ABSTRACT
During immune surveillance, CD8 T cells scan the surface of antigen-presenting cells using dynamic microvillar palpation and movements as well as by having their receptors preconcentrated into patches. Here, we use real-time lattice light-sheet microscopy to demonstrate the independence of microvillar and membrane receptor patch scanning. While T cell receptor (TCR) patches can distribute to microvilli, they do so stochastically and not preferentially as for other receptors such as CD62L. The distinctness of TCR patch movement from microvillar movement extends to many other receptors that form patches that also scan independent of the TCR. An exception to this is the CD8 coreceptor which largely comigrates in patches that overlap with or are closely adjacent to those containing TCRs. Microvilli that assemble into a synapse contain various arrays of the engaged patches, notably of TCRs and the inhibitory receptor PD-1, creating a pastiche of occupancies that vary from microvillar contact to contact. In summary, this work demonstrates that localization of receptor patches within the membrane and on microvillar projections is random prior to antigen detection and that such random variation may play into the generation of many individually composed receptor patch compositions at a single synapse.
Subject(s)
Antigen-Presenting Cells , CD8-Positive T-Lymphocytes , Microvilli , Receptors, Antigen, T-Cell , Antigen-Presenting Cells/cytology , CD8-Positive T-Lymphocytes/cytology , Cell Membrane/metabolism , Humans , Immunologic Surveillance , Immunological Synapses , Microvilli/metabolism , Receptors, Antigen, T-Cell/metabolismABSTRACT
Although much is known about the embryo during implantation, the architecture of the uterine environment in which the early embryo develops is not well understood. We employed confocal imaging in combination with 3D analysis to identify and quantify dynamic changes to the luminal structure of murine uterus in preparation for implantation. When applied to mouse mutants with known implantation defects, this method detected striking peri-implantation abnormalities in uterine morphology that cannot be visualized by histology. We revealed 3D organization of uterine glands and found that they undergo a stereotypical reorientation concurrent with implantation. Furthermore, we extended this technique to generate a 3D rendering of the cycling human endometrium. Analyzing the uterine and embryo structure in 3D for different genetic mutants and pathological conditions will help uncover novel molecular pathways and global structural changes that contribute to successful implantation of an embryo.
Subject(s)
Blastocyst/ultrastructure , Embryo Implantation/physiology , Embryo, Mammalian/ultrastructure , Endometrium/ultrastructure , Uterus/ultrastructure , Animals , Embryo, Mammalian/diagnostic imaging , Endometrium/diagnostic imaging , Endometrium/physiology , Female , Humans , Imaging, Three-Dimensional , Mice , Mice, Inbred C57BL , Uterus/diagnostic imaging , Uterus/physiology , Wnt-5a Protein/geneticsABSTRACT
We report a strategy for the optical determination of tip-substrate distance in nanoscale scanning electrochemical microscopy (SECM) using three-dimensional super-resolution fluorescence imaging. A phase mask is placed in the emission path of our dual SECM/optical microscope, generating a double helix point spread function at the image plane, which allows us to measure the height of emitting objects relative to the focus of the microscope. By exciting both a fluorogenic reaction at the nanoscale electrode tip as well as fluorescent nanoparticles at the substrate, we are able to calculate the tip-substrate distance as the tip approaches the surface with precision better than 25 nm. Attachment of a fluorescent particle to the insulating sheath of the SECM tip extends this technique to nonfluorogenic electrochemical reactions. Correlated electrochemical and optical determination of tip-substrate distance yielded excellent agreement between the two techniques. Not only does super-resolution imaging offer a secondary feedback mechanism for measuring the tip-sample gap during SECM experiments, it also enables facile tip alignment and a strategy for accounting for electrode tilt relative to the substrate.
ABSTRACT
We report electrogenerated chemiluminescence (ECL) generated at single gold nanowire electrodes supported on tin-doped indium oxide. Unlike other single nanoparticle electrochemical characterization techniques, ECL provides a massively parallel direct readout of electrochemical activity on individual nanoparticle electrodes without the need for extrinsic illumination or a scanning electrochemical probe. While ECL is not observed from as-purchased nanowires due to the surfactant layer, by removing the layer and coating the nanowires with a polymer blend, ECL from single nanowire electrodes is readily measured. With an increase in polymer thickness, an increase in ECL image quality and reproducibility over multiple redox cycles is observed. The polymer coating also provides a strategy for stabilizing gold nanoparticle electrodes against complete surface oxidation in aqueous environments.
