Improved spatial resolution in fluorescence focal modulation microscopy

Point defects in crystals provide important building blocks for quantum applications. Since we optically address these defect qubits, having an efficient optical interface is a highly important aspect. However, conventional confocal fluorescence microscopy of high-refractive-index crystals suffers from limited photon collection efficiency and spatial resolution. Here, we demonstrate high-resolution, high-contrast imaging of defects in diamonds using microsphere-assisted confocal microscopy. A microsphere provides an excellent optical interface for point defects with a magnified virtual image that increases the spatial resolution up to λ/5, as well as the optical signal-to-noise ratio by four times. These features enable individual optical addressing of single photons and single spins of multiple defects that are spatially unresolved in conventional confocal microscopy, with improved signal contrast. Combined with optical tweezers, this system also demonstrates the possibility of positioning or scanning the microspheres. The approach does not require any complicated fabrication or additional optical systems, but uses simple, off-the-shelf micro-optics. From these distinctive advantages of microspheres, our approach provides an efficient way to image and address closely spaced defects with much better resolution and sensitivity.

Proton CT (pCT) has the potential to accurately measure the electron density map of tissues at low doses but the spatial resolution is prohibitive if the curved paths of protons in matter is not accounted for. The authors propose to account for an estimate of the most likely path of protons in a filtered backprojection (FBP) reconstruction algorithm. The energy loss of protons is first binned in several proton radiographs at different distances to the proton source to exploit the depth-dependency of the estimate of the most likely path. This process is named the distance-driven binning. A voxel-specific backprojection is then used to select the adequate radiograph in the distance-driven binning in order to propagate in the pCT image the best achievable spatial resolution in proton radiographs. The improvement in spatial resolution is demonstrated using Monte Carlo simulations of resolution phantoms. The spatial resolution in the distance-driven binning depended on the distance of the objects from the source and was optimal in the binned radiograph corresponding to that distance. The spatial resolution in the reconstructed pCT images decreased with the depth in the scanned object but it was always better than previous FBP algorithms assuming straight line paths. In a water cylinder with 20 cm diameter, the observed range of spatial resolutions was 0.7 - 1.6 mm compared to 1.0 - 2.4 mm at best with a straight line path assumption. The improvement was strongly enhanced in shorter 200° scans. Improved spatial resolution was obtained in pCT images with filtered backprojection reconstruction using most likely path estimates of protons. The improvement in spatial resolution combined with the practicality of FBP algorithms compared to iterative reconstruction algorithms makes this new algorithm a candidate of choice for clinical pCT.

Acquisition time and optical sectioning capability are critical factors in fluorescence imaging. Confocal microscopy is a vital optical imaging method to inherently observe volumetric tissues with fine optical sectioning capability; however, point‐by‐point scanning is time‐consuming. Metasurfaces, a type of flat optics utilizing nano‐scale structures, provide diverse functionalities and extensive flexibility in controlling light wavefronts. Here, meta‐microlens‐array (meta‐MLA) for multifocal confocal fluorescence microscopy to enhance acquisition speed is introduced, reduce photo‐bleaching, and improve energy efficiency while remaining compatible with existing commercial scanning configurations. Point spread function (PSF) in the meta‐MLA confocal lateral and axial directions has been evaluated. Fast optically sectioned images of various samples, including pollen grains and biological tissue phantoms, are performed. Image quality is further enhanced by the Richardson–Lucy (RL) deconvolution method with total variation (TV). The trade‐off between spatial resolution and acquisition speed is overcome using deep neural network models, comparing performance metrics with a conventional confocal microscope. The combination of meta‐MLA confocal and deep learning with superior image quality and fast acquisition will likely extend the clinical applications of miniaturized optical imaging.

