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Acoustic transduction by plants has been proposed as a mechanism to enable just-in-time up-regulation of metabolically expensive defensive compounds. Although the mechanisms by which this "hearing" occurs are unknown, mechanosensation by elongated plant hair cells known as trichomes is suspected. To evaluate this possibility, we developed a theoretical model to evaluate the acoustic radiation force that an elongated cylinder can receive in response to sounds emitted by animals, including insect herbivores, and applied it to the long, cylindrical stem trichomes of the tomato plant Solanum lycopersicum. Based on perturbation theory and validated by finite element simulations, the model quantifies the effects of viscosity and frequency on this acoustic radiation force. Results suggest that acoustic emissions from certain animals, including insect herbivores, may produce acoustic radiation force sufficient to trigger stretch-activated ion channels.

Analysis of cellular pathways requires concentration measurements of dynamically interacting molecules within the three-dimensional (3D) space of single living cells. Förster resonance energy transfer (FRET) microscopy from widefield, from confocal, and potentially from superresolution microscopes can access this information; however, these measurements are distorted by the inherent 3D blurring of optical imaging, spectral overlap of fluorophores, and detection noise. We propose a mathematical model of these processes and demonstrate, through simulation, how these distortions limit the dynamic range and sensitivity of conventional FRET microscopy. Using this model, we devise and validate a new approach (called 3D-FRET stoichiometry reconstruction, 3DFSR) for reconstructing 3D distributions of bound and free fluorescent molecules. Previous attempts to reconstruct 3D-FRET data relied on sequential spectral unmixing and deconvolution, a process that corrupts the detection statistics. We demonstrate that 3DFSR is superior to these approaches since it simultaneously models spectral mixing, optical blurring, and detection noise. To achieve the full potential of this technique, we developed an instrument capable of acquiring 3D-FRET data rapidly and sensitively from single living cells. Compared with conventional FRET microscopy, our 3D-FRET reconstruction technique and new instrumentation provides orders of magnitude gains in both sensitivity and accuracy wherein sustained high-resolution four-dimensional (x,y,z,t) imaging of molecular interactions inside living cells was achieved. These results verify previous observations that Cdc42 signaling is localized to the advancing margins of forming phagosomes in macrophages.

Biochemical pathways are comprised of spatially organized interactions between multiple proteins. Fluorescent resonance energy transfer (FRET) microscopy techniques allow imaging of these interactions within living cells. Recently, these techniques have been extended to allow imaging of more than two fluorescently-tagged molecules, but these methods are limited to only two dimensions. Here, we explore the extension of multifluorophore FRET imaging to three-dimensional microscopy by image reconstruction methods. This work takes advantage of our previously developed three-dimensional FRET stoichiometry reconstruction (3D-FSR) and multifluorophore FRET microscopy (MFM). Three-dimensional multifluorophore FRET microscopy (3D-MFM) was utilized to observe the oligomerization of the HIV structural protein Gag, in the three-dimensional space of both fixed and living cells.

Our understanding of clathrin-mediated endocytosis is derived from analysis of live-cell protein recruitment kinetics in combination with static ultrastructure images of coated pit progression. These analyses however, cannot directly correlate membrane curvature dynamics with the arrival and activities of the endocytic machinery. Polarized Total Internal Reflection (pol-TIR) fluorescence microscopy can visualize membrane topology in cells labeled with lipophilic fluorophores whose dipoles align relative to the plasma membrane. We describe a new approach for creating s-pol and p-pol TIR fields in a commercial microscope utilizing a 2-dimensional scan head to position polarized lasers at orthogonal azimuthal positions in the back focal plane of a high numerical aperture TIR objective lens. This configuration reduces interference fringing of the coherent laser light. Ratio imaging of the two excitation polarizations permits visualization of subresolution membrane curvature with subsecond temporal resolution. By monitoring membrane topology relative to the arrival of chromosomally-tagged GFP-dynamin in skin melanoma cells, we were able to define the kinetics of membrane curvature relative to vesicle scission. Blocking the catalytic GTPase cycle of dynamin with the drug, Dynasore permitted visualization of striking curved invaginations, consistent with expected “frozen” clathrin coated pits. Tracking of single dynamin recruitment events provided a region in which to quantify membrane topology. Surprisingly, regions of high curvature were often observed adjacent to clathrin pits. This observation is consistent with a model for actin-mediated membrane protrusions that could drive pit closure. The role for actin was assessed using CFP-LifeAct, and perturbed with the drug, Latrunculin. These studies provide insight into the sequential topological changes during clathrin-mediated endocytosis and move toward a comprehensive understanding of this mechanism.