ABSTRACT
We report a method to study electro-active defects in passivated electrodes. This method couples fluorescence microscopy and electrochemistry to localize and size electro-active defects. The method was validated by comparison with a scanning probe technique, scanning electrochemical microscopy. We used our method for studying electro-active defects in thin TiO2 layers electrodeposited on 25 µm diameter Pt ultramicroelectrodes (UMEs). The permeability of the TiO2 layer was estimated by measuring the oxidation of ferrocenemethanol at the UME. Blocking of current ranging from 91.4 to 99.8% was achieved. Electro-active defects with an average radius ranging between 9 and 90 nm were observed in these TiO2 blocking layers. The distribution of electro-active defects over the TiO2 layer is highly inhomogeneous and the number of electro-active defect increases for lower degree of current blocking. The interest of the proposed technique is the possibility to quickly (less than 15 min) image samples as large as several hundreds of µm(2) while being able to detect electro-active defects of only a few tens of nm in radius.
ABSTRACT
Single-particle and single-molecule orientation determination plays a vital role in deciphering nanoscale motion in complex environments. Previous attempts to determine the absolute three-dimensional orientation of anisotropic particles rely on subjective pattern matching and are inherently plagued by high degrees of uncertainty. Herein, we describe a method utilizing total internal reflection scattering microscopy to determine the 3D orientation of gold nanorods with subdegree uncertainty. The method is then applied to the biologically relevant system of microtubule cargo loading. Finally, we demonstrate the method holds potential for identifying single particles versus proximate neighbors within the diffraction limited area.
Subject(s)
Gold/chemistry , Nanotechnology , Nanotubes/chemistry , Anisotropy , Microscopy , Nanoparticles/chemistryABSTRACT
The ability to directly follow three-dimensional rotational movement of anisotropic nanoparticles will greatly enhance our understanding of the way nanoparticles interact with surfaces. Herein, we demonstrate dual-color total internal reflection scattering microscopy as a tool to probe the interactions of plasmonic gold nanorods with functional surfaces. By taking advantage of both the short and long axis surface plasmon resonance scattering enhancement, we are able to decipher both in-plane and out-of-plane gold nanorod motion relative to the sample surface with equally high resolution. In combination with superlocalization through point spread function fitting, we overcome the four-quadrant angular degeneracy of gold nanorods in the focal plane of the objective and resolve conformations of surface-bound anisotropic nanoparticles in unprecedented detail.
ABSTRACT
The defocused orientation and position imaging (DOPI) and polarization-based in-focus imaging techniques have been widely used for detecting rotational motions with anisotropic gold nanorods (AuNRs) as orientation probes. However, these techniques have a number of significant limitations, such as the greatly reduced signal intensity and relatively low spatial and temporal resolutions for out-of-focus AuNRs and the angular degeneracy for in-focus AuNRs. Herein, we present a total internal reflection (TIR) scattering-based focused orientation and position imaging (FOPI) of AuNRs supported on a 50 nm thick gold film, which enables us to overcome the aforementioned limitations. Imaging AuNRs under the TIR scattering microscope provides excellent signal-to-noise ratio and results in no deteriorating images. The scattering patterns of AuNRs on the gold substrate are affected by the strong interaction of the excited dipole in the AuNR with the image dipole in the gold substrate. The doughnut-shaped scattering field distribution allows for high-throughput determination of the three-dimensional spatial orientation of in-focus AuNRs within a single frame without angular degeneracy. Therefore, the TIR scattering-based FOPI method is demonstrated to be an outstanding candidate for studying dynamics of functionalized nanoparticles on a large variety of functional surfaces.
ABSTRACT
T cells typically recognize their ligands using a defined cell biology-the scanning of their membrane microvilli (MV) to palpate their environment-while that same membrane scaffolds T cell receptors (TCRs) that can signal upon ligand binding. Chimeric antigen receptors (CARs) present both a therapeutic promise and a tractable means to study the interplay between receptor affinity, MV dynamics and T cell function. CARs are often built using single-chain variable fragments (scFvs) with far greater affinity than that of natural TCRs. We used high-resolution lattice lightsheet (LLS) and total internal reflection fluorescence (TIRF) imaging to visualize MV scanning in the context of variations in CAR design. This demonstrated that conventional CARs hyper-stabilized microvillar contacts relative to TCRs. Reducing receptor affinity, antigen density, and/or multiplicity of receptor binding sites normalized microvillar dynamics and synapse resolution, and effector functions improved with reduced affinity and/or antigen density, highlighting the importance of understanding the underlying cell biology when designing receptors for optimal antigen engagement.
