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Modified FlowCAM procedure for quantifying size distribution of zooplankton with sample recycling capacity

View Article: PubMed Central - PubMed

ABSTRACT

We have developed a modified FlowCAM procedure for efficiently quantifying the size distribution of zooplankton. The modified method offers the following new features: 1) prevents animals from settling and clogging with constant bubbling in the sample container; 2) prevents damage to sample animals and facilitates recycling by replacing the built-in peristaltic pump with an external syringe pump, in order to generate negative pressure, creates a steady flow by drawing air from the receiving conical flask (i.e. vacuum pump), and transfers plankton from the sample container toward the main flowcell of the imaging system and finally into the receiving flask; 3) aligns samples in advance of imaging and prevents clogging with an additional flowcell placed ahead of the main flowcell. These modifications were designed to overcome the difficulties applying the standard FlowCAM procedure to studies where the number of individuals per sample is small, and since the FlowCAM can only image a subset of a sample. Our effective recycling procedure allows users to pass the same sample through the FlowCAM many times (i.e. bootstrapping the sample) in order to generate a good size distribution. Although more advanced FlowCAM models are equipped with syringe pump and Field of View (FOV) flowcells which can image all particles passing through the flow field; we note that these advanced setups are very expensive, offer limited syringe and flowcell sizes, and do not guarantee recycling. In contrast, our modifications are inexpensive and flexible. Finally, we compared the biovolumes estimated by automated FlowCAM image analysis versus conventional manual measurements, and found that the size of an individual zooplankter can be estimated by the FlowCAM image system after ground truthing.

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Scatter plots illustrating the relationships between the biovolumes estimated with Area-Based-Diameter (VABD) from the FlowCAM versus the manual image measurements using ruler tools for the 7 dominant copepod morphotypes.A hundred individuals from each morphotype assemblage are randomly chosen. The biovolume of each individual was estimated by both VABD using the FlowCAM image analysis and “microscopic measurement” for each morphotype: (a) calanoid copepodite, (b) oithonid copepodite, (c) corycaeid copepodite, (d) oncaeid copepodite, (e) calanoid nauplius, (f) cyclopoid nauplius, and (g) harpacticoid nauplius. Linear regression analysis reveals significant correlations (p < 0.0001) between the biovolumes estimated with Area-Based-Diameter (VABD) versus microscopic measurement for all morphotypes.
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pone.0175235.g005: Scatter plots illustrating the relationships between the biovolumes estimated with Area-Based-Diameter (VABD) from the FlowCAM versus the manual image measurements using ruler tools for the 7 dominant copepod morphotypes.A hundred individuals from each morphotype assemblage are randomly chosen. The biovolume of each individual was estimated by both VABD using the FlowCAM image analysis and “microscopic measurement” for each morphotype: (a) calanoid copepodite, (b) oithonid copepodite, (c) corycaeid copepodite, (d) oncaeid copepodite, (e) calanoid nauplius, (f) cyclopoid nauplius, and (g) harpacticoid nauplius. Linear regression analysis reveals significant correlations (p < 0.0001) between the biovolumes estimated with Area-Based-Diameter (VABD) versus microscopic measurement for all morphotypes.

Mentions: To test the efficacy of VABD, the biovolumes of one hundred individuals of each morphotype were estimated by both “microscopic measurement” and VABD using the FlowCAM image analysis. The hundred individuals were randomly picked from pooled images of copepods sampled from the three stations. Data are provided in S1 Table. We used simple regression analysis to examine the relationship between the two measurements. Our comparison of VABD with the microscopic measurements indicates that VABD is a reasonable proxy for biovolume (Fig 5), although its efficacy is better for some taxa than others. We also provide conversion coefficients for each taxon (Table 1). This ground truthing procedure for converting VABD from FlowCAM image analysis to microscopic measurements is recommended for users targeting specific groups of organisms in specific regions. Thus, the biovolumes of zooplankton can be reasonably estimated using the regression relationships obtained from comparisons between VABD and microscopic measurements. Nevertheless, further image analysis developments may be required for improving the automatic estimation of plankton biovolume.


