The development of optical microscopy techniques for the advancement of single-particle studies

Single particle orientation and rotational tracking (SPORT) has recently become a powerful optical microscopy tool that can expose many molecular motions. Unfortunately, there is not yet a single microscopy technique that can decipher all particle motions in all environmental conditions, thus there are limitations to current technologies. Within, the two powerful microscopy tools of total internal reflection and interferometry are advanced to determine the position, orientation, and optical properties of metallic nanoparticles in a variety of environments. Total internal reflection is an optical phenomenon that has been applied to microscopy to produce either fluorescent or scattered light. The non-invasive far-field imaging technique is coupled with a near-field illumination scheme that allows for better axial resolution than confocal microscopy and epi-fluorescence microscopy. By controlling the incident illumination angle using total internal reflection fluorescence (TIRF) microscopy, a new type of imaging probe called non-blinking quantum dots (NBQDs) were super-localized in the axial direction to sub-10-nm precision. These particles were also used to study the rotational motion of microtubules being propelled by the motor protein kinesin across the substrate surface. The same instrument was modified to function under total internal reflection scattering (TIRS) microscopy to study metallic anisotropic nanoparticles and their dynamic interactions with synthetic lipid bilayers. Utilizing two illumination lasers with opposite polarization directions at wavelengths corresponding to the short and long axis surface plasmon resonance (SPR) of the nanoparticles, both the in-plane and out-of-plane movements of many particles could be tracked simultaneously. When combined with Gaussian point spread function (PSF) fitting for particle super-localization, the binding status and rotational movement could be resolved without degeneracy. TIRS microscopy was also used to find the 3D orientation of stationary metallic anisotropic nanoparticles utilizing only long-axis SPR enhancement. The polarization direction of the illuminating light was rotated causing the relative intensity of p-polarized and s-polarized light within the evanescent field to change. The interaction of the evanescent field with the particles is dependent on the orientation of the particle producing an intensity curve. This curve and the in-plane angle can be compared with simulations to accurately determine the 3D orientation. Differential interference contrast (DIC) microscopy is another non-invasive far-field technique based upon interferometry that does not rely on staining or other contrast enhancing techniques. In addition, high numerical aperture condensers and objectives can be used to give a very narrow depth of field allowing for the optical tomography of samples, which makes it an ideal candidate to study biological systems. DIC microscopy has also proven itself in determining the orientation of gold nanorods in both engineered environments and within cells. Many types of nanoparticles and nanostructures have been synthesized using lithographic techniques on silicon wafer substrates. Traditionally, reflective mode DIC microscopes have been developed and applied to the topographical study of reflective substrates and the imaging of chips on silicon wafers. Herein, a laser-illuminated reflected-mode DIC was developed for studying nanoparticles on reflective surfaces

Single Cell Optical Imaging and Spectroscopy

In his 1665 treatise, Micrographia, Robert Hooke described the many observations he had made using a microscope, including compartment-like structures in cork samples that he termed “cells

Clinical Sequencing Exploratory Research Consortium: Accelerating Evidence-Based Practice of Genomic Medicine

Despite rapid technical progress and demonstrable effectiveness for some types of diagnosis and therapy, much remains to be learned about clinical genome and exome sequencing (CGES) and its role within the practice of medicine. The Clinical Sequencing Exploratory Research (CSER) consortium includes 18 extramural research projects, one National Human Genome Research Institute (NHGRI) intramural project, and a coordinating center funded by the NHGRI and National Cancer Institute. The consortium is exploring analytic and clinical validity and utility, as well as the ethical, legal, and social implications of sequencing via multidisciplinary approaches; it has thus far recruited 5,577 participants across a spectrum of symptomatic and healthy children and adults by utilizing both germline and cancer sequencing. The CSER consortium is analyzing data and creating publically available procedures and tools related to participant preferences and consent, variant classification, disclosure and management of primary and secondary findings, health outcomes, and integration with electronic health records. Future research directions will refine measures of clinical utility of CGES in both germline and somatic testing, evaluate the use of CGES for screening in healthy individuals, explore the penetrance of pathogenic variants through extensive phenotyping, reduce discordances in public databases of genes and variants, examine social and ethnic disparities in the provision of genomics services, explore regulatory issues, and estimate the value and downstream costs of sequencing. The CSER consortium has established a shared community of research sites by using diverse approaches to pursue the evidence-based development of best practices in genomic medicine