This doctoral thesis addresses two novel approaches in fluorescence microscopy. The first part introduces a new method that can be used to enhance the effect of photoselection. This method, termed ExPAN (excitation polarization angle narrowing), exploits stimulated emission to narrow the angular distribution of excited fluorophores. ExPAN could be of significant interest for applications like fluorescence anisotropy measurements, which are commonly applied in life science, e.g. to study receptor-ligand interactions or investigate protein dynamics. In the context of this work, ExPAN was applied in conjunction with a new fluorescence microscopy approach that utilizes molecular orientation as a contrast mechanism. Using excitation light with continuously rotating polarization, it is possible to obtain information about the orientation of dye molecules. When excited in this manner, molecules with a preferred orientation exhibit a periodically modulated fluorescence signal as a result of photoselection. This work showed that the additional modulation information can be utilized to distinguish molecules with different orientations even when their signals overlap spatially. The approach was explored by experiments with single molecules on a surface using a modified widefield microscope and additionally by simulation studies. The results indicated that it is possible to distinguish signals from dye molecules with a distance down to 80 nm. It was demonstrated that ExPAN enhances the effect of photoselection and can be used to distinguish molecules even when their orientations are very similar. Furthermore, a modulated fluorescence signal was also observed in fixed biological samples and with surface labeled microparticles in aqueous solution. In the second part of this thesis a method is presented for improving the resolution of confocal laser scanning microscopes. This method, termed multi-detector scanning (MDS), is based on the concept of image scanning microscopy (ISM). Using ISM it is theoretically possible to double the resolution of a fluorescence microscope. Since ISM requires an array detector, most of the previously developed implementations use CCD or CMOS cameras for detection. Instead of a camera, several single-photon detectors that are combined to an array detector by a fiber optic bundle, are used in this work.This configuration allowed it for the first time to use the method in conjunction with Fluorescence-lifetime imaging microscopy (FLIM). FLIM has proven to be an important microscopy technique in life science and has been applied in a wide variety of studies including protein-protein interaction and cell metabolism research. Therefore, the improvement in spatial resolution of FLIM through ISM is of potential interest for numerous biological applications. Within the scope of this work, a multi-detector scanning microscope was constructed and characterized. Resolution improvements of 168 nm with MDS and 146 nm with MDS and deconvolution were achieved in fixed biological samples. Furthermore, the measurement of fluorescence lifetimes was demonstrated with the experimental setup. Thus, the application of MDS has the potential to increase the spatial resolution of FLIM and could serve as a tool for measuring biological systems in more detail.

In early studies of proton computed tomography (pCT), images were reconstructed with the fast and robust filtered backprojection (FBP) algorithm. Due to multiple Coulomb scattering of the protons within the object, the straight line path assumption of FBP resulted in poor spatial resolution. In an attempt to improve spatial resolution, a formalism to predict the proton path of maximum likelihood through the image space was created. The use of these paths with the iterative algebraic reconstruction technique (ART), have shown an improvement in spatial resolution, but also an increase in image noise, resulting in poor density resolution. In this work, we propose a reconstruction method that attempts to optimize both spatial and density resolution of pCT images. The new reconstruction approach makes use of the block-iterative diagonally relaxed orthogonal projections (DROP) algorithm with an initial FBP-reconstructed image estimate. Reconstruction of Monte Carlo simulated pCT data sets of spatial and density resolution phantoms demonstrated that the combined reconstruction approach resulted in better spatial resolution than the FBP algorithm alone and better density resolution than the DROP algorithm starting from a uniform initial image estimate.

Live-Cell Super-Resolution Fluorescence Imaging at High Spatiotemporal Resolutions

