Simultaneous Detection of Viability and Concentration of Microalgae Cells Based on Chlorophyll Fluorescence and Bright Field Dual Imaging

Abstract

Ship ballast water contains high concentration of plankton, bacteria, and other microorganisms. If the huge amount of ballast water is discharged without being inactivated, it will definitely spell disaster to the marine environment. Microalgae is the most common species exiting in ballast water, so the detection of the concentration and viability of microalgae is a very important issue. The traditional methods of detecting microalgae in ballast water were costly and need the help of bulky equipment. Herein, a novel method based on microalgae cell intracellular chlorophyll fluorescence (CF) imaging combines with cell bright field (BF) microscopy was proposed. The geometric features of microalgae cells were obtained by BF image, and the cell viability was obtained by CF image. The two images were fused through the classic image registration algorithm to achieve simultaneous detection of the viability and concentration of microalgae cells. Furthermore, a low-cost, miniaturized CF/BF microscopy imaging prototype system based on the above principles was designed. In order to verify the effectiveness of the proposed method, four typical microalgae in ballast water (Platymonas, Pyramimonas sp., Chrysophyta, and Prorocentrum lima) were selected as the samples. The experimental results show that the self-developed prototype can quickly and accurately determine the concentration and the viability of microalgae cells in ship ballast water based on the dual images of BF and CF, and the detection accuracy is equivalent to that of commercial microscope. It was the first time to simultaneously detect the viability and concentration of microalgae cells in ship ballast water using the method that combining the fluorescence and bright field images; moreover, a miniaturized microscopic imaging prototype was developed. Those findings expected to contribute to the microalgae detection and ship ballast water management.

Keywords: bright field; chlorophyll fluorescence; detection; microalgae; ship ballast water.

Figure 1

(a) Structure of the dual imaging microscopy system. (b) System design and the photograph. (c) Photographs of the light source module, the stage module and the imaging module.

Figure 2

(a) Positive resolution test target, the red line is a transect and that the adjacent plot shows gray scale modulation across the line pairs on the resolution test chart. (b) Field of view of the miniaturized microscopic system. (c–e) The BF images of the polystyrene particles (with the diameter of 2 µm) and two common microalgae cells (Platymonas and Prorocentrum lima) captured by the miniaturized microscope. (f–h) shows the comparison of those corresponding samples captured by a commercial microscope.

Figure 3

(a) Bright field (BF) and chlorophyll fluorescence (CF) images of the four samples captured by commercial microscope (10 μm particles, Chrysophyta, Prorocentrum lima, and Pyramimonas sp.). (b) CF images of the four samples captured by the miniaturized system. (c) Fluorescence intensity distribution curves of the Chrysophyta, Prorocentrum lima, and Pyramimonas sp.

Figure 4

(a) BF images of the five counting units of the hemocytometer (upper left, upper right, middle, lower left and lower right). (b) Concentration detection by the miniaturized system and a commercial microscope.

Figure 5

BF and CF images of the living and dead cells captured by commercial microscope and the miniaturized system. (a) Platymonas cells. (b) Chrysophyta cells.

Figure 6

(a) Fluorescence image of Prorocentrum lima cells treated with 6 different concentrations of NaClO captured by the miniaturized system. (b) Comparison of the mean fluorescence intensity calculated by the miniaturized system and the commercial microscope.

Figure 7

(a) BF image of the mixed samples (living and dead Platymonas cells) captured by the miniaturized system. (b) CF image of the mixed samples captured by this system. (c) Comparison of the inactivation rate calculated by this system and the real value.

Figure 8

(a) Fused image without registration. (b) Fused image after registration. (c) Comparison with commercial microscope in terms of concentration detection and inactivation rate.