The development of optical microscopy techniques for the advancement of single-particle studies

Single particle orientation and rotational tracking (SPORT) has recently become a powerful optical microscopy tool that can expose many molecular motions. Unfortunately, there is not yet a single microscopy technique that can decipher all particle motions in all environmental conditions, thus there are limitations to current technologies. Within, the two powerful microscopy tools of total internal reflection and interferometry are advanced to determine the position, orientation, and optical properties of metallic nanoparticles in a variety of environments. Total internal reflection is an optical phenomenon that has been applied to microscopy to produce either fluorescent or scattered light. The non-invasive far-field imaging technique is coupled with a near-field illumination scheme that allows for better axial resolution than confocal microscopy and epi-fluorescence microscopy. By controlling the incident illumination angle using total internal reflection fluorescence (TIRF) microscopy, a new type of imaging probe called "non-blinking" quantum dots (NBQDs) were super-localized in the axial direction to sub-10-nm precision. These particles were also used to study the rotational motion of microtubules being propelled by the motor protein kinesin across the substrate surface. The same instrument was modified to function under total internal reflection scattering (TIRS) microscopy to study metallic anisotropic nanoparticles and their dynamic interactions with synthetic lipid bilayers. Utilizing two illumination lasers with opposite polarization directions at wavelengths corresponding to the short and long axis surface plasmon resonance (SPR) of the nanoparticles, both the in-plane and out-of-plane movements of many particles could be tracked simultaneously. When combined with Gaussian point spread function (PSF) fitting for particle super-localization, the binding status and rotational movement could be resolved without degeneracy. TIRS microscopy was also used to find the 3D orientation of stationary metallic anisotropic nanoparticles utilizing only long-axis SPR enhancement. The polarization direction of the illuminating light was rotated causing the relative intensity of p-polarized and s-polarized light within the evanescent field to change. The interaction of the evanescent field with the particles is dependent on the orientation of the particle producing an intensity curve. This curve and the in-plane angle can be compared with simulations to accurately determine the 3D orientation. Differential interference contrast (DIC) microscopy is another non-invasive far-field technique based upon interferometry that does not rely on staining or other contrast enhancing techniques. In addition, high numerical aperture condensers and objectives can be used to give a very narrow depth of field allowing for the optical tomography of samples, which makes it an ideal candidate to study biological systems. DIC microscopy has also proven itself in determining the orientation of gold nanorods in both engineered environments and within cells. Many types of nanoparticles and nanostructures have been synthesized using lithographic techniques on silicon wafer substrates. Traditionally, reflective mode DIC microscopes have been developed and applied to the topographical study of reflective substrates and the imaging of chips on silicon wafers. Herein, a laser-illuminated reflected-mode DIC was developed for studying nanoparticles on reflective surfaces.</p

Three-Dimensional Orientation Determination of Stationary Anisotropic Nanoparticles with Sub-Degree Precision under Total Internal Reflection Scattering Microscopy

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

Three-Dimensional High-Resolution Rotational Tracking with Superlocalization Reveals Conformations of Surface-Bound Anisotropic Nanoparticles

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

Three-Dimensional High-Resolution Rotational Tracking with Superlocalization Reveals Conformations of Surface-Bound Anisotropic Nanoparticles

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

Focused Orientation and Position Imaging (FOPI) of Single Anisotropic Plasmonic Nanoparticles by Total Internal Reflection Scattering Microscopy

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

High-Precision Tracking with Non-blinking Quantum Dots Resolves Nanoscale Vertical Displacement

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.Reprinted (adapted) with permission from Journal of the American Chemical Society 134 (2012): 6108, doi:10.1021/ja301332t. Copyright 2012 American Chemical Society.</p

T cells use distinct topographical and membrane receptor scanning strategies that individually coalesce during receptor recognition

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