Sub-diffraction Limited Resolution Research Articles

Apparent self-heating of individual upconverting nanoparticle thermometers

Individual luminescent nanoparticles enable thermometry with sub-diffraction limited spatial resolution, but potential self-heating effects from high single-particle excitation intensities remain largely uninvestigated because thermal models predict negligible self-heating. Here, we report that the common “ratiometric” thermometry signal of individual NaYF4:Yb3+,Er3+ nanoparticles unexpectedly increases with excitation intensity, implying a temperature rise over 50 K if interpreted as thermal. Luminescence lifetime thermometry, which we demonstrate for the first time using individual NaYF4:Yb3+,Er3+ nanoparticles, indicates a similar temperature rise. To resolve this apparent contradiction between model and experiment, we systematically vary the nanoparticle’s thermal environment: the substrate thermal conductivity, nanoparticle-substrate contact resistance, and nanoparticle size. The apparent self-heating remains unchanged, demonstrating that this effect is an artifact, not a real temperature rise. Using rate equation modeling, we show that this artifact results from increased radiative and non-radiative relaxation from higher-lying Er3+ energy levels. This study has important implications for single-particle thermometry.

Super-Resolution Image Reconstruction for High-Density Three-Dimensional Single-Molecule Microscopy

Single-molecule localization based super-resolution microscopy, by localizing a sparse subset of stochastically activated emitters in each frame, achieves subdiffraction-limit spatial resolution. Its temporal resolution, however, is constrained by the maximal density of activated emitters that can be successfully reconstructed. The state-of-the-art three-dimensional (3-D) reconstruction algorithm based on compressed sensing suffers from high computational complexity and gridding error due to model mismatch. In this paper, we propose a novel super-resolution algorithm for 3-D image reconstruction, dubbed TVSTORM, which promotes the sparsity of activated emitters without discretizing their locations. Several strategies are pursued to improve the reconstruction quality under the Poisson noise model, and reduce the computational time by an order-of-magnitude. Numerical results on both simulated and cell imaging data are provided to validate the favorable performance of the proposed algorithm.

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina.

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.

The Detection of Nanoscale Membrane Bending with Polarized Localization Microscopy

The curvature of biological membranes at the nanometer scale is critically important for vesicle trafficking, organelle morphology, and disease propagation. The initiation of membrane bending occurs at a length scale that is irresolvable by most superresolution optical microscopy methods. Here, we report the development of polarized localization microscopy (PLM), a pointillist optical imaging technique for the detection of nanoscale membrane curvature in correlation with single-molecule dynamics and molecular sorting. PLM combines polarized total internal reflection fluorescence microscopy and single-molecule localization microscopy to reveal membrane orientation with subdiffraction-limited resolution without reducing localization precision by point spread function manipulation. Membrane curvature detection with PLM requires fewer localization events to detect curvature than three-dimensional single-molecule localization microscopy (e.g., photoactivated localization microscopy or stochastic optical reconstruction microscopy), which enables curvature detection 10× faster via PLM. With rotationally confined lipophilic fluorophores and the polarized incident fluorescence excitation, membrane-bending events are revealed with superresolution. Engineered hemispherical membrane curvature with a radius ≥24 nm was detected with PLM, and individual fluorophore localization precision was 13 ± 5 nm. Further, deciphering molecular mobility as a function of membrane topology was enabled. The diffusion coefficient of individual DiI molecules was 25 ± 5× higher in planar supported lipid bilayers than within nanoscale membrane curvature. Through the theoretical foundation and experimental demonstration provided here, PLM is poised to become a powerful technique for revealing the underlying biophysical mechanisms of membrane bending at physiological length scales.

