Biology subjects

Lidke, K. A.

Publications and source records attributed to Lidke, K. A..

15 recordsLinked to original sources

bioRxiv2025

Separation measurement of two freely rotating dipole emitters at near optimal precision

According to Rayleighs criterion, two incoherent emitters with a separation below the diffraction limit are not resolvable with a conventional fluorescence microscope. One method of Super-Resolution Microscopy (SRM) circumvents the diffraction-limited resolution by precisely estimating the position of spatiotemporally independent emitters. However, these methods of SRM techniques are not optimal for estimating the separation of two simultaneously excited emitters. Recently, a number of detection methods based on modal imaging have been developed to achieve the quantum Cramer-Rao lower bound (QCRB) to estimate the separations between two nearby emitters. The QCRB determines the minimum achievable precision for all possible detection methods. Current modal imaging techniques assume a scalar field generated from a point source, such as a distant source from an optical fiber or a pinhole. However, for fluorescently labeled samples, point emitters are single fluorophores that are modeled as dipole emitters and, in practice, are often freely rotating. Dipole radiation must be described by vectorial theory, and the assumption of a scalar field no longer holds. Here, we present a method to numerically calculate the QCRB for measuring the separation of two dipole emitters, incorporating the vectorial theory. Furthermore, we propose a near-quantum optimal detection scheme based on one of the modal imaging techniques, super-localization by image inversion interferometry (SLIVER), for estimating the separation of two freely rotating dipoles. In the proposed method, we introduce a vortex wave plate before the SLIVER detection to separate the radial and azimuthal components of the dipole radiation. With numerical simulations, we demonstrated that our method achieves non-divergent precision at any separation between two dipole emitters. We investigated several practical effects relevant to experimental measurements in super-resolution microscopy, including numerical aperture, detection bandwidth, number of estimation parameters, background, and misalignment on separation estimation. Our proposed measurement provides a near quantum-limited detection scheme for measuring the separation of two freely-rotating dipole emitters, such as fluorescently tagged molecules, which are commonly used in super-resolution microscopy.

biophysics↗

bioRxiv2024

Calibration-free estimation of field dependent aberrations for single molecule localization microscopy across large fields of view

Image quality in single molecule localization microscopy (SMLM) depends largely on the accuracy and precision of the localizations. While under ideal imaging conditions the theoretically obtainable precision and accuracy are achieved, in practice this changes if (field dependent) aberrations are present. Currently there is no simple way to measure and incorporate these aberrations into the Point Spread Function (PSF) fitting, therefore the aberrations are often taken constant or neglected all together. Here we introduce a model-based approach to estimate the field-dependent aberration directly from single molecule data without a calibration step. This is made possible by using nodal aberration theory to incorporate the field-dependency of aberrations into our fully vectorial PSF model. This results in a limited set of aberration fit parameters that can be extracted from the raw frames without a bead calibration measurement, also in retrospect. The software implementation is computationally efficient, enabling fitting of a full 2D or 3D dataset within a few minutes. We demonstrate our method on 2D and 3D localization data of microtubuli and nuclear pore complexes over fields of view (FOV) of up to 180 m and compare it with spline-based fitting and a deep learning based approach.

microbiology↗

bioRxiv2023

Universal inverse modelling of point spread functions for SMLM localization and microscope characterization

The point spread function (PSF) of a microscope describes the image of a point emitter. Knowing the accurate PSF model is essential for various imaging tasks, including single molecule localization, aberration correction and deconvolution. Here we present uiPSF (universal inverse modelling of Point Spread Functions), a toolbox to infer accurate PSF models from microscopy data, using either image stacks of fluorescent beads or directly images of blinking fluorophores, the raw data in single molecule localization microscopy (SMLM). The resulting PSF model enables accurate 3D super-resolution imaging using SMLM. Additionally, uiPSF can be used to characterize and optimize a microscope system by quantifying the aberrations, including field-dependent aberrations, and resolutions. Our modular framework is applicable to a variety of microscope modalities and the PSF model incorporates system or sample specific characteristics, e.g., the bead size, depth dependent aberrations and transformations among channels. We demonstrate its application in single or multiple channels or large field-of-view SMLM systems, 4Pi-SMLM, and lattice light-sheet microscopes using either bead data or single molecule blinking data.