Subject(s)
Microvilli , Receptors, Antigen, T-Cell , Receptors, Chimeric Antigen , T-Lymphocytes , Microvilli/metabolism , Receptors, Antigen, T-Cell/metabolism , Receptors, Chimeric Antigen/genetics , Receptors, Chimeric Antigen/metabolism , Single-Chain Antibodies/metabolism , Humans , AntigensABSTRACT
Novel non-blinking quantum dots (NBQDs) were utilized in three-dimensional super-localization, high-precision tracking applications under an automated scanning-angle total internal reflection fluorescence microscope (SA-TIRFM). NBQDs were randomly attached to stationary microtubules along the radial axis under gliding assay conditions. By automatically scanning through a wide range of incident angles with different evanescent-field layer thicknesses, the fluorescence intensity decay curves were obtained. Fit with theoretical decay functions, the absolute vertical positions were determined with sub-10-nm localization precision. The emission intensity profile of the NBQDs attached to kinesin-propelled microtubules was used to resolve the self-rotation of gliding microtubules within a small vertical distance of ~50 nm. We demonstrate the applicability of NBQDs in high-precision fluorescence imaging experiments.
Subject(s)
Quantum Dots , Animals , Automation , Biophysics/methods , Humans , Imaging, Three-Dimensional , Kinesins/metabolism , Microscopy, Fluorescence/methods , Microtubules/metabolism , Nanoparticles/chemistry , Nanotechnology/methods , Reproducibility of Results , SemiconductorsABSTRACT
T cell exhaustion is a major impediment to antitumor immunity. However, it remains elusive how other immune cells in the tumor microenvironment (TME) contribute to this dysfunctional state. Here, we show that the biology of tumor-associated macrophages (TAMs) and exhausted T cells (Tex) in the TME is extensively linked. We demonstrate that in vivo depletion of TAMs reduces exhaustion programs in tumor-infiltrating CD8+ T cells and reinvigorates their effector potential. Reciprocally, transcriptional and epigenetic profiling reveals that Tex express factors that actively recruit monocytes to the TME and shape their differentiation. Using lattice light sheet microscopy, we show that TAM and CD8+ T cells engage in unique, long-lasting, antigen-specific synaptic interactions that fail to activate T cells but prime them for exhaustion, which is then accelerated in hypoxic conditions. Spatially resolved sequencing supports a spatiotemporal self-enforcing positive feedback circuit that is aligned to protect rather than destroy a tumor.
Subject(s)
CD8-Positive T-Lymphocytes , Neoplasms , Cell Differentiation , Humans , Macrophages , Neoplasms/genetics , Tumor MicroenvironmentABSTRACT
How local interactions of actin regulators yield large-scale organization of cell shape and movement is not well understood. Here we investigate how the WAVE complex organizes sheet-like lamellipodia. Using super-resolution microscopy, we find that the WAVE complex forms actin-independent 230-nm-wide rings that localize to regions of saddle membrane curvature. This pattern of enrichment could explain several emergent cell behaviors, such as expanding and self-straightening lamellipodia and the ability of endothelial cells to recognize and seal transcellular holes. The WAVE complex recruits IRSp53 to sites of saddle curvature but does not depend on IRSp53 for its own localization. Although the WAVE complex stimulates actin nucleation via the Arp2/3 complex, sheet-like protrusions are still observed in ARP2-null, but not WAVE complex-null, cells. Therefore, the WAVE complex has additional roles in cell morphogenesis beyond Arp2/3 complex activation. Our work defines organizing principles of the WAVE complex lamellipodial template and suggests how feedback between cell shape and actin regulators instructs cell morphogenesis.
Subject(s)
Cell Membrane/metabolism , Cell Shape , Pseudopodia/metabolism , Wiskott-Aldrich Syndrome Protein Family/metabolism , Actin Cytoskeleton/genetics , Actin Cytoskeleton/metabolism , Actin-Related Protein 2-3 Complex/genetics , Actin-Related Protein 2-3 Complex/metabolism , Animals , Cell Membrane/genetics , Cell Membrane/ultrastructure , Cell Movement , HEK293 Cells , HL-60 Cells , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/ultrastructure , Humans , Macrophages/metabolism , Macrophages/ultrastructure , Melanoma, Experimental/genetics , Melanoma, Experimental/metabolism , Melanoma, Experimental/ultrastructure , Mice , Microscopy, Confocal , Microscopy, Electron, Transmission , Microscopy, Fluorescence , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Protein Transport , Pseudopodia/genetics , Pseudopodia/ultrastructure , Signal Transduction , Time Factors , Wiskott-Aldrich Syndrome Protein Family/geneticsABSTRACT
An automatic calibration and scanning-angle prism-type total internal reflection fluorescence microscope (TIRFM) was constructed and tested for the highest vertical resolution. The angle of the incident laser beam can be changed automatically and reliably from subcritical angles to nearly 90 degrees with intervals smaller than 0.2 degrees, and the laser illumination spot in the sample can be calibrated to automatically overlap with the center of the microscope's field of view. By scanning through a wide range of incident angles with different evanescent-field layer thicknesses, the fluorescence intensity decay curves of randomly distributed fluorescent nanospheres in agarose gel were obtained and fitted with the theoretical decay functions to determine their vertical positions. The best axial resolution was demonstrated to be better than 10 nm under the rigorous statistical analysis of confidence levels and by the Monte Carlo simulation. The new setup was further utilized to determine the tilting angle of the microtubules buried in agarose gel and to find the precise surface plasmon resonance (SPR) angle for gold film enhanced TIRFM. We demonstrate the new microscope's unique capability to find the best illumination configuration for complex systems automatically and reproducibly.