The spatial organization of protein-protein interactions inside of cells is an important component of cellular signaling. These interactions can be imaged using fluorescent resonance energy transfer (FRET) microscopy methods. We have described a generalized mathematical solution and method for any number of interacting FRET pairs (N-Way FRET). Here, we combine N-Way FRET with our three-dimensional FRET stoichiometry reconstruction (3D-FSR) to allow 3D-3Way-FRET image deconvolution. By developing a multi-camera instrument, we are able to acquire the 6 images needed per z-plane with sub-second time resolution, allowing observation of live cell dynamics. Furthermore, we implemented improved fast Fourier transforms and parallelized calculations to achieve a 10-fold decrease in the computational time required to reconstruct 3D-3Way-FRET data. This reduction has made it feasible to analyze dynamic 2Way and 3Way FRET data with 3D reconstructions, increase the number of reconstruction iterations, as well as explore stopping criteria for the algorithm.

Proteins can generate and recognize curved membranes that in turn, modulate protein clustering, diffusion and biochemical reactions. The biophysical mechanisms of this dynamic relationship influence numerous biologically important events including receptor signal transduction, endocytosis, exocytosis and viral assembly. New approaches are needed to measure the dynamics of membrane bending in conjunction with protein recruitment and assembly. Here, I will discuss our work using back focal plane (BFP) spinning total internal reflection fluorescence (360-TIRF) microscopy to create uniform evanescent fields for quantitative imaging of molecular recruitment and assembly. We have paired these methods with BFP positioning for polarized-TIRF microscopy of membrane-oriented lipophilic fluorophores to image the sub-resolution membrane curvature. Using these methods, we have imaged the membrane bending dynamics during assembly of clathrin and dynamin at single endocytic sites in living cells. Our data demonstrate that this method is capable of imaging sub-resolution endocytic events and within the context of their surrounding topography. Together, these methods are enabling new insights into the interplay between assembly, reaction dynamics and membrane topography in living cells.

Recent advances in fluorescence resonance energy transfer (FRET) microscopy have made it possible to measure simultaneously, multiple protein-protein interactions inside living cells. To further these methods, we integrated an improved multi-spectral imaging analysis method (N-way FRET) with optimized fluorophores for live-cell FRET microscopy. For 3-way FRET we determined the best cyan, yellow, and red (C-Y-R) FRET-trio by comparing the acceptor photobleaching FRET efficiencies of structurally similar linked fluorescent protein constructs. The pool of molecules examined was constrained to the available C, Y, R molecules with the best photophysical properties (CyPet/TFP1, mCitrine/YPet and TagRFP/mCherry, respectively). From this set, we determined that TFP, YPet or mCitrine and mCherry were the optimal FRET trio. For increasingly complex pathways, more fluorescent probes may be required. To extend this method to the analysis of 4-way FRET we examined fluorophore pairs with highly overlapping excitation and emission spectra (e.g. TFP and GFP or Citrine and GFP). These improvements should enhance the spatiotemporal resolution, the number of possible interacting probes and the reliability of FRET experiments leading to improved monitoring of multiple protein-protein interactions in complex pathways.

Induction of membrane curvature is an essential step in the formation of endocytic vesicles and viral particles. Axelrod and co-workers have shown that polarized total internal reflection fluorescence microscopy (pol-TIRFM) can be used to visualize membrane curvature in cells labeled with the lipophilic fluorophore DiI. We describe an approach for creating s-pol and p-pol TIRF fields using a commercial microscope system equipped with a 2-D scan head to position polarized laser beams at distinct azimuthal positions in the back focal plane (BFP) of a high numerical aperture TIRF lens. This configuration allows for reduction of interference fringing of the coherent light by alternating illumination at azimuthal positions separated by 180 degrees. Additional reduction of fringing could be obtained by sweeping the laser through small arcs in the BFP of about 8 degrees with minimal impact on the polarization of the TIRF fields. We investigated the performance of this pol-TIRFM approach for visualization of endocytosis and viral budding in cells labeled with DiI and either the endocytic marker clathrin-YFP or viral budding marker HIV Gag-YFP.

Current methods for fluorescence resonance energy transfer (FRET) microscopy of living cells involve taking a series of images with alternating excitation colors in separate camera exposures. Here we present a new FRET method based on polarization that requires only one camera exposure and thereby offers the possibility for better time resolution of dynamic associations among subcellular components. Polarized FRET (p-FRET) uses a simultaneous combination of excitation wavelengths from two orthogonally polarized sources, along with an emission channel tri-image splitter outfitted with appropriate polarizers, to concurrently excite and collect fluorescence from free donors, free acceptors, and FRET pairs. Based upon the throughput in each emission channel as premeasured on pure samples of each of the three species, decoupling of an unknown sample's three polarized fluorescence images can be performed to calculate the pixel-by-pixel concentrations of donor, acceptor, and FRET pairs. The theory of this approach is presented here, and its feasibility is experimentally confirmed by measurements on mixtures of cyan fluorescent protein (CFP), citrine ((Cit) a yellow fluorescent protein variant), and linked fusion proteins (CFP-L16-Cit, CFP-L7-Cit, CFP-L54-Cit) in living cells. The effects of shot noise, acceptor polarization, and FRET efficiency on the statistical accuracy of p-FRET experimental results are investigated by a noise-simulation program.