streptavidin in crude biofluids with a new, optimized biotin-fluorescein conjugate.

A new biotin-fluorescein conjugate with an ethylene diamine spacer was found to be the first fluorescent biotin derivative which truly mimicked d-biotin in terms of high affinity, fast association, and non-cooperative binding to avidin and streptavidin tetramers. These exceptional properties were attributed to the small size/length of the new ligand since all larger/longer biotin derivatives are known for their mutual steric hindrance and anti-cooperative binding in 4:1 complexes with avidin and streptavidin tetramers. Specific binding of the new biotin-fluorescein conjugate towards avidin and streptavidin was accompanied by 84-88% quenching of ligand fluorescence. In the accompanying study this effect was used for rapid estimation of avidin and streptavidin in a new 'single tube assay'. In the present study the strong quenching effect was utilized to accurately monitor stoichiometric titration of biotin-binding sites in samples with >/=200 pM avidin or streptavidin. The concentration was calculated from the consumption of fluorescent ligand up to the distinct breakpoint in the fluorescence titration profile which was marked by the abrupt appearance of strongly fluorescent ligands which were in excess. Due to this protocol the assay was not perturbed by background fluorescence or coloration in the unknown samples. The new fluorescence titration assay is particularly suited for quick checks on short notice because getting started only means to thaw an aliquot of a standardized stock solution of fluorescent ligand. No calibration is required for the individual assay and the ligand stock solution needs to be restandardized once per week (or once per year) when stored at -25 degrees C (or at -70 degrees C, respectively).

Rapid estimation of avidin and streptavidin by fluorescence quenching or fluorescence polarization.

A new biotin-carboxyfluorescein conjugate has been presented in the accompanying study (G. Kada et al., Biochim. Biophys. Acta 000 (1999) 000-000) which contains ethylene diamine as a 4-atom spacer. This so-called biotin-4-fluorescein showed exceptionally fast and tight binding to avidin and streptavidin, and binding was accompanied by strong quenching. In the present study the specific quenching of 'biotin-4-fluorescein' was utilized to measure (strept)avidin concentrations (0.2-2 nM) by the extent of fluorescence quenching at 8 nM ligand concentration. Adsorption of (strept)avidin to the assay tubes was suppressed by inclusion of bovine serum albumin (0.1 mg/ml). Virtually the same specific response to avidin and streptavidin was also observed with commercial 'fluorescein-biotin', except that >10 h incubation times were required. The slow association of 'fluorescein-biotin' was attributed to the anti-cooperative binding which is due to the much longer spacer as compared to 'biotin-4-fluorescein'. The third ligand tested in this study was 'biotin-4-FITC' which was analogous to 'biotin-4-fluorescein' except that carboxyfluorescein was replaced by the fluorescein isothiocyanate residue. Surprisingly, this probe was much less quenched by avidin but this was compensated by an exceptionally high fluorescence polarization in the avidin-bound state. In conclusion, the new ligand 'biotin-4-fluorescein' appeared to be the most general and convenient probe: quenching was most pronounced and linearly dependent on (strept)avidin concentrations, the dose response for streptavidin was almost the same as for avidin, and the association kinetics were fast enough to reach equilibrium within 30 min incubation time.

Accurate titration of avidin and streptavidin with biotin-fluorophore conjugates in complex, colored biofluids.

A new fluorimetric assay is presented for the specific and reliable quantitation of >/=2 nM avidin and streptavidin. The assay is based on pronounced changes in the fluorescence properties of commercial fluorescein-biotin, or of a newly synthesized biotin-poly(ethylene glycol)-pyrene conjugate, which occur upon binding to avidin and streptavidin. Accurate measurement of (strept)avidin in complex, colored biofluids, such as crude egg white or serum relies on a simple titration protocol. Only occasional recalibration of the reagent solution is required. Due to these merits the proposed assay is particularly suited for rapid measurement of few samples on short notice, for functional control of (strept)avidin-containing reagents after storage, and for the monitoring of (strept)avidin concentrations in large scale processes.