Two-photon polymerization for biological applications

Two-photon polymerization (2PP) leverages the two-photon absorption (TPA) of near-infrared (NIR) radiation for additive manufacturing with sub-diffraction limit resolution within the bulk of a photosensitive material. This technology draws heavily on photosensitive polymers from the microelectronics industry, which were not optimized for TPA or for biocompatibility. 2PP with sub 100 nm resolution has been repeatedly demonstrated; however, this level of fabrication resolution comes at the expense of long fabrication times. Manufacturing of medical devices beyond surface texturing would be prohibitively slow using the current state of the art 2PP technology. Current research into TPA-sensitive photopolymers with good biocompatibility and holographic projections using spatial light modulators address current technological limitations by providing materials specifically formulated for biological applications and by making better use of available laser power for applications in which nanoscale resolution is not required.

A Method For Achieving Super Resolution Vibrational Sum-Frequency Generation Microscopy By Structured Illumination

A scheme was proposed to achieve wide-field vibrational sum-frequency generation (SFG) microscopy with subdiffraction-limited resolution in one dimension. In this approach, samples are illuminated with a structured visible field and a uniform IR field to induce vibrational sum-frequency generation. With five raw images acquired at five different phases of the visible stripe pattern, a super-resolved vibrational SFG (SR-SFG) image with the resolution triple to that of the original can be reconstructed. Theoretical framework describing the coherent image formation and reconstruction scheme for the SR-SFG imaging system was derived and carried out with numerical simulations to investigate its imaging performance. With a typical imaging system, the lateral resolution was improved from 390 nm of the conventional SFG imaging system, to around 130 nm of the SR-SFG imaging system.

SAVI: Synthetic apertures for long-range, subdiffraction-limited visible imaging using Fourier ptychography.

Synthetic aperture radar is a well-known technique for improving resolution in radio imaging. Extending these synthetic aperture techniques to the visible light domain is not straightforward because optical receivers cannot measure phase information. We propose to use macroscopic Fourier ptychography (FP) as a practical means of creating a synthetic aperture for visible imaging to achieve subdiffraction-limited resolution. We demonstrate the first working prototype for macroscopic FP in a reflection imaging geometry that is capable of imaging optically rough objects. In addition, a novel image space denoising regularization is introduced during phase retrieval to reduce the effects of speckle and improve perceptual quality of the recovered high-resolution image. Our approach is validated experimentally where the resolution of various diffuse objects is improved sixfold.

Three-dimensional wide-field pump-probe structured illumination microscopy.

We propose a new structured illumination scheme for achieving depth resolved wide-field pump-probe microscopy with sub-diffraction limit resolution. By acquiring coherent pump-probe images using a set of 3D structured light illumination patterns, a 3D super-resolution pump-probe image can be reconstructed. We derive the theoretical framework to describe the coherent image formation and reconstruction scheme for this structured illumination pump-probe imaging system and carry out numerical simulations to investigate its imaging performance. The results demonstrate a lateral resolution improvement by a factor of three and providing 0.5 µm level axial optical sectioning.

Super-Resolution Imaging and Plasmonics.

This review describes the growing partnership between super-resolution imaging and plasmonics, by describing the various ways in which the two topics mutually benefit one another to enhance our understanding of the nanoscale world. First, localization-based super-resolution imaging strategies, where molecules are modulated between emissive and nonemissive states and their emission localized, are applied to plasmonic nanoparticle substrates, revealing the hidden shape of the nanoparticles while also mapping local electromagnetic field enhancements and reactivity patterns on their surface. However, these results must be interpreted carefully due to localization errors induced by the interaction between metallic substrates and single fluorophores. Second, plasmonic nanoparticles are explored as image contrast agents for both superlocalization and super-resolution imaging, offering benefits such as high photostability, large signal-to-noise, and distance-dependent spectral features but presenting challenges for localizing individual nanoparticles within a diffraction-limited spot. Finally, the use of plasmon-tailored excitation fields to achieve subdiffraction-limited spatial resolution is discussed, using localized surface plasmons and surface plasmon polaritons to create confined excitation volumes or image magnification to enhance spatial resolution.

