Simultaneous spectral differentiation of multiple fluorophores in super-resolution imaging using a glass phase plate.

By engineering the point-spread function (PSF) of single molecules, different fluorophore species can be imaged simultaneously and distinguished by their unique PSF patterns. Here, we insert a silicon-dioxide phase plate at the Fourier plane of the detection path of a wide-field fluorescence microscope to produce distinguishable PSFs (X-PSFs) at different wavelengths. We demonstrate that the resulting PSFs can be localized spatially and spectrally using a maximum-likelihood estimation algorithm and can be utilized for hyper-spectral super-resolution microscopy of biological samples. We produced superresolution images of fixed U2OS cells using X-PSFs for dSTORM imaging with simultaneous illumination of up to three fluorophore species. The species were distinguished only by the PSF pattern. We achieved ∼21-nm lateral localization precision (FWHM) and ∼17-nm axial precision (FWHM) with an average of 1,800 - 3,500 photons per PSF and a background as high as 130 - 400 photons per pixel. The modified PSF distinguished fluorescent probes with ∼80 nm separation between spectral peaks.

Heterogeneous optoelectronic characteristics of Si micropillar arrays fabricated by metal-assisted chemical etching.

Recent progress achieved in metal-assisted chemical etching (MACE) has enabled the production of high-quality micropillar arrays for various optoelectronic applications. Si micropillars produced by MACE often show a porous Si/SiOx shell on crystalline pillar cores introduced by local electrochemical reactions. In this paper, we report the distinct optoelectronic characteristics of the porous Si/SiOx shell correlated to their chemical compositions. Local photoluminescent (PL) images obtained with an immersion oil objective lens in confocal microscopy show a red emission peak (≈ 650 nm) along the perimeter of the pillars that is threefold stronger compared to their center. On the basis of our analysis, we find an unexpected PL increase (≈ 540 nm) at the oil/shell interface. We suggest that both PL enhancements are mainly attributed to the porous structures, a similar behavior observed in previous MACE studies. Surface potential maps simultaneously recorded with topography reveal a significantly high surface potential on the sidewalls of MACE-synthesized pillars (+ 0.5 V), which is restored to the level of planar Si control (- 0.5 V) after removing SiOx in hydrofluoric acid. These distinct optoelectronic characteristics of the Si/SiOx shell can be beneficial for various sensor architectures.

Models and Impacts of Science Research Experiences: A Review of the Literature of CUREs, UREs, and TREs.

In efforts to increase scientific literacy and enhance the preparation of learners to pursue careers in science, there are growing opportunities for students and teachers to engage in scientific research experiences, including course-based undergraduate research experiences (CUREs), undergraduate research experiences (UREs), and teacher research experiences (TREs). Prior literature reviews detail a variety of models, benefits, and challenges and call for the continued examination of program elements and associated impacts. This paper reports a comprehensive review of 307 papers published between 2007 and 2017 that include CURE, URE, and TRE programs, with a special focus on research experiences for K-12 teachers. A research-supported conceptual model of science research experiences was used to develop a coding scheme, including participant demographics, theoretical frameworks, methodology, and reported outcomes. We summarize recent reports on program impacts and identify gaps or misalignments between goals and measured outcomes. The field of biology was the predominant scientific disciplinary focus. Findings suggest a lack of studies explicitly targeting 1) participation and outcomes related to learners from underrepresented populations, 2) a theoretical framework that guides program design and analysis, and, for TREs, 3) methods for translation of research experiences into K-12 instructional practices, and 4) measurement of impact on K-12 instructional practices.

Interference based localization of single emitters.

The ability to localize precisely a single optical emitter is important for particle tracking applications and super resolution microscopy. It is known that for a traditional microscope the ability to localize such an emitter is limited by the photon count. Here we analyze the ability to improve such localization by imposing interference fringes. We show here that a simple grating interferometer can introduce such improvement in certain circumstances and analyze what is required to increase the localization precision further.

Increased localization precision by interference fringe analysis.

We report a novel optical single-emitter-localization methodology that uses the phase induced by path length differences in a Mach-Zehnder interferometer to improve localization precision. Using information theory, we demonstrate that the localization capability of a modified Fourier domain signal generated by photon interference enables a more precise localization compared to a standard Gaussian intensity distribution of the corresponding point-spread function. The calculations were verified by numerical simulations and an exemplary experiment, where the centers of metal nanoparticles were localized to a precision of 3 nm.

Using a sharp metal tip to control the polarization and direction of emission from a quantum dot.

Optical antennas can be used to manipulate the direction and polarization of radiation from an emitter. Usually, these metallic nanostructures utilize localized plasmon resonances to generate highly directional and strongly polarized emission, which is determined predominantly by the antenna geometry alone, and is thus not easily tuned. Here we show experimentally that the emission polarization can be manipulated using a simple, nonresonant scanning probe consisting of the sharp metallic tip of an atomic force microscope; finite element simulations reveal that the emission simultaneously becomes highly directional. Together, the measurements and simulations demonstrate that interference between light emitted directly into the far field with that elastically scattered from the tip apex in the near field is responsible for this control over polarization and directionality. Due to the relatively weak emitter-tip coupling, the tip must be positioned very precisely near the emitter, but this weak coupling also leads to highly tunable emission properties with a similar degree of polarization and directionality compared to resonant antennas.

Asymmetric packaging of polymerases within vesicular stomatitis virus.

