Assessment of holographic microscopy for quantifying marine particle size and concentration

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 (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.

Plankton classification with high-throughput submersible holographic microscopy and transfer learning

“…Holography has certain technical challenges for capturing high-quality plankton features, owing first to the need for numerical reconstruction of a sample volume, followed by object detection and autofocusing. In assessing the HoloSea, Walcutt et al [ 41 ] observed two notable biases underlying particle size and density estimates, including the attenuated light intensity from the point source, both radially and axially across the sample volume and secondly, that foreground objects inevitably shade the volume background. Although this study is concerned with classification, both biases are present in this study.…”

“…Although this study is concerned with classification, both biases are present in this study. Several modifications offered by Walcutt et al [ 41 ] apply here: Adjusting the point source-to-camera distance to expand sample space illumination and create a more uniform light intensity, scaling object detection probability based on pixel intensity, and local adaptive thresholding to improve ROIs detection consistency at the dimmed hologram edges—as opposed to the fixed, global thresholding algorithm used here. Because objects are less likely to be detected at the hologram edges, only a fraction of the particle field is consistently imaged.…”

“…Because objects are less likely to be detected at the hologram edges, only a fraction of the particle field is consistently imaged. The total volume imaged, calculated as the product of the number of holograms and the volume of each hologram (maximally 0.1 mL), should be corrected by the actual illuminated proportion of the sample volume: For the HoloSea, Walcutt et al [ 41 ] empirically derived the working image volume at 0.063 mL per hologram. The digital corrections are likely simpler and should be implemented in future quantitative assessments, unless the increasing ability of deep learning algorithms in holographic reconstruction, enhancing depth-of-field and autofocusing can outperform instrument-specific corrections [ 17 ].…”

“…General DIHM designs for biological applications are reviewed in Garcia-Sucerquia et al [ 6 ] and Xu et al [ 20 ]. A similar submersible DIHM, the 4-Deep HoloSea S5 1 (92 × 351 mm, 2.6 kg), first introduced by Walcutt et al [ 41 ], was used here to image plankton cells. Its principal advantage is a simple lensless, in-flow configuration with 0.1 mL per frame and high frame rates (> 20 s −1 ) that support a maximum flow rate > 130 mL min −1 .…”

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

“…They suggest that in particle fields dominated by non-spherical particles, the LISST-100X might report a single large particle as several individual particles, thus skewing the PSD. While the above studies use holography as a standard to compare other instrumentation, a study by Walcutt et al (2020) is also pertinent to this discussion. Here, a 4Deep holographic microscope was simultaneously used along with a FlowCam, Imaging Flow Cytobot (IFCB), and standard microscope, to compare PSDs.…”