3D nano surface profilometry by combining the photonic nanojet with interferometry

Interference microscopy has become a standard technique for measuring microscopic surface roughness, being rapid, non-contact and having a high nanometric axial resolution. Nonetheless, a limitation is the lateral resolution of λ/2 due to diffraction. Amongst the many recently developed techniques for improving the lateral resolution in unlabelled far field imaging, the use of microspheres placed on the sample has recently been extended from 2D imaging to 3D measurement by combining it with interferometry. In the present work, results are shown of combining the high lateral resolution of the photonic nanojet produced by the microsphere with interference microscopy to achieve sub-diffraction limited lateral resolution with nanometric axial resolution. This is achieved by imaging through a microsphere placed on top of the sample in front of the interference objective in a Linnik setup. Results of the 3D measurements of narrow gratings through the microsphere are presented, that are not observable with the same objective in air since they are below the resolution limit. Simulations of the interaction between the photonic jet and the grating leads to a basic understanding of the image formation. This new technique opens new possibilities for high resolution characterization in nanomaterials and the biological sciences.

Ultrasensitive Detection of α-Fetoprotein by Total Internal Reflection Scattering-Based Super-Resolution Microscopy for Superlocalization of Nano-Immunoplasmonics.

Superlocalization of immunoplasmonic nanotags on antibody-bound gold-nanoislands (GNIs) along the x and y coordinates was determined using total internal reflection scattering-based super-resolution microscopy (TIRS-SRM) at subdiffraction limit resolution. Individual immunoplasmonic nanotags (20 nm silver nanoparticles) and 100 nm GNIs were selectively acquired in the evanescent field layer by wavelength-dependent plasmonic scattering using two illumination lasers (405 and 635 nm, respectively). α-Fetoprotein (AFP), a liver cancer-related model protein, was immobilized as a target molecule on the GNI arrays. The centroid position of a localized immunoplasmonic nanotag on the GNI was resolved at less than 10 nm of spatial resolution by applying 2D Gaussian fitting to its point spread function. This method showed enhanced sensitive quantification with a limit of detection (LOD) of 7.04 zM (1-2 molecules of AFP/GNI), which was 100-5000000000 times lower than detection limits obtained with previous AFP detection methods. Furthermore, the method was also successfully applied to quantify AFP molecules at the single-molecule level in human serum samples. The wavelength-dependent TIRS-SRM method was demonstrated to be an effective tool for superlocalizing individual protein molecules and interactions in nanoscale regions and was a reliable method for the ultrasensitive quantitative detection of disease-related protein molecules as a nanosensor and for diagnosis at the single-molecule level.

Augmented 3D super-resolution of fluorescence-free nanoparticles using enhanced dark-field illumination based on wavelength-modulation and a least-cubic algorithm.

Augmented three-dimensional (3D) subdiffraction-limited resolution of fluorescence-free single-nanoparticles was achieved with wavelength-dependent enhanced dark-field (EDF) illumination and a least-cubic algorithm. Various plasmonic nanoparticles on a glass slide (i.e., gold nanoparticles, GNPs; silver nanoparticles, SNPs; and gold nanorods, GNRs) were imaged and sliced in the z-direction to a thickness of 10 nm. Single-particle images were then compared with simulation data. The 3D coordinates of individual GNP, SNP, and GNR nanoparticles (x, y, z) were resolved by fitting the data with 3D point spread functions using a least-cubic algorithm and collation. Final, 3D super-resolution microscopy (SRM) images were obtained by resolving 3D coordinates and their Cramér-Rao lower bound-based localization precisions in an image space (530 nm × 530 nm × 300 nm) with a specific voxel size (2.5 nm × 2.5 nm × 5 nm). Compared with the commonly used least-square method, the least-cubic method was more useful for finding the center in asymmetric cases (i.e., nanorods) with high precision and accuracy. This novel 3D fluorescence-free SRM technique was successfully applied to resolve the positions of various nanoparticles on glass and gold nanospots (in vitro) as well as in a living single cell (in vivo) with subdiffraction limited resolution in 3D.

