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Register a new userThe point spread function in interferometric scattering microscopy (iSCAT). I. Aberrations in defocusing and axial localization
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Added by
Reza Gholami Mahmoodabadi
Publication date
2020
fields
Physics
and research's language is
English
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Interferometric scattering (iSCAT) microscopy is an emerging label-free technique optimized for the sensitive detection of nano-matter. Previous iSCAT studies have approximated the point spread function in iSCAT by a Gaussian intensity distribution. However, recent efforts to track the mobility of nanoparticles in challenging speckle environments and over extended axial ranges has necessitated a quantitative description of the interferometric point spread function (iPSF). We present a robust vectorial diffraction model for the iPSF in tandem with experimental measurements and rigorous FDTD simulations. We examine the iPSF under various imaging scenarios to understand how aberrations due to the experimental configuration encode information about the nanoparticle. We show that the lateral shape of the iPSF can be used to achieve nanometric three-dimensional localization over an extended axial range on the order of 10$,mu$m either by means of a fit to an analytical model or calibration-free unsupervised machine learning. Our results have immediate implications for three-dimensional single particle tracking in complex scattering media.
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In coherent X-ray diffraction microscopy the diffraction pattern generated by a sample illuminated with coherent x-rays is recorded, and a computer algorithm recovers the unmeasured phases to synthesize an image. By avoiding the use of a lens the resolution is limited, in principle, only by the largest scattering angles recorded. However, the imaging task is shifted from the experiment to the computer, and the algorithms ability to recover meaningful images in the presence of noise and limited prior knowledge may produce aberrations in the reconstructed image. We analyze the low order aberrations produced by our phase retrieval algorithms. We present two methods to improve the accuracy and stability of reconstructions.
Interferometric scattering microscopy has been a very promising technology for highly sensitive label-free imaging of a broad spectrum of biological nanoparticles from proteins to viruses in a high-throughput manner. Although it can reveal the specimens size and shape information, the chemical composition is inaccessible in interferometric measurements. Infrared spectroscopic imaging provides chemical specificity based on inherent chemical bond vibrations of specimens but lacks the ability to image and resolve individual nanoparticles due to long infrared wavelengths. Here, we describe a bond-selective interferometric scattering microscope where the mid-infrared induced photothermal signal is detected by a visible beam in a wide-field common-path interferometry configuration. A thin film layered substrate is utilized to reduce the reflected light and provide a reference field for the interferometric detection of the weakly scattered field. A pulsed mid-IR laser is employed to modulate the interferometric signal. Subsequent demodulation via a virtual lock-in camera offers simultaneous chemical information about tens of micro- or nano-particles. The chemical contrast arises from a minute change in the particles scattered field in consequence of the vibrational absorption at the target molecule. We characterize the system with sub-wavelength polymer beads and highlight biological applications by chemically imaging several microorganisms including Staphylococcus aureus, Escherichia coli, and Candida albicans. A theoretical framework is established to extend bond-selective interferometric scattering microscopy to a broad range of biological micro- and nano-particles.
Localization microscopy is an imaging technique in which the positions of individual nanoscale point emitters (e.g. fluorescent molecules) are determined at high precision from their images. This is the key ingredient in single/multiple-particle-tracking and several super-resolution microscopy approaches. Localization in three-dimensions (3D) can be performed by modifying the image that a point-source creates on the camera, namely, the point-spread function (PSF). The PSF is engineered using additional optical elements to vary distinctively with the depth of the point-source. However, localizing multiple adjacent emitters in 3D poses a significant algorithmic challenge, due to the lateral overlap of their PSFs. Here, we train a neural network to receive an image containing densely overlapping PSFs of multiple emitters over a large axial range and output a list of their 3D positions. Furthermore, we then use the network to design the optimal PSF for the multi-emitter case. We demonstrate our approach numerically as well as experimentally by 3D STORM imaging of mitochondria, and volumetric imaging of dozens of fluorescently-labeled telomeres occupying a mammalian nucleus in a single snapshot.
One of the problems often encountered in X-ray mirror manufacturing is setting proper manufacturing tolerances to guarantee an angular resolution - often expressed in terms of Point Spread Function (PSF) - as needed by the specific science goal. To do this, we need an accurate metrological apparatus, covering a very broad range of spatial frequencies, and an affordable method to compute the PSF from the metrology dataset. [...] However, the separation between these spectral ranges is difficult do define exactly, and it is also unclear how to affordably combine the PSFs, computed with different methods in different spectral ranges, into a PSF expectation at a given X-ray energy. For this reason, we have proposed a method entirely based on the Huygens-Fresnel principle to compute the diffracted field of real Wolter-I optics, including measured defects over a wide range of spatial frequencies. Owing to the shallow angles at play, the computation can be simplified limiting the computation to the longitudinal profiles, neglecting completely the effect of roundness errors. Other authors had already proposed similar approaches in the past, but only in far-field approximation, therefore they could not be applied to the case of Wolter-I optics, in which two reflections occur in sequence within a short range. The method we suggest is versatile, as it can be applied to multiple reflection systems, at any X-ray energy, and regardless of the nominal shape of the mirrors in the optical system. The method has been implemented in the WISE code, successfully used to explain the measured PSFs of multilayer-coated optics for astronomic use, and of a K-B optical system in use at the FERMI free electron laser.
Here, we report analysis and summary of research in the field of localization microscopy for optical imaging. We introduce the basic elements of super-resolved localization microscopy methods for PALM and STORM, commonly used both in vivo and in vitro, discussing the core essentials of background theory, instrumentation and computational algorithms. We discuss the resolution limit of light microscopy and the mathematical framework for localizing fluorescent dyes in space beyond this limit, including the precision obtainable as a function of the amount of light emitted from a dye, and how it leads to a fundamental compromise between spatial and temporal precision. The properties of a good dye are outlined, as are the features of PALM and STORM super-resolution microscopy and adaptations that may need to be made to experimental protocols to perform localization determination. We analyse briefly some of the methods of modern super-resolved optical imaging that work through reshaping point spread functions and how they utilize aspects of localization microscopy, such as stimulated depletion (STED) methods and MINFLUX, and summarize modern methods that push localization into 3D using non-Gaussian point spread functions. We report on current methods for analyzing localization data including determination of 2D and 3D diffusion constants, molecular stoichiometries, and performing cluster analysis with cutting-edge techniques, and finally discuss how these techniques may be used to enable important insight into a range of biological processes.
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