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1. Multimodal illumination platform for 3D single-molecule super-resolution imaging throughout mammalian cells

   https://doi.org/10.1364/BOE.521362  

   Nelson, Tyler; Vargas-Hernández, Sofía; Freire, Margareth; Cheng, Siyang; Gustavsson, Anna-Karin (April 2024, Biomedical Optics Express)

   Single-molecule super-resolution imaging is instrumental in investigating cellular architecture and organization at the nanoscale. Achieving precise 3D nanometric localization when imaging structures throughout mammalian cells, which can be multiple microns thick, requires careful selection of the illumination scheme in order to optimize the fluorescence signal to background ratio (SBR). Thus, an optical platform that combines different wide-field illumination schemes for target-specific SBR optimization would facilitate more precise 3D nanoscale studies of a wide range of cellular structures. Here, we demonstrate a versatile multimodal illumination platform that integrates the sectioning and background reduction capabilities of light sheet illumination with homogeneous, flat-field epi- and TIRF illumination. Using primarily commercially available parts, we combine the fast and convenient switching between illumination modalities with point spread function engineering to enable 3D single-molecule super-resolution imaging throughout mammalian cells. For targets directly at the coverslip, the homogenous intensity profile and excellent sectioning of our flat-field TIRF illumination scheme improves single-molecule data quality by providing low fluorescence background and uniform fluorophore blinking kinetics, fluorescence signal, and localization precision across the entire field of view. The increased contrast achieved with LS illumination, when compared with epi-illumination, makes this illumination modality an excellent alternative when imaging targets that extend throughout the cell. We validate our microscopy platform for improved 3D super-resolution imaging by two-color imaging of paxillin – a protein located in the focal adhesion complex – and actin in human osteosarcoma cells. 

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2. Annular illumination in 2D quantitative phase imaging: a systematic evaluation

   https://doi.org/10.1364/AO.452325  

   Kulkarni, Pranav_P; Bao, Yijun; Gaylord, Thomas_K (April 2022, Applied Optics)

   Quantitative phase imaging (QPI) is an invaluable microscopic technology for definitively imaging phase objects such as biological cells and optical fibers. Traditionally, the condenser lens in QPI produces disk illumination of the object. However, it has been realized by numerous investigators that annular illumination can produce higher-resolution images. Although this performance improvement is impressive and well documented, the evidence presented has invariably been qualitative in nature. Recently, a theoretical basis for annular illumination was presented by Baoet al.[Appl. Opt.58,137(2019)APOPAI0003-693510.1364/AO.58.000137]. In our current work, systematic experimental QPI measurements are made with a reference phase mask to rigorously document the performance of annular illumination. In both theory and experiment, three spatial-frequency regions are identified: low, mid, and high. The low spatial-frequency region response is very similar for disk and annular illumination, both theoretically and experimentally. Theoretically, the high spatial-frequency region response is predicted to be much better for the annular illumination compared to the disk illumination––and is experimentally confirmed. In addition, the mid-spatial-frequency region response is theoretically predicted to be less for annular illumination than for disk illumination. This theoretical degradation of the mid-spatial-frequency region is only slightly experimentally observed. This bonus, although not well understood, further elevates the performance of annular illumination over disk illumination. 

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3. Subcellular three-dimensional imaging deep through multicellular thick samples by structured illumination microscopy and adaptive optics

   https://doi.org/10.1038/s41467-021-23449-6  

   Lin, Ruizhe; Kipreos, Edward T.; Zhu, Jie; Khang, Chang Hyun; Kner, Peter (May 2021, Nature Communications)

   Abstract Structured Illumination Microscopy enables live imaging with sub-diffraction resolution. Unfortunately, optical aberrations can lead to loss of resolution and artifacts in Structured Illumination Microscopy rendering the technique unusable in samples thicker than a single cell. Here we report on the combination of Adaptive Optics and Structured Illumination Microscopy enabling imaging with 150 nm lateral and 570 nm axial resolution at a depth of 80 µm throughCaenorhabditis elegans. We demonstrate that Adaptive Optics improves the three-dimensional resolution, especially along the axial direction, and reduces artifacts, successfully realizing 3D-Structured Illumination Microscopy in a variety of biological samples. 

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4. Circumventing the optical diffraction limit with customized speckles

   https://doi.org/10.1364/OPTICA.411007  

   Bender, Nicholas; Sun, Mengyuan; Yılmaz, Hasan; Bewersdorf, Joerg; Cao, Hui (January 2021, Optica)

   Speckle patterns have been used widely in imaging techniques such as ghost imaging, dynamic speckle illumination microscopy, structured illumination microscopy, and photoacoustic fluctuation imaging. Recent advances in the ability to control the statistical properties of speckles has enabled the customization of speckle patterns for specific imaging applications. In this work, we design and create special speckle patterns for parallelized nonlinear pattern-illumination microscopy based on fluorescence photoswitching. We present a proof-of-principle experimental demonstration where we obtain a spatial resolution three times higher than the diffraction limit of the illumination optics in our setup. Furthermore, we show that tailored speckles vastly outperform standard speckles. Our work establishes that customized speckles are a potent tool in parallelized super-resolution microscopy. 

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5. Human-Centered Drone LED Illumination System: A Proof-of-Concept Prototype

   https://doi.org/10.1177/1071181321651124  

   Hao, Shuai; Yerebakan, Mustafa Ozkan; Hu, Boyi (September 2021, Proceedings of the Human Factors and Ergonomics Society Annual Meeting)

   Recent years have seen a rapid increase in drone usage in both commercial and personal use, due to recent changes in guidelines by the Federal Aviation Administration (FAA). In those guidelines, however, there seems to be very few requirements in terms of illumination requirements, apart from the need to use a visible strob ing anti-collision light for nighttime operations. Hence in this study, wereviewed existing LED illumination systems in off-the-shelf drones to determine what type of configurations they have and how is the LED illumination system generally used. We also introduced a customizable LED illumination system and tested it in a human in the loop study. Our p reliminary findings have revealed that the colors that are preferred by the participants did not match the most used colors in existing LED illumination systems in most off-the-shelf drones. We also observed a possible relationship between the color preferred and the weather conditions. 

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