Vesicular stomatitis virus (VSV) is a prototypic negative sense single-stranded RNA virus. The bullet-shape appearance of the virion results from tightly wound helical turns of the nucleoprotein encapsidated RNA template (N-RNA) around a central cavity. Transcription and replication require polymerase complexes, which include a catalytic subunit L and a template-binding subunit P. L and P are inferred to be in the cavity, however lacking direct observation, their exact position has remained unclear. Using super-resolution fluorescence imaging and atomic force microscopy (AFM) on single VSV virions, we show that L and P are packaged asymmetrically towards the blunt end of the virus. The number of L and P proteins varies between individual virions and they occupy 57 ± 12 nm of the 150 nm central cavity of the virus. Our finding positions the polymerases at the opposite end of the genome with respect to the only transcriptional promoter.

Improved localization accuracy in stochastic super-resolution fluorescence microscopy by K-factor image deshadowing.

Localization of a single fluorescent particle with sub-diffraction-limit accuracy is a key merit in localization microscopy. Existing methods such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) achieve localization accuracies of single emitters that can reach an order of magnitude lower than the conventional resolving capabilities of optical microscopy. However, these techniques require a sparse distribution of simultaneously activated fluorophores in the field of view, resulting in larger time needed for the construction of the full image. In this paper we present the use of a nonlinear image decomposition algorithm termed K-factor, which reduces an image into a nonlinear set of contrast-ordered decompositions whose joint product reassembles the original image. The K-factor technique, when implemented on raw data prior to localization, can improve the localization accuracy of standard existing methods, and also enable the localization of overlapping particles, allowing the use of increased fluorophore activation density, and thereby increased data collection speed. Numerical simulations of fluorescence data with random probe positions, and especially at high densities of activated fluorophores, demonstrate an improvement of up to 85% in the localization precision compared to single fitting techniques. Implementing the proposed concept on experimental data of cellular structures yielded a 37% improvement in resolution for the same super-resolution image acquisition time, and a decrease of 42% in the collection time of super-resolution data with the same resolution.

Using the near-field coupling of a sharp tip to tune fluorescence-emission fluctuations during quantum-dot blinking.

We demonstrate that the cycling between internal states of quantum dots during fluorescence blinking can be used to tune the near-field coupling with a sharp tip. In particular, the fluorescence emission from states with high quantum yield is quenched due to energy transfer, while that from low-yield states is elevated due to field enhancement. Thus, as a quantum dot blinks, its emission fluctuations are progressively suppressed upon approach of a tip.

Enhancing long-range exciton guiding in molecular nanowires by H-aggregation lifetime engineering.

Excitonic transitions in organic semiconductors are associated with large oscillator strength that limits the excited-state lifetime and can in turn impede long-range exciton migration. We present perylene-based emissive H-aggregate nanowires where the lowest energy state is only weakly coupled to the ground state, thus dramatically enhancing lifetime. Exciton migration occurs by thermally activated hopping, leading to luminescence quenching on topological wire defects. An atomic force microscope tip can introduce local topological quenchers by distorting the H-aggregate structure, demonstrating long-range exciton migration at room temperature and offering a potential route to writing fluorescent "nanobarcodes" and excitonic circuits.

Energy transfer from an individual quantum dot to a carbon nanotube.

Precision measurements of resonant energy transfer from isolated quantum dots (QDs) to individual carbon nanotubes (CNTs) exhibit unique features due to the one-dimensional nature of CNTs. In particular, excitons can be created at varying distances from the QD at different locations along the CNT length. This leads to large variations in energy transfer length scales for different QDs and a novel saturation of the energy transfer efficiency at ∼96%, seemingly independent of CNT chirality.

Three-dimensional mapping of near-field interactions via single-photon tomography.

We demonstrate a near-field tomography method for investigating the coupling between a nanoscopic probe and a fluorescent sample. By correlating the arrival of single fluorescence photons with the lateral and vertical position of an oscillating tip, a complete three-dimensional analysis of the near-field coupling is achieved. The technique is used to reveal a number of interesting three-dimensional near-field features and to improve image contrast in tip-enhanced fluorescence microscopy.

Resolving single fluorophores within dense ensembles: contrast limits of tip-enhanced fluorescence microscopy.

We investigate the limits of one-photon fluorescence as a contrast mechanism in nanoscale-resolution tip-enhanced optical microscopy. Specifically, we examine the magnitude of tip-induced signal enhancement needed to resolve individual fluorophores within densely-packed ensembles. Modulation of fluorescence signals induced by an oscillating tip followed by demodulation with a lock-in amplifier increases image contrast by nearly two orders of magnitude. A theoretical model of this simple modulation/ demodulation scheme predicts an optimal value for the tip-oscillation amplitude that agrees with experimental measurements. Further, as an important step toward the eventual application of tip-enhanced fluorescence microscopy to the nanoscale structural analysis of biomolecular systems, we show that requisite signal enhancement factors are within the capabilities of commercially available silicon tips.

Fluorescence near-field microscopy of DNA at sub-10 nm resolution.

We demonstrate apertureless near-field microscopy of single molecules at sub-10 nm resolution. With a novel phase filter, near-field images of single organic fluorophores were obtained with approximately sixfold improvement in the signal-to-noise ratio. The improvement allowed pairs of molecules separated by approximately 15 nm to be reliably and repeatedly resolved, thus demonstrating the first true Rayleigh resolution test for near-field images of single molecules. The potential of this technique for biological applications was demonstrated with an experiment that measured the helical rise of A-form DNA.

Tip-enhanced fluorescence microscopy at 10 nanometer resolution.

We demonstrate unambiguously that the field enhancement near the apex of a laser-illuminated silicon tip decays according to a power law that is moderated by a single parameter characterizing the tip sharpness. Oscillating the probe in intermittent contact with a semiconductor nanocrystal strongly modulates the fluorescence excitation rate, providing robust optical contrast and enabling excellent background rejection. Laterally encoded demodulation yields images with <10 nm spatial resolution, consistent with independent measurements of tip sharpness.