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1. Shot noise-mitigated secondary electron imaging with ion count-aided microscopy

   https://doi.org/10.1073/pnas.2401246121  

   Agarwal, Akshay; Kasaei, Leila; He, Xinglin; Kitichotkul, Ruangrawee; Hitit, Oğuz Kağan; Peng, Minxu; Schultz, J Albert; Feldman, Leonard C; Goyal, Vivek K (July 2024, Proceedings of the National Academy of Sciences)

   Modern science is dependent on imaging on the nanoscale, often achieved through processes that detect secondary electrons created by a highly focused incident charged particle beam. Multiple types of measurement noise limit the ultimate trade-off between the image quality and the incident particle dose, which can preclude useful imaging of dose-sensitive samples. Existing methods to improve image quality do not fundamentally mitigate the noise sources. Furthermore, barriers to assigning a physically meaningful scale make the images qualitative. Here, we introduce ion count-aided microscopy (ICAM), which is a quantitative imaging technique that uses statistically principled estimation of the secondary electron yield. With a readily implemented change in data collection, ICAM substantially reduces source shot noise. In helium ion microscopy, we demonstrate 3 $\times$dose reduction and a good match between these empirical results and theoretical performance predictions. ICAM facilitates imaging of fragile samples and may make imaging with heavier particles more attractive. 

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2. Combined speckle- and propagation-based single shot two-dimensional phase retrieval method

   https://doi.org/10.1364/OE.531269  

   Leong, Andrew_F_T; Hodge, Daniel_S; Kurzer-Ogul, Kelin; Marchesini, Stefano; Pandolfi, Silvia; Liu, Yanwei; Barber, John_L; Li, Kenan; Sakdinawat, Anne; Galtier, Eric_C; et al (December 2024, Optics Express)

   Single-shot two-dimensional (2D) phase retrieval (PR) can recover the phase shift distribution within an object from a single 2D x-ray phase contrast image (XPCI). Two competing XPCI imaging modalities often used for single-shot 2D PR to recover material properties critical for predictive performance capabilities are: speckle-based (SP-XPCI) and propagation-based (PB-XPCI) XPCI imaging. However, PR from SP-XPCI and PB-XPCI images are, respectively, limited to reconstructing accurately slowly and rapidly varying features due to noise and differences in their contrast mechanisms. Herein, we consider a combined speckle- and propagation-based XPCI (SPB-XPCI) image by introducing a mask to generate a reference pattern and imaging in the near-to-holographic regime to induce intensity modulations in the image. We develop a single-shot 2D PR method for SPB-XPCI images of pure phase objects without imposing restrictions such as object support constraints. It is compared against PR methods inspired by those developed for SP-XPCI and PB-XPCI on simulated and experimental images of a thin glass shell before and during shockwave compression. Reconstructed phase maps show improvements in quantitative scores of root-mean-square error and structural similarity index measure using our proposed method. 

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3. Experimentally validated x-ray image simulations of 50 μm x-ray PIV tracer particles

   https://doi.org/10.1088/1361-6501/ac4c0d  

   Parker, Jason T; Mäkiharju, Simo A (February 2022, Measurement Science and Technology)

   Abstract We evaluate Beer–Lambert (BL) ray-tracing and Monte Carlo N-Particle (MCNP) photon tracking simulations for prediction and comparison of x-ray imaging system performance. These simulation tools can aid the methodical design of laboratory-scale x-ray particle image velocimetry (XPIV) experiments and tracer particles by predicting image quality. Particle image signal-to-noise ratio (SNR) is used as the metric of system performance. Simulated and experiment data of hollow, silver-coated, glass sphere tracer particles (AGSF-33) are compared. As predicted by the simulations, the AGSF-33 particles are visible with a SNR greater than unity in 100 ms exposure time images, demonstrating their potential as x-ray PIV or particle tracking velocimetry (XPTV) tracers. The BL approach predicts the image contrast, is computationally inexpensive, and enables the exploration of a vast parameter space for system design. MCNP simulations, on the other hand, predict experiment images slightly more accurately, but are more than an order of magnitude more computationally expensive than BL simulations. For most practical XPIV system design applications, the higher computational expense of MCNP is likely not justified by the modest accuracy improvement compared to BL. 

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4. Deep-SMOLM: deep learning resolves the 3D orientations and 2D positions of overlapping single molecules with optimal nanoscale resolution

   https://doi.org/10.1364/OE.470146  

   Wu, Tingting; Lu, Peng; Rahman, Md_Ashequr; Li, Xiao; Lew, Matthew_D (September 2022, Optics Express)

   Dipole-spread function (DSF) engineering reshapes the images of a microscope to maximize the sensitivity of measuring the 3D orientations of dipole-like emitters. However, severe Poisson shot noise, overlapping images, and simultaneously fitting high-dimensional information–both orientation and position–greatly complicates image analysis in single-molecule orientation-localization microscopy (SMOLM). Here, we report a deep-learning based estimator, termed Deep-SMOLM, that achieves superior 3D orientation and 2D position measurement precision within 3% of the theoretical limit (3.8° orientation, 0.32 sr wobble angle, and 8.5 nm lateral position using 1000 detected photons). Deep-SMOLM also demonstrates state-of-art estimation performance on overlapping images of emitters, e.g., a 0.95 Jaccard index for emitters separated by 139 nm, corresponding to a 43% image overlap. Deep-SMOLM accurately and precisely reconstructs 5D information of both simulated biological fibers and experimental amyloid fibrils from images containing highly overlapped DSFs at a speed ~10 times faster than iterative estimators. 

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5. Real-Time 3D Single Particle Tracking: Towards Active Feedback Single Molecule Spectroscopy in Live Cells

   https://doi.org/10.3390/molecules24152826  

   Hou, Shangguo; Johnson, Courtney; Welsher, Kevin (August 2019, Molecules)

   Single molecule fluorescence spectroscopy has been largely implemented using methods which require tethering of molecules to a substrate in order to make high temporal resolution measurements. However, the act of tethering a molecule requires that the molecule be removed from its environment. This is especially perturbative when measuring biomolecules such as enzymes, which may rely on the non-equilibrium and crowded cellular environment for normal function. A method which may be able to un-tether single molecule fluorescence spectroscopy is real-time 3D single particle tracking (RT-3D-SPT). RT-3D-SPT uses active feedback to effectively lock-on to freely diffusing particles so they can be measured continuously with up to photon-limited temporal resolution over large axial ranges. This review gives an overview of the various active feedback 3D single particle tracking methods, highlighting specialized detection and excitation schemes which enable high-speed real-time tracking. Furthermore, the combination of these active feedback methods with simultaneous live-cell imaging is discussed. Finally, the successes in real-time 3D single molecule tracking (RT-3D-SMT) thus far and the roadmap going forward for this promising family of techniques are discussed. 

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