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1. Assessment of holographic microscopy for quantifying marine particle size and concentration

   https://doi.org/10.1002/lom3.10379  

   Walcutt, Noah L.; Knörlein, Benjamin; Cetinić, Ivona; Ljubesic, Zrinka; Bosak, Suncica; Sgouros, Tom; Montalbano, Amanda L.; Neeley, Aimee; Menden‐Deuer, Susanne; Omand, Melissa M. (August 2020, Limnology and Oceanography: Methods)

   Abstract Holographic microscopy has emerged as a tool for in situ imaging of microscopic organisms and other particles in the marine environment: appealing because of the relatively larger sampling volume and simpler optical configuration compared to other imaging systems. However, its quantitative capabilities have so far remained uncertain, in part because hologram reconstruction and image recognition have required manual operation. Here, we assess the quantitative skill of our automated hologram processing pipeline (CCV Pipeline), to evaluate the size and concentration measurements of environmental and cultured assemblages of marine plankton particles, and microspheres. Over 1 million particles, ranging from 10 to 200 μm in equivalent spherical diameter, imaged by the 4‐Deep HoloSea digital inline holographic microscope (DIHM) are analyzed. These measurements were collected in parallel with a FlowCam (FC), Imaging FlowCytobot (IFCB), and manual microscope identification. Once corrections for particle location and nonuniform illumination were developed and applied, the DIHM showed an underestimate in ESD of about 3% to 10%, but successfully reproduced the size spectral slope from environmental samples, and the size distribution of cultures (Dunaliella tertiolecta,Heterosigma akashiwo, andProrocentrum micans) and microspheres. DIHM concentrations (order 1 to 1000 particles ml⁻¹) showed a linear agreement (r²= 0.73) with the other instruments, but individual comparisons at times had large uncertainty. Overall, we found the DIHM and the CCV Pipeline required extensive manual correction, but once corrected, provided concentration and size estimates comparable to the other imaging systems assessed in this study. Holographic cameras are mechanically simple, autonomous, can operate at very high pressures, and provide a larger sampling volume than comparable lens‐based tools. Thus, we anticipate that these characterization efforts will be rewarded with novel discovery in new oceanic environments. 

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2. Laser Scanning Microscope Based Digital Holography for biomedical application

   https://doi.org/10.1364/DH.2023.HTu5C.4  

   Zakharov, Iurii; Muravyeva, Maria; Perelman, Lev T. (January 2023, Optica Imaging Congress (3D, COSI, DH, FLatOptics, IS, pcAOP) Technical Digest Series)

   Digital holographic system is built on the base of Zeiss LSM-510 microscope. Using created algorithm and software for correct hologram reconstruction and quantitative phase imaging, reflection and phase map of cell cultures and tissues are obtained with other regular microscopic images. 

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3. Microscopic Object Classification through Passive Motion Observations with Holographic Microscopy

   https://doi.org/10.3390/life11080793  

   Rouzie, Devan; Lindensmith, Christian; Nadeau, Jay (August 2021, Life)

   null (Ed.)

   Digital holographic microscopy provides the ability to observe throughout a volume that is large compared to its resolution without the need to actively refocus to capture the entire volume. This enables simultaneous observations of large numbers of small objects within such a volume. We have constructed a microscope that can observe a volume of 0.4 µm × 0.4 µm × 1.0 µm with submicrometer resolution (in xy) and 2 µm resolution (in z) for observation of microorganisms and minerals in liquid environments on Earth and on potential planetary missions. Because environmental samples are likely to contain mixtures of inorganics and microorganisms of comparable sizes near the resolution limit of the instrument, discrimination between living and non-living objects may be difficult. The active motion of motile organisms can be used to readily distinguish them from non-motile objects (live or inorganic), but additional methods are required to distinguish non-motile organisms and inorganic objects that are of comparable size but different composition and structure. We demonstrate the use of passive motion to make this discrimination by evaluating diffusion and buoyancy characteristics of cells, styrene beads, alumina particles, and gas-filled vesicles of micron scale in the field of view. 

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4. A Review of Holography in the Aquatic Sciences: In situ Characterization of Particles, Plankton, and Small Scale Biophysical Interactions

   https://doi.org/10.3389/fmars.2020.572147  

   Nayak, Aditya R.; Malkiel, Ed; McFarland, Malcolm N.; Twardowski, Michael S.; Sullivan, James M. (January 2021, Frontiers in Marine Science)

   null (Ed.)

   The characterization of particle and plankton populations, as well as microscale biophysical interactions, is critical to several important research areas in oceanography and limnology. A growing number of aquatic researchers are turning to holography as a tool of choice to quantify particle fields in diverse environments, including but not limited to, studies on particle orientation, thin layers, phytoplankton blooms, and zooplankton distributions and behavior. Holography provides a non-intrusive, free-stream approach to imaging and characterizing aquatic particles, organisms, and behavior in situ at high resolution through a 3-D sampling volume. Compared to other imaging techniques, e.g., flow cytometry, much larger volumes of water can be processed over the same duration, resolving particle sizes ranging from a few microns to a few centimeters. Modern holographic imaging systems are compact enough to be deployed through various modes, including profiling/towed platforms, buoys, gliders, long-term observatories, or benthic landers. Limitations of the technique include the data-intensive hologram acquisition process, computationally expensive image reconstruction, and coherent noise associated with the holograms that can make post-processing challenging. However, continued processing refinements, rapid advancements in computing power, and development of powerful machine learning algorithms for particle/organism classification are paving the way for holography to be used ubiquitously across different disciplines in the aquatic sciences. This review aims to provide a comprehensive overview of holography in the context of aquatic studies, including historical developments, prior research applications, as well as advantages and limitations of the technique. Ongoing technological developments that can facilitate larger employment of this technique toward in situ measurements in the future, as well as potential applications in emerging research areas in the aquatic sciences are also discussed. 

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5. What You Net Depends on if You Grab: A Meta-analysis of Sampling Method’s Impact on Measured Aquatic Microplastic Concentration

   https://doi.org/10.1021/acs.est.1c03019  

   Watkins, Lisa; Sullivan, Patrick J.; Walter, M. Todd (January 2022, Environmental Science & Technology)

   Microplastic pollution is measured with a variety of sampling methods. Field experiments indicate that commonly used sampling methods, including net, pump, and grab samples, do not always result in equivalent measured concentration. We investigate the comparability of these methods through a meta-analysis of 121 surface water microplastic studies. We find systematic relationships between measured concentration and sampled volume, method of collection, mesh size used for filtration, and waterbody sampled. Most significantly, a strong log−linear relationship exists between sample volume and measured concentration, with small-volume grab samples measuring up to 104 particles/L higher concentrations than larger volume net samples, even when sampled concurrently. Potential biasing factors explored included filtration size (±102 particles/L), net volume overestimation (±101 particles/L), fiber loss through net mesh (unknown magnitude), intersample variability (±101 particles/L), and contamination, the potential factor with an effect large enough (±103 particles/L) to explain the observed differences. On the basis of these results, we caution against comparing concentrations across multiple studies or combining multiple study results to identify regional patterns. Additionally, we emphasize the importance of contamination reduction and quantification strategies, namely that blank samples from all stages of field sampling be collected and reported as a matter of course for all studies. 

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