Abstract
High-content biological microscopy targets high-resolution imaging across large fields-of-view, often achieved by computational imaging approaches. Previously, we demonstrated 2D multimodal high-content microscopy via structured illumination microscopy (SIM) with resolution the diffraction limit, using speckle illumination from Scotch tape. In this work, we extend the method to 3D by leveraging the fact that the speckle illumination is in fact a 3D structured pattern. We use both a coherent and an incoherent imaging model to develop algorithms for joint retrieval of the 3D super-resolved fluorescent and complex-field distributions of the sample. Our reconstructed images resolve features beyond the physical diffraction-limit set by the system's objective and demonstrate 3D multimodal imaging with m3 resolution over a volume of m3.
Highlights
High-content optical microscopy is a driving force for large-scale biological study in fields such as drug discovery and systems biology
We propose a method for 3D SR quantitative phase and fluorescence microscopy using speckle illumination
The coherent and fluorescent light have different wavelengths and are optically separated by a dichroic mirror (Thorlabs, DMLP550R), after which the fluorescence is further spectrally filtered before being imaged onto Sensor-F (PCO.edge 5.5)
Summary
High-content optical microscopy is a driving force for large-scale biological study in fields such as drug discovery and systems biology. In contrast with slide scanning, these strategies often employ a low-NA imaging objective to acquire low-resolution (large-FOV) measurements, use computational techniques like synthetic aperture [10,11,12] and super-resolution (SR) [13,14,15,16,17,18] to digitally reconstruct a high-resolution image This eliminates the requirement for large-distance mechanical scanning in high-content imaging, which results in faster acquisition and more cost-effective optical setups, while relaxing the sample’s auto-refocusing requirements due to the low-NA objective’s longer depth-of-field (DOF) [19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36]. 3D high-content imaging capability has only been demonstrated in the coherent imaging context (quantitative phase and absorption) by Fourier ptychography [25, 27]
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