biophysics↗

bioRxiv2023

Antigen Geometry Tunes Mast Cell Signaling Through Distinct FcϵRI Aggregation andStructural Changes

Fc receptors containing immunoreceptor tyrosine-based activation motifs (ITAMs) are critical components of the innate immune system that bridge adaptive antibody recognition to cellular effector responses. In allergic responses, the high-affinity IgE receptor, Fc{varepsilon}RI, is activated when multivalent antigens crosslink receptor-bound IgE, yet the molecular mechanisms linking antigen structure to signaling output remain incompletely understood. Here, we compare two antigens presenting identical IgE-binding haptens but differing in geometry: the high-valency, heterogeneous DNP-BSA and the defined trivalent antigen DF3. We find that these ligands elicit distinct patterns of degranulation and Fc{varepsilon}RI {gamma}-chain phosphorylation, correlating with differences in the recruitment of the inhibitory lipid phosphatase SHIP1. Monte Carlo simulations predicted that each antigen generates receptor aggregates with distinct size, complexity, and inter-receptor spacing. Using direct stochastic optical reconstruction microscopy (dSTORM) and Bayesian Grouping of Localizations (BaGoL) analysis, we directly visualized the nanoscale aggregate geometry and found that DF3 induced smaller, more linear aggregates with tighter receptor spacing than DNP-BSA. Together, our results show that antigen properties, including size, valency, and epitope spacing, modulate Fc{varepsilon}RI aggregate architecture and tune the balance of positive and negative signaling to ultimately shape mast cell outcomes. Statement of SignificanceAllergic immune responses are initiated when multivalent antigens aggregate IgE-bound Fc{varepsilon}RI on mast cells, yet the relationship between antigen structure and signaling strength remains unclear. This study combines biochemical assays, Monte Carlo modeling, and super-resolution imaging to show that allergen properties, specifically valency and nanoscale epitope spacing, govern the geometry of Fc{varepsilon}RI aggregates and the balance of activating and inhibitory signals. These findings establish a direct mechanistic link between antigen structure and immune receptor output, providing new insight into how physical antigen features encode mast cell responses.

biophysics↗

bioRxiv2023

Signal Improved ultra-Fast Light-sheet Microscope (SIFT) for large tissue imaging

Light-sheet fluorescence microscopy (LSFM) in conjunction with tissue clearing techniques enables morphological investigation of large tissues faster and with excellent optical sectioning. Recently, cleared tissue axially swept light-sheet microscope (ctASLM) demonstrated three-dimensional isotropic resolution in millimeter-scaled tissues. But ASLM based microscopes suffer from low detection signal and slow imaging speed. Here we report a simple and efficient imaging platform that employs precise control of two fixed distant light-sheet foci to carry out ASLM. This allowed us to carry out full field of view (FOV) imaging at 40 frames per second (fps) which is a four-fold improvement compared to the current state-of-the-art. In addition, in a particular frame rate, our method doubles the signal compared to the current ASLM technique. To augment the overall imaging performance, we also developed a deep learning based tissue information classifier that enables faster determination of tissue boundary. We demonstrated the performance of our imaging platform on various cleared tissue samples and demonstrated its robustness over a wide range of clearing protocols.

biophysics↗

bioRxiv2023

Distinct interactions stabilize EGFR dimers and higher-order oligomers in cell membranes

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (RTK) with important roles in many cellular processes as well as cancer and other diseases. EGF binding promotes EGFR dimerization and autophosphorylation through interactions that are well understood structurally. However, it is not clear how these dimers relate to higher-order EGFR oligomers detected at the cell surface. We used single-particle tracking (SPT) and Forster resonance energy transfer (FRET) imaging to examine how each domain within EGFR contributes to receptor dimerization and the rate of its diffusion in the cell membrane. We show that the EGFR extracellular region is sufficient to drive receptor dimerization, but that the EGF-induced EGFR slow-down seen by SPT requires formation of higher order oligomers, mediated in part by the intracellular tyrosine kinase domain - but only when in its active conformation. Our data thus provide important insight into higher-order EGFR interactions required for EGF signaling.