To evaluate the spatial resolution of proton CT using both a prototype proton CT scanner and Monte Carlo simulations. A custom cylindrical edge phantom containing twelve tissue-equivalent inserts with four different compositions at varying radial displacements from the axis of rotation was developed for measuring the modulation transfer function (MTF) of a prototype proton CT scanner. Two scans of the phantom, centered on the axis of rotation, were obtained with a 200 MeV, low-intensity proton beam: one scan with steps of 4°, and one scan with the phantom continuously rotating. In addition, Monte Carlo simulations of the phantom scan were performed using scanners idealized to various degrees. The data were reconstructed using an iterative projection method with added total variation superiorization based on individual proton histories. Edge spread functions in the radial and azimuthal directions were obtained using the oversampling technique. These were then used to obtain the modulation transfer functions. The spatial resolution was defined by the 10% value of the modulation transfer function (MTF10%) in units of line pairs per centimeter (lp/cm). Data from the simulations were used to better understand the contributions of multiple Coulomb scattering in the phantom and the scanner hardware, as well as the effect of discretization of proton location. The radial spatial resolution of the prototype proton CT scanner depends on the total path length, W, of the proton in the phantom, whereas the azimuthal spatial resolution depends both on W and the position, u-, at which the most-likely path uncertainty is evaluated along the path. For protons contributing to radial spatial resolution, W varies with the radial position of the edge, whereas for protons contributing to azimuthal spatial resolution, W is approximately constant. For a pixel size of 0.625 mm, the radial spatial resolution of the image reconstructed from the fully idealized simulation data ranged between 6.31 ± 0.36 lp/cm for W = 197 mm i.e., close to the center of the phantom, and 13.79 ± 0.36 lp/cm for W = 97 mm, near the periphery of the phantom. The azimuthal spatial resolution ranged from 6.99 ± 0.23 lp/cm at u- = 75 mm (near the center) to 11.20 ± 0.26 lp/cm at u- = 20 mm (near the periphery). Multiple Coulomb scattering limits the radial spatial resolution for path lengths greater than approximately 130 mm, and the azimuthal spatial resolution for positions of evaluation greater than approximately 40 mm for W = 199 mm. The radial spatial resolution of the image reconstructed from data from the 4° stepped experimental scan ranged from 5.11 ± 0.61 lp/cm for W = 197 mm to 8.58 ± 0.50 lp/cm for W = 97 mm. In the azimuthal direction, the spatial resolution ranged from 5.37 ± 0.40 lp/cm at u- = 75 mm to 7.27 ± 0.39 lp/cm at u- = 20 mm. The continuous scan achieved the same spatial resolution as that of the stepped scan. Multiple Coulomb scattering in the phantom is the limiting physical factor of the achievable spatial resolution of proton CT; additional loss of spatial resolution in the prototype system is associated with scattering in the proton tracking system and inadequacies of the proton path estimate used in the iterative reconstruction algorithm. Improvement in spatial resolution may be achievable by improving the most likely path estimate by incorporating information about high and low density materials, and by minimizing multiple Coulomb scattering in the proton tracking system.

Thermoresistive probes are increasingly popular in thermal conductivity characterization using scanning thermal microscopy (SThM). A systematic analysis of the thermal conductivity measurement performance (sensitivity and spatial resolution) of thermoresistive SThM probe configurations that are available commercially is of interest to practitioners. In this work, the authors developed and validated 3D finite element models of noncontact SThM with self-heated thermoresistive probes under ambient conditions with the probe–sample heat transfer in transition heat conduction regime for the four types of SThM probe configurations resembling commercially available products: Wollaston wire (WW) type probe, Kelvin nanotechnology (KNT) type probe, doped silicon (DS) type probe and nanowire (NW) type probe. These models were then used to investigate the sensitivity and spatial resolution of the WW, KNT, DS and NW type probes for thermal conductivity measurements in noncontact mode in ambient conditions. The comparison of the SThM probes performance for measuring sample thermal conductivity and for the specific operating conditions investigated here show that the NW type probe has the best spatial resolution while the DS type probe has the best thermal conductivity measurement sensitivity in the range between 2 and 10 W·m−1·K−1. The spatial resolution is negatively affected by large probe diameters or by the presence of the cantilever in close proximity to the sample surface which strongly affects the probe–sample heat transfer in ambient conditions. An example of probe geometry configuration optimization was illustrated for the WW probe by investigating the effect of probe wire diameter on the thermal conductivity measurement sensitivity, showing ∼20% improvement in spatial resolution at the diameter with maximum thermal conductivity measurement sensitivity.