Near-Field Imaging of Cell Membranes in Liquid Enabled by Active Scanning Probe Mechanical Resonance Control

Despite the power of far-field super-resolution microscopies for three-dimensional imaging of biomolecular structures and processes, its application is challenged in dense and crowded samples and for certain surface and membrane studies. Although near-field imaging with its ability to provide intrinsic subdiffraction limited spatial resolution at any optical modality, its application to biological systems has remained limited because of the difficulties of routine operation in liquid environments. Here we demonstrate stable and sensitive near-field scanning optical microscopy (NSOM) in a liquid based on a new mechanical resonance control and an optimization of the tip length, achieving a high quality factor (>2800) force sensing of the near-field probe. Through near-field imaging of the spatial distribution of epidermal growth factor receptors (EGFRs) on the membrane of A431 cancer cells as an example, we reveal nanoscale correlations between surface EGFR and intracellular organelle structures with ∼50 nm...

Dual-color STED microscopy reveals a sandwich structure of Bassoon and Piccolo in active zones of adult and aged mice.

Presynaptic active zones play a pivotal role as synaptic vesicle release sites for synaptic transmission, but the molecular architecture of active zones in mammalian neuromuscular junctions (NMJs) at sub-diffraction limited resolution remains unknown. Bassoon and Piccolo are active zone specific cytosolic proteins essential for active zone assembly in NMJs, ribbon synapses, and brain synapses. These proteins are thought to colocalize and share some functions at active zones. Here, we report an unexpected finding of non-overlapping localization of these two proteins in mouse NMJs revealed using dual-color stimulated emission depletion (STED) super resolution microscopy. Piccolo puncta sandwiched Bassoon puncta and aligned in a Piccolo-Bassoon-Piccolo structure in adult NMJs. P/Q-type voltage-gated calcium channel (VGCC) puncta colocalized with Bassoon puncta. The P/Q-type VGCC and Bassoon protein levels decreased significantly in NMJs from aged mouse. In contrast, the Piccolo levels in NMJs from aged mice were comparable to levels in adult mice. This study revealed the molecular architecture of active zones in mouse NMJs at sub-diffraction limited resolution, and described the selective degeneration mechanism of active zone proteins in NMJs from aged mice. Interestingly, the localization pattern of active zone proteins described herein is similar to active zone structures described using electron microscope tomography.

Characterizing Transcription and Splicing Kinetics by 3D Orbital Tracking

We describe our recent application of two photon laser scanning microscopy to characterize transcription and splicing rates in living cells with sub-diffraction limited spatial resolution and second to hour long temporal resolution by the application of 3D orbital tracking[1] and two color in vivo RNA labelling[2]. We find that measurements agree with previous studies utilizing widefield volumetric imaging but increase the temporal resolution 100 fold and the duration of each experiment by 10 fold characterizing protein-RNA binding, RNA synthesis and splicing and long timescale transcriptional bursting in a single measurement. This technique may have future applications for the study of both natural and synthetic gene regulation in eukaryotes.1. Levi V, Ruan Q, Gratton E. 3-D particle tracking in a two-photon microscope: application to the study of molecular dynamics in cells. Biophys J. 2005;88: 2919-2928. http://dx.doi.org/10.1529/biophysj.104.0442302. Coulon A, Ferguson ML, de Turris V, Palangat M, Chow CC, Larson DR. Kinetic competition during the transcription cycle results in stochastic RNA processing. Elife. 2014;3. http://dx.doi.org/10.7554/eLife.03939.

Femtosecond Nanostructuring of Glass with Optically Trapped Microspheres and Chemical Etching.

Laser processing with optically trapped microspheres is a promising tool for nanopatterning at subdiffraction-limited resolution in a wide range of technological and biomedical applications. In this paper, we investigate subdiffraction-limited structuring of borosilicate glass with femtosecond pulses in the near-field of optically trapped microspheres combined with chemical postprocessing. The glass surface was processed by single laser pulses at 780 nm focused by silica microspheres and then subjected to selective etching in KOH, which produced pits in the laser-affected zones (LAZs). Chemical postprocessing allowed obtaining structures with better resolution and reproducibility. We demonstrate production of reproducible pits with diameters as small as 70 nm (λ/11). Complex two-dimensional structures with 100 nm (λ/8) resolution were written on the glass surface point by point with microspheres manipulated by optical tweezers. Furthermore, the mechanism of laser modification underlying selective etching was investigated with mass spectrum analysis. We propose that the increased etching rate of laser-treated glass results from changes in its chemical composition and oxygen deficiency.

