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, and Prorocentrum 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.

中文翻译：

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全息显微镜评估以量化海洋颗粒大小和浓度

全息显微镜已成为海洋环境中微生物和其他微粒原位成像的一种工具：之所以吸引人，是因为与其他成像系统相比，采样量相对较大且光学配置更简单。然而，到目前为止，其定量能力仍不确定，部分原因是全息图重建和图像识别需要手动操作。在这里，我们评估了自动化全息图处理管道（CCV管道）的定量技术，以评估海洋浮游生物颗粒和微球的环境和培养组合的大小和浓度测量。超过一百万个粒子，范围从10到 200μ分析了4深HoloSea数字在线全息显微镜（DIHM）成像的等效球面直径m。这些测量是与FlowCam（FC），Imaging FlowCytobot（IFCB）和手动显微镜识别并行收集的。一旦开发出并应用了对颗粒位置和不均匀照明的校正方法，DIHM的ESD就会被低估约3％至10％，但可以成功地从环境样品中复制出尺寸光谱的斜率以及培养物的尺寸分布（杜氏藻，杜氏藻，杂种akashiwo和Prorocentrum micans）和微球。DIHM浓度（1到1000个颗粒ml ^-1阶^数）显示出线性一致性（r ²= 0.73），但有时个别比较的不确定性很大。总体而言，我们发现DIHM和CCV管道需要进行大量的手动校正，但是一旦校正，其浓度和尺寸估算值可与本研究中评估的其他成像系统相媲美。全息相机在机械上简单，自主，可以在非常高的压力下运行，并且比基于镜头的工具提供更大的采样量。因此，我们预计这些表征工作将在新的海洋环境中得到新发现而得到回报。