Subject(s)
Microscopy, Fluorescence/instrumentation , Calibration , Equipment Design , Microscopy, Fluorescence/methods , Microtubules/ultrastructure , Surface Plasmon ResonanceSubject(s)
Optical Imaging/methods , Single-Cell Analysis , Apoptosis/physiology , Fluorescence Resonance Energy Transfer , Fluorescent Dyes/chemistry , Lipid Metabolism/physiology , Microscopy, Confocal , Microscopy, Fluorescence, Multiphoton/instrumentation , Microscopy, Fluorescence, Multiphoton/methods , Microscopy, Interference , Mitosis/physiology , Nanoparticles/chemistry , Optical Imaging/instrumentation , Plant Cells/pathologyABSTRACT
Generation of tumor-infiltrating lymphocytes begins when tumor antigens reach the lymph node (LN) to stimulate T cells, yet we know little of how tumor material is disseminated among the large variety of antigen-presenting dendritic cell (DC) subsets in the LN. Here, we demonstrate that tumor proteins are carried to the LN within discrete vesicles inside DCs and are then transferred among DC subsets. A synapse is formed between interacting DCs and vesicle transfer takes place in the absence of free exosomes. DCs -containing vesicles can uniquely activate T cells, whereas DCs lacking them do not. Understanding this restricted sharing of tumor identity provides substantial room for engineering better anti-tumor immunity.
Subject(s)
Antigen Presentation/immunology , Antigens, Neoplasm/immunology , Dendritic Cells/immunology , Melanoma, Experimental/immunology , Myeloid Cells/immunology , Synapses/immunology , T-Lymphocytes/immunology , Animals , Dendritic Cells/cytology , Dendritic Cells/metabolism , Male , Melanoma, Experimental/metabolism , Melanoma, Experimental/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Myeloid Cells/cytology , Myeloid Cells/metabolism , Receptors, CCR2/physiology , Receptors, CCR7/physiology , Synapses/metabolism , Synapses/pathology , T-Lymphocytes/cytology , T-Lymphocytes/metabolismABSTRACT
During immune surveillance, T cells survey the surface of antigen-presenting cells. In searching for peptide-loaded major histocompatibility complexes (pMHCs), they must solve a classic trade-off between speed and sensitivity. It has long been supposed that microvilli on T cells act as sensory organs to enable search, but their strategy has been unknown. We used lattice light-sheet and quantum dot-enabled synaptic contact mapping microscopy to show that anomalous diffusion and fractal organization of microvilli survey the majority of opposing surfaces within 1 minute. Individual dwell times were long enough to discriminate pMHC half-lives and T cell receptor (TCR) accumulation selectively stabilized microvilli. Stabilization was independent of tyrosine kinase signaling and the actin cytoskeleton, suggesting selection for avid TCR microclusters. This work defines the efficient cellular search process against which ligand detection takes place.
Subject(s)
Microscopy/methods , Microvilli/chemistry , T-Lymphocytes/metabolism , Actin Cytoskeleton/metabolism , Animals , Antigens/immunology , Fractals , Ligands , Mice , Microvilli/metabolism , Quantum Dots , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/cytology , T-Lymphocytes/immunologyABSTRACT
An automatic calibration and angle-scanning prism-type total internal reflection fluorescence microscope (TIRFM) was modified to function in both TIRFM and pseudo-TIRFM modes. When the incident angle of the excitation laser beam was controlled to be larger than the critical angle, the instrument served as a variable-angle TIRFM. A homemade computer program automatically calibrates the laser illumination spot in the sample to overlap with the center of the microscope's field of view. Then, by measuring the fluorescence intensities at different incident angles, the z-positions of fluorescent nanospheres close to the cell basolateral membrane can be extracted. When the incident angle is reduced to be in the subcritical range, the instrument works as a pseudo-TIRFM. The whole cell body from bottom to top can be imaged in a vertical scan process. Furthermore, the illumination field depth in the pseudo-TIRFM can be controlled by changing the incident angle or the horizontal position of the laser spot.