biophysics↗

bioRxiv2021

Robust, Fiducial-Free Drift Correction for Super-resolution Imaging

We describe a robust, fiducial-free method of drift correction for use in single molecule localization-based super-resolution methods. The method combines periodic 3D registration of the sample using brightfield images with a fast post-processing algorithm that corrects residual registration errors and drift between registration events. The method is robust to low numbers of collected localizations, requires no specialized hardware, and provides stability and drift correction for an indefinite time period.

biophysics↗

bioRxiv2020

Comparing Lifeact and Phalloidin for Super-resolution Imaging of Actin in Fixed Cells

Visualizing actin filaments in fixed cells is of great interest for a variety of topics in cell biology such as cell division, cell movement, and cell signaling. We investigated the possibility of replacing phalloidin, the standard reagent for super-resolution imaging of F-actin in fixed cells, with the actin binding peptide lifeact. We compared the labels for use in single molecule based super-resolution microscopy, where AlexaFluor 647 labeled phalloidin was used in a (d)STORM modality and Atto 655 labeled lifeact was used in a single molecule imaging, reversible binding modality. We found that imaging with lifeact had a comparable resolution in reconstructed images and provided several advantages over phalloidin including lower costs, the ability to image multiple regions of interest on a coverslip without degradation, simplified sequential super-resolution imaging, and more continuous labeling of thin filaments.

cell biology↗

bioRxiv2020

EGFR transactivates RON to drive oncogenic crosstalk

SUMMARYCrosstalk between disparate membrane receptors is thought to drive oncogenic signaling and allow for therapeutic resistance. EGFR and RON are members of two unique receptor tyrosine kinase (RTK) subfamilies that engage in crosstalk through unknown mechanisms. We combined high resolution imaging with biochemical studies and structural mutants to understand how EGFR and RON communicate. We found that EGF stimulation results in EGFR-dependent RON phosphorylation. Crosstalk is unidirectional, since MSP stimulation of RON does not trigger EGFR phosphorylation. Two-color single particle tracking captured the formation of complexes between RON and EGFR, supporting a role for direct interactions in propagating crosstalk. We further show that RON is a substrate for EGFR kinase, and transactivation of RON requires the formation of a signaling competent EGFR dimer. These results identify critical structural features of EGFR/RON crosstalk and provide new mechanistic insights into therapeutic resistance.

cancer biology↗

bioRxiv2019

Sub-Nanometer Precision using Bayesian Grouping of Localizations

Single-molecule localization microscopy super-resolution methods such as DNA-PAINT and (d)STORM can generate multiple observed localizations over the time course of data acquisition from each dye or binding site that are not a priori assigned to those specific dyes or binding sites. We describe a Bayesian method of grouping and combining localizations from multiple blinking/binding events that can improve localization precision to better than one nanometer. The known statistical distribution of the number of binding/blinking events per dye/docking strand along with the precision of each localization event are used to estimate the true number and location of emitters in closely-spaced clusters.

biophysics↗

bioRxiv2019

Bayesian Multiple Emitter Fitting using Reversible Jump Markov Chain Monte Carlo

In single molecule localization-based super-resolution imaging, high labeling density or the desire for greater data collection speed can lead to clusters of overlapping emitter images in the raw super-resolution image data. We describe a Bayesian inference approach to multiple-emitter fitting that uses Reversible Jump Markov Chain Monte Carlo to identify and localize the emitters in dense regions of data. This formalism can take advantage of any prior information, such as emitter intensity and density. The output is both a posterior probability distribution of emitter locations that includes uncertainty in the number of emitters and the background structure, and a set of coordinates and uncertainties from the most probable model.

biophysics↗

bioRxiv2017

Sequential Super-Resolution Imaging using DNA Strand Displacement

Sequential labeling and imaging in fluorescence microscopy allows the imaging of multiple structures in the same cell using a single fluorophore species. In super-resolution applications, the optimal dye suited to the method can be chosen, the optical setup can be simpler and there are no chromatic aberrations between images of different structures. We describe a method based on DNA strand displacement that can be used to quickly and easily perform the labeling and removal of the fluorophores during each sequence. Site-specific tags are conjugated with unique and orthogonal single stranded DNA. Labeling for a particular structure is achieved by hybridization of antibody-bound DNA with a complimentary dye-labeled strand. After imaging, the dye is removed using toehold-mediated strand displacement, in which an invader strand competes off the dye-labeled strand than can be subsequently washed away. Labeling and removal of each DNA-species requires only a few minutes. We demonstrate the concept using sequential dSTORM super-resolution for multiplex imaging of subcellular structures.