Modified FlowCAM procedure for quantifying size distribution of zooplankton with sample recycling capacity
Scatter plots illustrating the relationships between the biovolumes estimated with Area-Based-Diameter (VABD) from the FlowCAM versus the manual image measurements using ruler tools for the 7 dominant copepod morphotypes.A hundred individuals from each morphotype assemblage are randomly chosen. The biovolume of each individual was estimated by both VABD using the FlowCAM image analysis and “microscopic measurement” for each morphotype: (a) calanoid copepodite, (b) oithonid copepodite, (c) corycaeid copepodite, (d) oncaeid copepodite, (e) calanoid nauplius, (f) cyclopoid nauplius, and (g) harpacticoid nauplius. Linear regression analysis reveals significant correlations (p < 0.0001) between the biovolumes estimated with Area-Based-Diameter (VABD) versus microscopic measurement for all morphotypes.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5383268&req=5

pone.0175235.g005: Scatter plots illustrating the relationships between the biovolumes estimated with Area-Based-Diameter (VABD) from the FlowCAM versus the manual image measurements using ruler tools for the 7 dominant copepod morphotypes.A hundred individuals from each morphotype assemblage are randomly chosen. The biovolume of each individual was estimated by both VABD using the FlowCAM image analysis and “microscopic measurement” for each morphotype: (a) calanoid copepodite, (b) oithonid copepodite, (c) corycaeid copepodite, (d) oncaeid copepodite, (e) calanoid nauplius, (f) cyclopoid nauplius, and (g) harpacticoid nauplius. Linear regression analysis reveals significant correlations (p < 0.0001) between the biovolumes estimated with Area-Based-Diameter (VABD) versus microscopic measurement for all morphotypes.
Mentions: To test the efficacy of VABD, the biovolumes of one hundred individuals of each morphotype were estimated by both “microscopic measurement” and VABD using the FlowCAM image analysis. The hundred individuals were randomly picked from pooled images of copepods sampled from the three stations. Data are provided in S1 Table. We used simple regression analysis to examine the relationship between the two measurements. Our comparison of VABD with the microscopic measurements indicates that VABD is a reasonable proxy for biovolume (Fig 5), although its efficacy is better for some taxa than others. We also provide conversion coefficients for each taxon (Table 1). This ground truthing procedure for converting VABD from FlowCAM image analysis to microscopic measurements is recommended for users targeting specific groups of organisms in specific regions. Thus, the biovolumes of zooplankton can be reasonably estimated using the regression relationships obtained from comparisons between VABD and microscopic measurements. Nevertheless, further image analysis developments may be required for improving the automatic estimation of plankton biovolume.

View Article: PubMed Central - PubMed

ABSTRACT

We have developed a modified FlowCAM procedure for efficiently quantifying the size distribution of zooplankton. The modified method offers the following new features: 1) prevents animals from settling and clogging with constant bubbling in the sample container; 2) prevents damage to sample animals and facilitates recycling by replacing the built-in peristaltic pump with an external syringe pump, in order to generate negative pressure, creates a steady flow by drawing air from the receiving conical flask (i.e. vacuum pump), and transfers plankton from the sample container toward the main flowcell of the imaging system and finally into the receiving flask; 3) aligns samples in advance of imaging and prevents clogging with an additional flowcell placed ahead of the main flowcell. These modifications were designed to overcome the difficulties applying the standard FlowCAM procedure to studies where the number of individuals per sample is small, and since the FlowCAM can only image a subset of a sample. Our effective recycling procedure allows users to pass the same sample through the FlowCAM many times (i.e. bootstrapping the sample) in order to generate a good size distribution. Although more advanced FlowCAM models are equipped with syringe pump and Field of View (FOV) flowcells which can image all particles passing through the flow field; we note that these advanced setups are very expensive, offer limited syringe and flowcell sizes, and do not guarantee recycling. In contrast, our modifications are inexpensive and flexible. Finally, we compared the biovolumes estimated by automated FlowCAM image analysis versus conventional manual measurements, and found that the size of an individual zooplankter can be estimated by the FlowCAM image system after ground truthing.

No MeSH data available.


Related in: MedlinePlus