Single particle tracking has provided a wealth of information about biophysical processes such as motor protein transport and diffusion in cell membranes. However, motion out of the plane of the microscope or blinking of the fluorescent probe used as a label generally limits observation times to several seconds. Here, we overcome these limitations by using novel non-blinking quantum dots as probes and employing a custom 3D tracking microscope to actively follow motion in three dimensions (3D) in live cells. Signal-to-noise is improved in the cellular milieu through the use of pulsed excitation and time-gated detection.
The ability to follow and observe single molecules as they function in live cells represents a major milestone
for molecular-cellular biology. Here we present a tracking microscope that is able to track quantum dots in
three dimensions and simultaneously record time-resolved emission statistics from a single dot. This innovative
microscopy approach is based on four spatial filters and closed loop feedback to constantly keep a single quantum
dot in the focal spot. Using this microscope, we demonstrate the ability to follow quantum dot labeled IgE
antibodies bound to FcεRI membrane receptors in live RBL-2H3 cells. The results are consistent with prior
studies of two dimensional membrane diffusion (Andrews et al., Nat. Cell Biol., 10, 955, 2008). In addition, the
microscope captures motion in the axial (Z) direction, which permits tracking of diffusing receptors relative to
the "hills and valleys" of the dynamically changing membrane landscape. This approach is uniquely capable of
following single molecule dynamics on live cells with three dimensional spatial resolution.
The development of colloidal quantum dots (QDs) for biological imaging has brought a new level of sensitivity to live
cell imaging. Single particle tracking (SPT) techniques in particular benefit from the superior photostability, high
extinction coefficient and distinct emission spectra of QDs. Here we describe the use of QDs for SPT to study the
dynamics of membrane proteins in living cells. We work with the RBL-2H3 mast cell model that signals through the
high affinity IgE receptor, Fc&Vegr;RI. Using wide field or Total Internal Reflection Fluorescence (TIRF) microscopy we
have achieved simultaneous imaging of two spectrally distinct QDs with frame rates of up to 750 frames/s and
localization accuracy of ~10 nm. We also describe the imaging and analysis of QDs using a novel hyperspectral
microscope and multivariate curve resolution analysis for multi-color QD tracking. The same QD-tag used for SPT is
used to localize proteins at <10 nm resolution by electron microscopy (EM) on fixed membrane sheets.
Innovations in fluorescence microscopy of live cells involving new reagents and techniques reveal dynamic processes that were not previously observable and therefore unknown. Water soluble, biofunctionalized semiconductor quantum dots (QDs) provide advantages of much greater photostability compared to conventional fluorescent dyes, and, as a consequence, single QDs can be easily detected. QDs coupled to growth factor ligands behave similarly as the natural ligand and serve as highly fluorescent probes of the erbB family of tyrosine kinase receptors in living cells. Continuous confocal laser scanning microscopy and flow cytometry measurements of QDs combined with visible fluorescent fusions of the receptors have elucidated individual steps in the signaling cascades initiated by these receptors. This report highlights advantages and some disadvantages of QDs, such as size and blinking behavior that complicate some live cell imaging applications. The new class of noble metal nanodots constitute an attractive alternative to QDs in that they are not only highly fluorescent and photostable, but also, much smaller and nontoxic. We present a new synthesis method for the production of Au nanodots. We demonstrate that electrochemical synthesis allows the reproducible control of cluster size. The resulting clusters are more monodisperse than those formed by other methods and are stable over many months. We report their characterization using MALDI-TOF mass spectrometry and UV-VIS spectroscopy.
Fluorescence anisotropy, a measure of the polarization state of fluorescence emission, is a sensitive measure of molecular rotational motion and of resonance energy transfer (RET). We report here the formalism and application of dynamic and static fluorescence anisotropy measurements primarily intended for implementation in imaging systems. These include confocal lasre scanning microscopes (CLSM) as well as wide-field instruments, in the latter case adapted for anisotropy-based dynamic frequency domain fluorescence lifetime imaging microscopy (FLIM), a method we denote as rFLIM. Anisotropy RET is one of the modalities used for fluorescence RET (FRET) determinations of the association, and proximity of cellular proteins in vivo. A requirement is the existence of intrinsic or extrinsic probes exhibiting homotransfer FRET (in our nomenclature, energy migration or emFRET) between like fluorophores. This phenomenon is particularly useful in studies of the activation and processing of transmembrane receptor tyrosine kinases involved in signal transduction and expressed as fusions with Visible Fluorescence Proteins (VFPs).
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