Achieving diffraction-limited resolution in soft-X-ray Fourier-transform holography

The spatial resolution of microscopic images acquired via X-ray Fourier-transform holography is limited by the source size of the reference wave and by the numerical aperture of the detector. We analyze the interplay between both influences and show how they are matched in practice. We further identify, how high spatial frequencies translate to imaging artifacts in holographic reconstructions where mainly the reference beam limits the spatial resolution. As a solution, three methods are introduced based on numerical post-processing of the reconstruction. The methods comprise apodization of the hologram, refocusing via wave propagation, and deconvolution using the transfer function of the imaging system. In particular for the latter two, we demonstrate that image details smaller than the source size of the reference beam can be recovered up to the diffraction limit of the hologram. Our findings motivate the intentional application of a large reference-wave source enhancing the image contrast in applications with low photon numbers such as single-shot experiments at free-electron lasers or imaging at laboratory sources.BMBF, 05K10KTB, Verbundprojekt: FSP 301 - FLASH: Nanoskopische Systeme. Teilprojekt 1.1: Universelle Experimentierkammer für Streuexperimente mit kohärenten Femtosekunden-Röntgenpulsen Multi Purpose Coherent Scattering Chamber for FLASH and XFEL 'MPscatt

Fundamentals of 3D imaging and displays: a tutorial on integral imaging, light-field, and plenoptic systems

There has been great interest in researching and implementing effective technologies for the capture, processing, and display of 3D images. This broad interest is evidenced by widespread international research and activities on 3D technologies. There is a large number of journal and conference papers on 3D systems, as well as research and development efforts in government, industry, and academia on this topic for broad applications including entertainment, manufacturing, security and defense, and biomedical applications. Among these technologies, integral imaging is a promising approach for its ability to work with polychromatic scenes and under incoherent or ambient light for scenarios from macroscales to microscales. Integral imaging systems and their variations, also known as plenoptics or light-field systems, are applicable in many fields, and they have been reported in many applications, such as entertainment (TV, video, movies), industrial inspection, security and defense, and biomedical imaging and displays. This tutorial is addressed to the students and researchers in different disciplines who are interested to learn about integral imaging and light-field systems and who may or may not have a strong background in optics. Our aim is to provide the readers with a tutorial that teaches fundamental principles as well as more advanced concepts to understand, analyze, and implement integral imaging and light-field-type capture and display systems. The tutorial is organized to begin with reviewing the fundamentals of imaging, and then it progresses to more advanced topics in 3D imaging and displays. More specifically, this tutorial begins by covering the fundamentals of geometrical optics and wave optics tools for understanding and analyzing optical imaging systems. Then, we proceed to use these tools to describe integral imaging, light-field, or plenoptics systems, the methods for implementing the 3D capture procedures and monitors, their properties, resolution, field of view, performance, and metrics to assess them. We have illustrated with simple laboratory setups and experiments the principles of integral imaging capture and display systems. Also, we have discussed 3D biomedical applications, such as integral microscopy

Remote refocusing light-sheet fluorescence microscopy for high-speed 2D and 3D imaging of calcium dynamics in cardiomyocytes

The high prevalence and poor prognosis of heart failure are two key drivers for research into cardiac electrophysiology and regeneration. Dyssynchrony in calcium release and loss of structural organization within individual cardiomyocytes (CM) has been linked to reduced contractile strength and arrhythmia. Correlating calcium dynamics and cell microstructure requires multidimensional imaging with high spatiotemporal resolution. In light-sheet fluorescence microscopy (LSFM), selective plane illumination enables fast optically sectioned imaging with lower phototoxicity, making it suitable for imaging subcellular dynamics. In this work, a custom remote refocusing LSFM system is applied to studying calcium dynamics in isolated CM, cardiac cell cultures and tissue slices. The spatial resolution of the LSFM system was modelled and experimentally characterized. Simulation of the illumination path in Zemax was used to estimate the light-sheet beam waist and confocal parameter. Automated MATLAB-based image analysis was used to quantify the optical sectioning and the 3D point spread function using Gaussian fitting of bead image intensity distributions. The results demonstrated improved and more uniform axial resolution and optical sectioning with the tighter focused beam used for axially swept light-sheet microscopy. High-speed dual-channel LSFM was used for 2D imaging of calcium dynamics in correlation with the t-tubule structure in left and right ventricle cardiomyocytes at 395 fps. The high spatio-temporal resolution enabled the characterization of calcium sparks. The use of para-nitro-blebbistatin (NBleb), a non-phototoxic, low fluorescence contraction uncoupler, allowed 2D-mapping of the spatial dyssynchrony of calcium transient development across the cell. Finally, aberration-free remote refocusing was used for high-speed volumetric imaging of calcium dynamics in human induced pluripotent stem-cell derived cardiomyocytes (hiPSC-CM) and their co-culture with adult-CM. 3D-imaging at up to 8 Hz demonstrated the synchronization of calcium transients in co-culture, with increased coupling with longer co-culture duration, uninhibited by motion uncoupling with NBleb.Open Acces