Park, Jungsu
(Water Quality Research Center, Korea Water Resources Corporation)
,
Kim, Yongje
(Department of Civil, Environmental and Construction Engineering, University of Central Florida)
,
Kim, Minjae
(School of Life Science, Kyungbook National University)
,
Lee, Woo Hyoung
(Department of Civil, Environmental and Construction Engineering, University of Central Florida)
Microcystis sp. is one of the most common harmful cyanobacteria that release toxic substances. Counting algal cells is often used for effective control of harmful algal blooms. However, Microcystis sp. is commonly observed as a colony, so counting individual cells is challenging, as it requires sign...
Microcystis sp. is one of the most common harmful cyanobacteria that release toxic substances. Counting algal cells is often used for effective control of harmful algal blooms. However, Microcystis sp. is commonly observed as a colony, so counting individual cells is challenging, as it requires significant time and labor. It is urgent to develop an accurate, simple, and rapid method for counting algal cells for regulatory purposes, estimating the status of blooms, and practicing proper management of water resources. The flow cytometer and microscope (FlowCAM), which is a dynamic imaging particle analyzer, can provide a promising alternative for rapid and simple cell counting. However, there is no accurate method for counting individual cells within a Microcystis colony. Furthermore, cell counting based on two-dimensional images may yield inaccurate results and underestimate the number of algal cells in a colony. In this study, a three-dimensional cell counting approach using a novel model algorithm was developed for counting individual cells in a Microcystis colony using a FlowCAM. The developed model algorithm showed satisfactory performance for Microcystis sp. cell counting in water samples collected from two rivers, and can be used for algal management in fresh water systems.
Microcystis sp. is one of the most common harmful cyanobacteria that release toxic substances. Counting algal cells is often used for effective control of harmful algal blooms. However, Microcystis sp. is commonly observed as a colony, so counting individual cells is challenging, as it requires significant time and labor. It is urgent to develop an accurate, simple, and rapid method for counting algal cells for regulatory purposes, estimating the status of blooms, and practicing proper management of water resources. The flow cytometer and microscope (FlowCAM), which is a dynamic imaging particle analyzer, can provide a promising alternative for rapid and simple cell counting. However, there is no accurate method for counting individual cells within a Microcystis colony. Furthermore, cell counting based on two-dimensional images may yield inaccurate results and underestimate the number of algal cells in a colony. In this study, a three-dimensional cell counting approach using a novel model algorithm was developed for counting individual cells in a Microcystis colony using a FlowCAM. The developed model algorithm showed satisfactory performance for Microcystis sp. cell counting in water samples collected from two rivers, and can be used for algal management in fresh water systems.
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문제 정의
80, respectively. This study demonstrated the practical applicability of the FlowCAM for rapid and unbiased analysis of algal blooms, which is important for the proper management of water quality in freshwater systems and to provide safe water to the public. Further improvement of the precision of the FlowCAM library and the model algorithm for cell counting in algal colonies, as well as the extension of the library to other algae species, were left for future research.
This study was funded and supported by K-water (Korea Water Resources Corporation) to develop an innovative method for the effective management of harmful algal blooms in freshwater systems.
제안 방법
Water samples at the Daecheong Dam were randomly collected from five different sites between 30 and 36 km upstream of the dam body. Five milliliters of each sample were analyzed using the FlowCAM, and the number of cells (cells/mL) was compared between the FlowCAM method and visual inspection with a conventional microscope to determine the accuracy of the model algorithm developed in this study.
To the best of our knowledge, there is no standard procedure for counting the individual cells in a 3D Microcystis colony using a FlowCAM. In this study, we developed a novel method for automated quantification of Microcystis cells in fresh water using a FlowCAM. The purpose of this study was to verify the applicability of the method in practical cell counting of Microcystis colonies for water quality management rather than to provide specific parameters of the developed method.
Second, in this study, a filter tube with a diameter of 300 μm coupled with a microscope of ×200 magnification was used for algal cell imaging and counting.
image library, which is individually developed by users. The development of an improved library could improve the applicability of the algorithm developed in this study to count the number of cells in Microcystis colonies.
In this study, we developed a novel method for automated quantification of Microcystis cells in fresh water using a FlowCAM. The purpose of this study was to verify the applicability of the method in practical cell counting of Microcystis colonies for water quality management rather than to provide specific parameters of the developed method. First, Microcystis colonies within a water sample were identified using a FlowCAM with a local FlowCAM image library of Microcystis colonies collected in this study from fresh water of rivers and reservoirs in South Korea.
