Keith A. Lidke

Measuring image resolution in optical nanoscopy

Resolution in optical nanoscopy (or super-resolution microscopy) depends on the localization uncertainty and density of single fluorescent labels and on the samples spatial structure. Currently there is no integral, practical resolution measure that accounts for all factors. We introduce a measure based on Fourier ring correlation (FRC) that can be computed directly from an image. We demonstrate its validity and benefits on two-dimensional (2D) and 3D localization microscopy images of tubulin and actin filaments. Our FRC resolution method makes it possible to compare achieved resolutions in images taken with different nanoscopy methods, to optimize and rank different emitter localization and labeling strategies, to define a stopping criterion for data acquisition, to describe image anisotropy and heterogeneity, and even to estimate the average number of localizations per emitter. Our findings challenge the current focus on obtaining the best localization precision, showing instead how the best image resolution can be achieved as fast as possible.

Actin restricts Fc|[epsiv]|RI diffusion and facilitates antigen-induced receptor immobilization

The actin cytoskeleton has been implicated in restricting diffusion of plasma membrane components. Here, simultaneous observations of quantum dot-labelled FcɛRI motion and GFP-tagged actin dynamics provide direct evidence that actin filament bundles define micron-sized domains that confine mobile receptors. Dynamic reorganization of actin structures occurs over seconds, making the location and dimensions of actin-defined domains time-dependent. Multiple FcɛRI often maintain extended close proximity without detectable correlated motion, suggesting that they are co-confined within membrane domains. FcɛRI signalling is activated by crosslinking with multivalent antigen. We show that receptors become immobilized within seconds of crosslinking. Disruption of the actin cytoskeleton results in delayed immobilization kinetics and increased diffusion of crosslinked clusters. These results implicate actin in membrane partitioning that not only restricts diffusion of membrane proteins, but also dynamically influences their long-range mobility, sequestration and response to ligand binding.

Reaching out for signals: filopodia sense EGF and respond by directed retrograde transport of activated receptors

ErbB1 receptors situated on cellular filopodia undergo systematic retrograde transport after binding of the epidermal growth factor (EGF) and activation of the receptor tyrosine kinase. Specific inhibitors of the erbB1 receptor tyrosine kinase as well as cytochalasin D, a disruptor of the actin cytoskeleton, abolish transport but not free diffusion of the receptor–ligand complex. Diffusion constants and transport rates were determined with single molecule sensitivity by tracking receptors labeled with EGF conjugated to fluorescent quantum dots. Retrograde transport precedes receptor endocytosis, which occurs at the base of the filopodia. Initiation of transport requires the interaction and concerted activation of at least two liganded receptors and proceeds at a constant rate mediated by association with actin. These findings suggest a mechanism by which filopodia detect the presence and concentration of effector molecules far from the cell body and mediate cellular responses via directed transport of activated receptors.

Simultaneous multiple-emitter fitting for single molecule super-resolution imaging

Single molecule localization based super-resolution imaging techniques require repeated localization of many single emitters. We describe a method that uses the maximum likelihood estimator to localize multiple emitters simultaneously within a single, two-dimensional fitting sub-region, yielding an order of magnitude improvement in the tolerance of the analysis routine with regards to the single-frame active emitter density. Multiple-emitter fitting enables the overall performance of single-molecule super-resolution to be improved in one or more of several metrics that result in higher single-frame density of localized active emitters. For speed, the algorithm is implemented on Graphics Processing Unit (GPU) architecture, resulting in analysis times on the order of minutes. We show the performance of multiple emitter fitting as a function of the single-frame active emitter density. We describe the details of the algorithm that allow robust fitting, the details of the GPU implementation, and the other imaging processing steps required for the analysis of data sets.

