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SUMMARY Crosslinking of IgE-bound FcεRI triggers mast cell degranulation. Previous FRAP and phosphorescent anisotropy studies suggested that FcεRI must immobilize to signal. Here, single quantum dot (QD) tracking and hyperspectral microscopy methods are used to redefine relationships between receptor mobility and signaling. QD-IgE-FcεRI aggregates of at least three receptors remain highly mobile over extended times at low concentrations of antigen that induce Syk kinase activation and near-maximal secretion. Multivalent antigen, presented as DNP-QD, also remains mobile at low doses that support secretion. FcεRI immobilization is marked at intermediate and high antigen concentrations, correlating with increases in cluster size and rates of receptor internalization. The kinase inhibitor PP2 blocks secretion without affecting immobilization or internalization. We propose that immobility is a feature of highly crosslinked immunoreceptor aggregates, is a trigger for receptor internalization, and is not required for tyrosine kinase activation leading to secretion.

The quantitative prediction abilities of four multivariate calibration methods for spectral analyses are compared by using extensive Monte Carlo simulations. The calibration methods compared Include Inverse least-squares (ILS), classical least-squares (CLS), partial least-squares (PLS), and principal component regression (PCR) methods. ILS is a frequencylimited method while the latter three are capable of fullspectrum calibration. The simulations were performed assuming Beer's law holds and that spectral measurement errors and concentration errors associated with the reference method are normally distributed. Eight different factors that could affect the relative performance of the calibration methods were varied In a two-level, eight-factor experimental design In order to evaluate their effect on the prediction abilities of the four methods. It Is found that each of the three full-spectrum methods has its range of superior performance.The frequency-limited ILS method was never the best method, although In the presence of relatively large concentration errors it sometimes yields comparable analysis precision to the full-spectrum methods for the major spectral component. The Importance of each factor In the absolute and relative performances of the four methods is compared. A relatively simple model Involving the mean squared prediction errors Is developed for estimating the prediction errors for each calibration method over the range of variation of the factors considered. These results offer the analyst guidelines to be used In evaluating which multivariate calibration method will provide the best predictions when applied to a given spectral data set. In the absence of specific Information about the data set, we would recommend the use of PLS since It Is usually optimal or close to optimal.

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Hyperspectral confocal fluorescence imaging provides the opportunity to obtain individual fluorescence emission spectra in small (Ϸ0.03-m 3 ) volumes. Using multivariate curve resolution, individual fluorescence components can be resolved, and their intensities can be calculated. Here we localize, in vivo, photosynthesis-related pigments (chlorophylls, phycobilins, and carotenoids) in wild-type and mutant cells of the cyanobacterium Synechocystis sp. PCC 6803. Cells were excited at 488 nm, exciting primarily phycobilins and carotenoids. Fluorescence from phycocyanin, allophycocyanin, allophycocyanin-B/terminal emitter, and chlorophyll a was resolved. Moreover, resonance-enhanced Raman signals and very weak fluorescence from carotenoids were observed. Phycobilin emission was most intense along the periphery of the cell whereas chlorophyll fluorescence was distributed more evenly throughout the cell, suggesting that fluorescing phycobilisomes are more prevalent along the outer thylakoids. Carotenoids were prevalent in the cell wall and also were present in thylakoids. Two chlorophyll fluorescence components were resolved: the shortwavelength component originates primarily from photosystem II and is most intense near the periphery of the cell; and the long-wavelength component that is attributed to photosystem I because it disappears in mutants lacking this photosystem is of higher relative intensity toward the inner rings of the thylakoids. Together, the results suggest compositional heterogeneity between thylakoid rings, with the inner thylakoids enriched in photosystem I. In cells depleted in chlorophyll, the amount of both chlorophyll emission components was decreased, confirming the accuracy of the spectral assignments. These results show that hyperspectral fluorescence imaging can provide unique information regarding pigment organization and localization in the cell.cyanobacteria ͉ photosynthetic pigments ͉ multivariate curve resolution C yanobacteria convert light energy to chemical energy by means of photosynthesis, using water as a source of electrons for CO 2 reduction and O 2 production. A key part of the photosynthesis process is light absorption (harvesting) by pigments, followed by excitation transfer to reaction center chlorophyll (Chl) a of photosystems (PS) II and I (1). These processes take place in thylakoid membranes that in cyanobacteria generally form an extensive internal membrane complex of several layers along the periphery of the cytoplasm, with thylakoids found less frequently toward the center of the cell (2).The pigments associated with the photosynthetic apparatus are bound to thylakoid proteins, modifying their spectral properties and providing a spatial distribution that aids in the efficiency of light harvesting and energy transfer to reaction center Chls. Pigments bound to integral membrane proteins in reaction center complexes in thylakoids of cyanobacteria include Chl a [Ϸ40 per PS II (3) and Ϸ100 per PS I (4)] and carotenoids; the latter act in photoprotection and 3 Chl quenchin...

We have developed a new, high performance, hyperspectral microscope for biological and other applications. For each voxel within a three-dimensional specimen, the microscope simultaneously records the emission spectrum from 500 nm to 800 nm, with better than 3 nm spectral resolution. The microscope features a fully confocal design to ensure high spatial resolution and high quality optical sectioning. Optical throughput and detection efficiency are maximized through the use of a custom prism spectrometer and a backside thinned electron multiplying charge coupled device (EMCCD) array. A custom readout mode and synchronization scheme enable 512-point spectra to be recorded at a rate of 8300 spectra per second. In addition, the EMCCD readout mode eliminates curvature and keystone artifacts that often plague spectral imaging systems. The architecture of the new microscope is described in detail, and hyperspectral images from several specimens are presented.