Far-field optical microscopy using focused light is an important tool in a number of scientific disciplines including chemical, (bio)physical and biomedical research, particularly with respect to the study of living cells and organisms. Unfortunately, the applicability of the optical microscope is limited, since the diffraction of light imposes limitations on the spatial resolution of the image. Consequently the details of, for example, cellular protein distributions, can be visualized only to a certain extent. Fortunately, recent years have witnessed the development of ‘super-resolution’ far-field optical microscopy (nanoscopy) techniques such as stimulated emission depletion (STED), ground state depletion (GSD), reversible saturated optical (fluorescence) transitions (RESOLFT), photoactivation localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), structured illumination microscopy (SIM) or saturated structured illumination microscopy (SSIM), all in one way or another addressing the problem of the limited spatial resolution of far-field optical microscopy. While SIM achieves a two-fold improvement in spatial resolution compared to conventional optical microscopy, STED, RESOLFT, PALM/STORM, or SSIM have all gone beyond, pushing the limits of optical image resolution to the nanometer scale. Consequently, all super-resolution techniques open new avenues of biomedical research. Because the field is so young, the potential capabilities of different super-resolution microscopy approaches have yet to be fully explored, and uncertainties remain when considering the best choice of methodology. Thus, even for experts, the road to the future is sometimes shrouded in mist. The super-resolution optical microscopy roadmap of Journal of Physics D: Applied Physics addresses this need for clarity. It provides guidance to the outstanding questions through a collection of short review articles from experts in the field, giving a thorough discussion on the concepts underlying super-resolution optical microscopy, the potential of different approaches, the importance of label optimization (such as reversible photoswitchable proteins) and applications in which these methods will have a significant impact.Mark Bates, Christian Eggeling

Single photon emission computed tomography (SPECT) imaging inherits some limitations, i.e., due to scattered gamma photons which degrade spatial resolution causes poor image quality. This study attempts to reduce a fraction of scattered gamma photons before reaching gamma camera detector by using Fe sheet (0.35 mm and 0.40 mm) as a physical filter. Also investigate the effects on spectra of Tc-99m, spatial resolution, system volume sensitivity and uniformity. The thickness of Fe physical filter is selected on the basis of percentage attenuation calculations of different gamma ray energies by various thicknesses of material. Data were acquired using Philip ADAC forte dual-head gamma camera without and with physical filter with LEHR collimator installed. For spectra, uniformity and system volume sensitivity, a cylindrical source tank filled with water added with Tc-99m was scanned. Uniformity and system volume sensitivity images were reconstructed with FBP method by applying Butterworth filter of order 5, cut-off frequency 0.35 cycles/cm and Chang's attenuation correction method using 0.13 cm−1 linear attenuation coefficient. Spatial resolution study was done by scanning a line source (0.8 mm inner diameter) of Tc-99m at various source-to-collimator distances in air and in scattering medium without and with physical filter. A substantial reduction in count rate from Compton and photopeak regions of Tc-99m spectra with physical filter is recorded. Improvement in spatial resolution with physical filter up to 4 cm source-to-collimator distance is obtained. System volume sensitivity was reduced and no improvement in uniformity. These thicknesses of physical filter may be tested further by scanning different planar/SPECT phantoms in Tc-99m imaging.

Improving the spatial resolution in CZT detectors using charge sharing effect and transient signal analysis: Simulation study