ErbB1 dimerization is promoted by domain co-confinement and stabilized by ligand binding

The extent to which ligand occupancy and dimerization contribute to erbB1 signaling is controversial. To examine this, we used two-color quantum-dot tracking for visualization of the homodimerization of human erbB1 and quantification of the dimer off-rate (koff) on living cells. Kinetic parameters were extracted using a three-state hidden Markov model to identify transition rates between free, co-confined and dimerized states. We report that dimers composed of two ligand-bound receptors are long-lived and their koff is independent of kinase activity. By comparison, unliganded dimers have a more than four times faster koff. Transient co-confinement of receptors promotes repeated encounters and enhances dimer formation. Mobility decreases more than six times when ligand-bound receptors dimerize. Blockade of erbB1 kinase activity or disruption of actin networks results in faster diffusion of receptor dimers. These results implicate both signal propagation and the cortical cytoskeleton in reduced mobility of signaling-competent erbB1 dimers.

One- and two-photon photoactivation of a paGFP-fusion protein in live Drosophila embryos

We constructed a photoactivatable Drosophila histone 2 A variant green fluorescent fusion protein (H2AvD‐paGFP) for tracking chromatin loci in living Drosophila embryos. Activation of paGFP was achieved by irradiation from a single‐photon diode laser at 408 nm, but activated nuclei failed to divide. Photoconversion could also be achieved by two‐photon fs pulses in the range of 780–840 nm. Viability in whole‐mount embryos could only be maintained at 820 nm, at which we could activate, simultaneously track and quantitate the mobility of multiple fluorescent loci. This report constitutes the first demonstration of two‐photon activation of paGFP and the use of a paGFP‐fusion protein in investigations of whole organisms.

Multi-Color Quantum Dot Tracking Using a High-Speed Hyperspectral Line-Scanning Microscope

Many cellular signaling processes are initiated by dimerization or oligomerization of membrane proteins. However, since the spatial scale of these interactions is below the diffraction limit of the light microscope, the dynamics of these interactions have been difficult to study on living cells. We have developed a novel high-speed hyperspectral microscope (HSM) to perform single particle tracking of up to 8 spectrally distinct species of quantum dots (QDs) at 27 frames per second. The distinct emission spectra of the QDs allows localization with ∼10 nm precision even when the probes are clustered at spatial scales below the diffraction limit. The capabilities of the HSM are demonstrated here by application of multi-color single particle tracking to observe membrane protein behavior, including: 1) dynamic formation and dissociation of Epidermal Growth Factor Receptor dimers; 2) resolving antigen induced aggregation of the high affinity IgE receptor, FcεR1; 3) four color QD tracking while simultaneously visualizing GFP-actin; and 4) high-density tracking for fast diffusion mapping.

Dual-color superresolution microscopy reveals nanoscale organization of mechanosensory podosomes

Podosomes are multimolecular mechanosensory structures with a protrusive actin core and an adhesive ring of integrins and adaptor proteins. Dual-color direct stochastic optical reconstruction microscopy is used to reveal the nanoscale localization of the ring components αMβ2 integrin, talin, and vinculin with respect to the actin core.

Imaging Quantum Dots Switched On and Off by Photochromic Fluorescence Resonance Energy Transfer (pcFRET)

ABSTRACT The reversible modulation of the emission of CdSe/ZnS semiconductor nanocrystals (quantum dots) was achieved by binding photochromic diheteroarylethenes and switchable acceptors for fluorescence resonance energy transfer. A biotinylated diheteroarylethene derivative was bound to quantum dots bearing conjugated streptavidin, leading to an intensity decrease as a consequence of energy transfer to the closed form of the acceptor. Interconversion between the open and closed forms by irradiation with 365 and 546 nm light enabled deactivation and activation, respectively, of the FRET process with a corresponding modulation of quantum dot emission, observed both in solution and by sequential wide-field imaging.

The role of photon statistics in fluorescence anisotropy imaging

Anisotropy imaging can be used to image resonance energy transfer between pairs of identical fluorophores and, thus, constitutes a powerful tool for monitoring protein homo-association in living single cells. The requirement for only a single fluorophore significantly simplifies biological preparation and interpretation. We use quantitative methods for the acquisition and image processing of anisotropy data that return the expected error of the anisotropy per pixel based on photon statistics. The analysis methods include calibration procedures and allow for a balance in spatial, anisotropy, and temporal resolution. They are featured here with anisotropy images of fluorescent calibration beads and enhanced green fluorescent protein complexes in live cells.