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, and Prorocentrum micans) and microspheres. DIHM concentrations (order 1 to 1000 particles ml−1) showed a linear agreement (r 2 = 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.
Highlights
Quantitive particle measurements, such as size spectral shape and particle concentration, provides insights into ecological community composition, abundance, and diversity (Cavender-Bares et al 2001)
Our methodology for performing this assessment focussed on laboratory and field-based inter-calibration with commercially available particle sizers as well as with traditional microscope counting methods
Concentration measurements tend to be 5% higher overall than the ensemble average size of those measured by all instruments combined
Summary
Quantitive particle measurements, such as size spectral shape and particle concentration, provides insights into ecological community composition, abundance, and diversity (Cavender-Bares et al 2001). Depth-resolved collection by nets and bottles that concentrate small particles from seawater have been conducted extensively since the early 20th century (Gutkowska et al 2012), spatio-temporal sampling coverage has been limited by time- and effort-intensive collection and analysis protocols which often employed manual microscope analysis. This method can often damage the more delicate forms. Aldridge (1972) circumvented this challenge through manual, in situ measurements of marine particles using underwater photography by divers, implementing on broad scales is prohibitive. The in situ Video Plankton Recorder (Davis et al 2005) and the Underwater Vision Profiler (Picheral et al 2010) sample larger particles, more than 1mm and more
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