bioengineering↗

bioRxiv2017

Single Molecule Pull-down for Quantification of Membrane Receptor Multisite Phosphorylation

The Epidermal Growth Factor Receptor (EGFR/ErbB1/HER1) plays an important role in both physiological and cancer-related processes. To study the factors that influence receptor phosphorylation, we have coupled Single Molecule Pull-down (SiMPull) measurements with rule-based modeling of EGFR signaling. Using SiMPull, we quantified the phosphorylation state of thousands of individual receptors. These measurements enabled the first direct detection of multisite phosphorylation on full-length EGFR and revealed that the extent of phosphorylation varies by tyrosine site and is dependent on the relative abundance of signaling partners that limit access by tyrosine phosphatases. We also evaluated the impact of oncogenic mutations and ligands with varying affinity on phosphorylation kinetics. Simulations highlight the importance of dimer lifetimes on EGFR phosphorylation and signaling output.

cell biology↗

bioRxiv2017

Mannan molecular sub-structures control nanoscale glucan exposure in Candida

N-linked mannans (N-mannans) in the cell wall of Candida albicans are thought to mask {beta}-(1,3)-glucan from recognition by Dectin-1, contributing to innate immune evasion. Lateral cell wall exposures of glucan on Candida albicans are predominantly single receptor-ligand interaction sites and are restricted to nanoscale geometries. Candida species exhibit a range of basal glucan exposures and their mannans also vary in size and complexity at the molecular level. We used super resolution fluorescence imaging and a series of protein mannosylation mutants in C. albicans and C. glabrata to investigate the role of specific N-mannan features in regulating the nanoscale geometry of glucan exposure. Decreasing acid labile mannan abundance and -(1,6)-mannan backbone length correlated most strongly with increased density and nanoscopic size of glucan exposures in C. albicans and C. glabrata, respectively. Additionally, a C. albicans clinical isolate with high glucan exposure produced similarly perturbed N-mannan structures and exhibited similar changes to nanoscopic glucan exposure geometry. We conclude that acid labile N-mannan controls glucan exposure geometry at the nanoscale. Furthermore, variations in glucan nanoexposure characteristics are clinically relevant and are likely to impact the nature of the pathogenic surface presented to innate immunocytes at dimensions relevant to receptor engagement, aggregation and signaling.

microbiology↗

bioRxiv2017

Differential Mast Cell Outcomes Are Sensitive to FcϵRI-Syk Binding Kinetics

Crosslinking of IgE-bound Fc{varepsilon}RI triggers multiple cellular responses, including degranulation and cytokine production. Signaling is dependent on recruitment of Syk via docking of its dual SH2 domains to phosphorylated tyrosines within the Fc{varepsilon}RI immunoreceptor tyrosine-based activation motifs. Using single molecule imaging in live cells, we directly visualized and quantified the binding of individual mNeonGreen-tagged Syk molecules as they associated with the plasma membrane after Fc{varepsilon}RI activation. We found that Syk colocalizes transiently to Fc{varepsilon}RI and that Syk-Fc{varepsilon}RI binding dynamics are independent of receptor aggregate size. Substitution of glutamic acid for tyrosine between the Syk SH2 domains (SykY130E) led to an increased Syk-Fc{varepsilon}RI off-rate, loss of site-specific Syk autophosphorylation, and impaired downstream signaling. CRISPR-Cas9 engineered cells expressing only SykY130E were deficient in antigen-stimulated calcium release, degranulation and production of some cytokines (TNF-a, IL-3) but not others (MCP-1, IL-4). We propose that kinetic discrimination along the Fc{varepsilon}RI signaling pathway occurs at the level of Syk-Fc{varepsilon}RI interactions, with key outcomes dependent upon sufficiently long-lived Syk binding events.\n\nSummarySchwartz et al. use single molecule imaging to quantify the transient nature of Fc{varepsilon}RI-Syk interactions in live mast cells. A functional mutation that increases Syk off-rate leads to loss of site-specific Syk phosphorylation and impaired signaling, highlighting the importance of finely tuned protein interactions in directing cellular outcomes.

cell biology↗