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1. Deep-3D microscope: 3D volumetric microscopy of thick scattering samples using a wide-field microscope and machine learning

   https://doi.org/10.1364/BOE.444488  

   Li, Bowen; Tan, Shiyu; Dong, Jiuyang; Lian, Xiaocong; Zhang, Yongbing; Ji, Xiangyang; Veeraraghavan, Ashok (December 2021, Biomedical Optics Express)

   Confocal microscopy is a standard approach for obtaining volumetric images of a sample with high axial and lateral resolution, especially when dealing with scattering samples. Unfortunately, a confocal microscope is quite expensive compared to traditional microscopes. In addition, the point scanning in confocal microscopy leads to slow imaging speed and photobleaching due to the high dose of laser energy. In this paper, we demonstrate how the advances in machine learning can be exploited to teach a traditional wide-field microscope, one that’s available in every lab, into producing 3D volumetric images like a confocal microscope. The key idea is to obtain multiple images with different focus settings using a wide-field microscope and use a 3D generative adversarial network (GAN) based neural network to learn the mapping between the blurry low-contrast image stacks obtained using a wide-field microscope and the sharp, high-contrast image stacks obtained using a confocal microscope. After training the network with widefield-confocal stack pairs, the network can reliably and accurately reconstruct 3D volumetric images that rival confocal images in terms of its lateral resolution, z-sectioning and image contrast. Our experimental results demonstrate generalization ability to handle unseen data, stability in the reconstruction results, high spatial resolution even when imaging thick (∼40 microns) highly-scattering samples. We believe that such learning-based microscopes have the potential to bring confocal imaging quality to every lab that has a wide-field microscope. 

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2. Precise and accurate speed measurements in rapidly flowing dense suspensions

   https://doi.org/10.3389/fphy.2024.1480376  

   Moghimi, Esmaeel; Blair, Daniel L; Urbach, Jeffrey S (October 2024, Frontiers in Physics)

   We introduce a method for precise and accurate measurements of particle speeds in dense suspensions flowing at high rates and demonstrate the utility of the approach for revealing complex flow fluctuations during shearing in a setup that combines imaging with a confocal microscope and shearing with a rheometer. We scan the focal point in one dimension, aligned with direction of flow, producing absolute measurements of speed that are independent of suspension structure and particle shape. We compare this flow-direction line scanning approach with a complementary method we introduced previously, measuring speed using line scanning in the vorticity direction. By comparing results in various flow conditions, including shear-thinning and thickening regimes, we demonstrate the efficacy of our new approach. We find that both approaches exhibit qualitatively similar flow profiles, but a comparative analysis reveals a 15%–25% overestimation in speed measurement using vorticity line scanning, with discrepancies generated by anisotropic suspension microstructure under flow. Moreover, in the thickening regime where complex flow fields are present, both approaches capture local speed fluctuations. However, line scanning in the flow direction reveals and precisely captures stagnation and backflows, a capability not achievable with vorticity line scanning. The approach introduced here not only provides a refined technique for speed measurement in fast-flowing suspensions but also emphasizes the significance of accurate measurement techniques in advancing our understanding of flow behavior in dense suspensions, particularly in contexts where strong non-affine flows are prevalent. 

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3. Fixation and staining of gemmule-hatched Ephydatia muelleri for fluorescence microscopy v1

   https://doi.org/10.17504/protocols.io.j8nlkwnx6l5r/v1  

   Nichols, Scott (February 2023, Protocols.io)

   This protocol is intended for the preparation of gemmule-hatched freshwater sponges for imaging with an inverted scanning confocal microscope. 

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4. Hyperspectral confocal microscopy in the short-wave infrared range

   https://doi.org/10.1364/OL.498290  

   Sung, Yongjin; Wang, Weizhong (July 2023, Optics Letters)

   We demonstrate hyperspectral confocal microscopy in the short-wave infrared (SWIR) range of 1100–1600 nm using a wavelength-scanning laser in tandem with laser scanning confocal microscopy. Confocal microscopy in the SWIR range allows for high-resolution inspection of an integrated circuit (IC) chip, while hyperspectral imaging, together with a chemometric analysis, enables us to identify functional circuit block groups in the acquired image. With the extended capability, the developed instrument can be potentially used for inline inspection and non-invasive failure analysis of IC chips. 

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5. 2D Au-Coated Resonant MEMS Scanner for NIR Fluorescence Intraoperative Confocal Microscope

   https://doi.org/10.3390/mi10050295  

   Yao, Cheng-You; Li, Bo; Qiu, Zhen (May 2019, Micromachines)

   The electrostatic MEMS scanner plays an important role in the miniaturization of the microscopic imaging system. We have developed a new two-dimensional (2D) parametrically-resonant MEMS scanner with patterned Au coating (>90% reflectivity at an NIR 785-nm wavelength), for a near-infrared (NIR) fluorescence intraoperative confocal microscopic imaging system with a compact form factor. A silicon-on-insulator (SOI)-wafer based dicing-free microfabrication process has been developed for mass-production with high yield. Based on an in-plane comb-drive configuration, the resonant MEMS scanner performs 2D Lissajous pattern scanning with a large mechanical scanning angle (MSA, ±4°) on each axis at low driving voltage (36 V). A large field-of-view (FOV) has been achieved by using a post-objective scanning architecture of the confocal microscope. We have integrated the new MEMS scanner into a custom-made NIR fluorescence intraoperative confocal microscope with an outer diameter of 5.5 mm at its distal-end. Axial scanning has been achieved by using a piezoelectric actuator-based driving mechanism. We have successfully demonstrated ex vivo 2D imaging on human tissue specimens with up to five frames/s. The 2D resonant MEMS scanner can potentially be utilized for many applications, including multiphoton microendoscopy and wide-field endoscopy. 

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