A review on the processing accuracy of two-photon polymerization

Two-photon polymerization (TPP) is a powerful and potential technology to fabricate true three-dimensional (3D) micro/nanostructures of various materials with subdiffraction-limit resolution. And it has been applied to microoptics, electronics, communications, biomedicine, microfluidic devices, MEMS and metamaterials. These applications, such as microoptics and photon crystals, put forward rigorous requirements on the processing accuracy of TPP, including the dimensional accuracy, shape accuracy and surface roughness and the processing accuracy influences their performance, even invalidate them. In order to fabricate precise 3D micro/nanostructures, the factors influencing the processing accuracy need to be considered comprehensively and systematically. In this paper, we review the basis of TPP micro/nanofabrication, including mechanism of TPP, experimental set-up for TPP and scaling laws of resolution of TPP. Then, we discuss the factors influencing the processing accuracy. Finally, we summarize the methods reported lately to improve the processing accuracy from improving the resolution and changing spatial arrangement of voxels.

Nonlinear spectroscopy in the near-field: time resolved spectroscopy and subwavelength resolution non-invasive imaging

AbstractThe combination of near-field microscopy along with nonlinear optical spectroscopic techniques is presented here. The scanning near-field imaging technique can be integrated with nonlinear spectroscopic techniques to improve spatial and axial resolution of the images. Additionally, ultrafast dynamics can be probed down to nano-scale dimension. The review shows some examples for this combination, which resulted in an exciton map and vibrational contrast images with sub-wavelength resolution. Results of two-color femtosecond time-resolved pump-probe experiments using scanning near-field optical microscopy (SNOM) on thin films of the organic semiconductor 3,4,9,10 Perylenetetracarboxylic dianhydride (PTCDA) are presented. While nonlinear Raman techniques have been used to obtain highly resolved images in combination with near-field microscopy, the use of femtosecond laser pulses in electronic resonance still constitutes a big challenge. Here, we present our first results on coherent anti-Stokes Raman scattering (fs-CARS) with femtosecond laser pulses detected in the near-field using SNOM. We demonstrate that highly spatially resolved images can be obtained from poly(3-hexylthiophene) (P3HT) nano-structures where the fs-CARS process was in resonance with the P3HT absorption and with characteristic P3HT vibrational modes without destruction of the samples. Sub-diffraction limited lateral resolution is achieved. Especially the height resolution clearly surpasses that obtained with standard microCARS. These results will be the basis for future investigations of mode-selective dynamics in the near-field.

Super Resolution Microscopy with Low Power CW Lasers

We demonstrate a new and experimentally straightforward method for obtaining sub-diffraction limit resolution in fluorescence microscopy with low on-sample laser powers. The technique involves the analysis of the time evolution of fluorescence images in the presence of weak and unstructured continuous wave (CW) stimulated emission. A sub-diffraction limited point spread function (PSF) is obtained by the recombination of the time segments of the evolving image. Theoretical modelling presented here shows how manipulation of the fluorescence lifetime results in modification of the spatial distribution of fluorescence on a nanosecond timescale, and how photon noise impacts upon this. A computational method for image reconstruction using a genetic algorithm is also described. The experimental set-up is straightforward and consists of a commercial fluorescence lifetime imaging microscope with a low cost, low power CW laser co-linear with the pulsed excitation laser. On-sample laser power is typically <10mW. Preliminary results are presented for imaging and subsequent reconstruction of 20nm fluorescent beads, both isolated and absorbed into living HEK-293 cells. Sub-diffraction limit resolution is readily observed, and neither morphology nor viability of the HEK-293 cells appears to be compromised.