To achieve good spatial resolution, small loop size is required in the traditional loop probe. However, the smaller loop size will lead to lower sensitivity for the probe. In addition, loop size is always limited by the minimum line spacing of the fabrication process. Another problem is that the asymmetric electric field coupling into a probe will not be canceled perfectly even if the structure of this probe is symmetric. To circumvent these problems, a space difference magnetic near-field probe with three kinds of spatial resolutions is proposed in this paper. The probe head including a one-turn loop and a two-turn loop is manufactured in low temperature co-fired ceramics (LTCC). The one-turn loop is clamped with the two-turn loop. Two loops are covered with two shielding ground plates to form a tri-plate structure. The received signals from these two loops are outputted with two SMA connectors through two striplines. The flip-chip junctions with low loss and good shielding capability are used between the probe head and the striplines. The probe characteristics are measured using a 436- μm-wide microstrip line with impedance of 50 Ω. Two output ports have different spatial resolutions because two different loops are located above the microstrip line at different height. The proposed probe will have higher spatial resolution when the received signals are outputted in difference. These results are also verified by measuring a 2000- μm-wide straight and a 500- μm-wide meander microstrip line. The experiment results have good agreement with the simulation results and show the resolution improvement of the difference output of the proposed probe for different lines.

Super‐resolution fluorescence imaging provides critically improved information about the composition, organization, and dynamics of subcellular structures. Quantum dot triexciton imaging (QDTI) has been introduced as an easy‐to‐use sub‐diffraction imaging method that achieves an almost 2‐fold improvement in resolution when used with conventional confocal microscopes. Here, we report an overall 3‐fold increase in lateral and axial resolution compared to conventional confocal microscopes by combining QDTI with state‐of‐the‐art commercial laser scanning microscope systems.

Background: For many years, air quality researchers have focused heavily on improving the spatial resolution of outdoor pollution estimates. This has been accomplished through improved satellite remote sensing technologies combined with complex models, currently producing surface-level estimates down to the kilometer and sub-kilometer scales. However, as resolutions grow increasingly refined, it is important to evaluate how much new information additional resolution provides and compare the relative importance of spatial and temporal variability in assigning pollution exposures to study populations. Methods: Daily PM2.5 estimates at a 1 km2 resolution, derived from NASA’s MODIS satellite instrument and the MAIAC algorithm, were estimated for 2015-2018 and linked to California ZIP codes using spatial interpolation weighting factors. Within- and between-ZIP code spatial and temporal variations were calculated for all cities with at least three ZIP codes, then aggregated and analyzed using time-series and GIS analyses. Results: Variation of PM2.5 concentrations within ZIP codes is negligible, with much greater variation observed between ZIP codes in the same city. Within and between ZIP code variation did show strong seasonal influences with greater variation observed during the cooler months of the year, particularly in Northern California which is heavily influenced by local emissions sources. In all cases, temporal variability was much greater than spatial variability at the sub-urban spatial resolutions included in this study. Conclusions: For much of the year, there is little additional information gained by using spatial resolutions more refined than ZIP code level estimates in California during the study period. However, during specific seasons there may be additional value in assigning exposures based on resolutions down to 1 km2. As air quality research moves forward, there may be diminishing returns from further improvements in spatial resolution for PM2.5 and efforts may be better allocated to other areas of exposure assessment.

Aperture synthesis technique can provide high spatial resolution without requiring very large and massive real aperture. However, for a large aperture synthesis system, which will involve hundreds of antennas and thousands of correlators, system and its calibration are very complicated. In this letter, mirrored interferometric aperture synthesis (MIAS) is proposed, which can achieve the same spatial resolution as a large traditional aperture synthesis system but needs fewer antennas. First, the imaging principle of MIAS is presented. A new concept, named Cosine Visibility, is proposed to replace the visibility function used in traditional aperture synthesis. The relationship between the brightness temperature and the cosine visibility is a Cosine Transform. Inverse Cosine Transform can be applied to reconstruct the image of brightness temperature. Then, an example for MIAS is given, and a method is proposed to handle the problems of baselines missing and the rank defect associated with the underdetermined linear equations which relate the cross correlation between signals collected by pairs of antennas with the cosine visibility. Moreover, MIAS with a small array can provide higher and higher spatial resolution by combining more and more linear equations with the help of adjusting the distance from the array to the reflector. The simulation results demonstrate the validity of MIAS and the improvement of spatial resolution. The study in this letter provides a means to reduce the complexity of system and calibration of large traditional aperture synthesis systems.