One challenge is counting cells in a 3D colony using a 2D analytical method. This study is the first to provide a systematic procedure and to develop a model algorithm for cell counts of Microcystis colonies considering the 3D form of the colony. The cross-validation of the developed model algorithm with unknown samples using the FlowCAM showed satisfactory performance for the estimation of Microcystis sp.
03 in this study) need to be developed initially for the FlowCAM use and the results may be different depending on sites. Using the proposed model algorithm and analytical procedures from this study, one can easily develop their own FlowCAM library and algorithm and utilize the FlowCAM for accurate cell counting for different sites and watersheds. Geomorphological comparison of a 3D ball and a 2D circle and further analytical analysis of the size of individual Microcystis sp.
Water samples collected from the Daecheong Dam (Site 4) in the Geum River between August 16 and 17, 2017 were used to develop a model algorithm for the cell count of Microcystis colonies (Fig. 1). A total of 55 Microcystis colonies were analyzed to determine the relationship between the 2D surface area of a colony and the number of cells in each colony.
대상 데이터
A total of 2,520 photo images of Microcystis colonies were obtained using a FlowCAM from the samples that were randomly collected from four sites in the Nakdong River, namely the Dalsung Weir (July 25, 2017), Hapcheon-changnyeong Weir (June 27, 2017), Changnyeong-haman Weir (June 27, 2017), Busan (July 19, 2017), and one site at about 35 km upstream of the Daecheong Dam (August 16, 2017) in the Geum River (Fig. 2). The FlowCAM identifies algal images when a water sample flows through a filter tube inside the device where the diameter of the filter tube is coupled with a specific magnification (e.
The colonies larger than 300 μm in diameter were filtered to prevent clogging in the FlowCAM filter tube, and thus were excluded from the library. Approximately 60% of the photo images (1,500) were taken from the samples collected in the Nakdong River, and the rest were from the samples collected in the Daecheong Dam. The 2,520 photo images were utilized for the development of a library of FlowCAM images that was used for the identification and classification of Microcystis colonies in unknown water samples.
1. Research sites and sampling locations in the Nakdong River and Geum River in South Korea.
1 and Table 1(a)), where algal blooms have been an important issue in water quality management [29]. The water samples were collected from the surface of the two rivers.
For weirs, water samples were collected about 500 m upstream of each weir body. Water samples at the Daecheong Dam were randomly collected from five different sites between 30 and 36 km upstream of the dam body. Five milliliters of each sample were analyzed using the FlowCAM, and the number of cells (cells/mL) was compared between the FlowCAM method and visual inspection with a conventional microscope to determine the accuracy of the model algorithm developed in this study.
이론/모형
within the water sample. The accuracy of the model algorithm in counting the total number of individual cells in Microcystis colonies within the water sample was then evaluated by comparing the result from the FlowCAM with the result from a conventional microscopic method for algal cell counting.
성능/효과
In this study, a novel method for cell counting in Microcystis colonies was developed from water samples collected from two major watersheds in South Korea. The number of cells in Microcystis colonies obtained from the model algorithm was evaluated by cross-validation with three statistical methods, where the average values of RSR, NSE, and R2 of the FlowCAM model were 0.44, 0.80, and 0.80, respectively. This study demonstrated the practical applicability of the FlowCAM for rapid and unbiased analysis of algal blooms, which is important for the proper management of water quality in freshwater systems and to provide safe water to the public.
65 [32, 33]. Therefore, it was concluded that the developed model algorithm for cell number counting using the FlowCAM in this study was well calibrated, and a strong correlation was achieved between the observed and predicted cell numbers for the 28 samples.
후속연구
This study demonstrated the practical applicability of the FlowCAM for rapid and unbiased analysis of algal blooms, which is important for the proper management of water quality in freshwater systems and to provide safe water to the public. Further improvement of the precision of the FlowCAM library and the model algorithm for cell counting in algal colonies, as well as the extension of the library to other algae species, were left for future research.
Larger colonies can be detected using a next available filter tube larger than 300 μm which is a 600 μm filter tube with a microscope of ×200 magnification. However, the use of different filter tubes with different magnifications for sample analysis can make the system more complicated with increased uncertainties, requiring further study. In practice, 300 μm tube filter with a microscope of ×400 magnification is typically used as it covers most dominant sizes of colonies found in water.
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