A method for accurately counting Microcystis blooms using live fluorescence

Abstract

The present invention discloses a method for accurately counting Microcystis aeruginosa in vivo by fluorescence, belonging to the field of environmental science and engineering. In the existing counting methods, the pretreatment process is complicated, the time span is too long, and Microcystis aeruginosa in the actual water body overlaps and aggregates together, making it impossible to quickly and accurately count under the condition of live algae. The present invention proposes to add Fe<supgt;2+< / supgt; to the collected stock solution to change the cell morphology of Microcystis aeruginosa cells that are easily broken, so that the Microcystis aeruginosa cells can be completely dispersed without aggregation under the high power of 100w of the cell crusher and still maintain the integrity of the cells; then, using the spontaneous fluorescence characteristics of chlorophyll a in live algal cells, each Microcystis aeruginosa cell emits a fluorescence point, and the number of Microcystis aeruginosa can be accurately obtained by counting the number of fluorescence points after being processed by the digital image processing algorithm, realizing the accurate counting of live Microcystis aeruginosa at the cell level, and can be used for on-site online analysis of actual water bodies.

Description

Technical Field

[0001] The invention belongs to the field of environmental science and engineering, and in particular relates to a method for accurately counting water bloom Microcystis by living fluorescence. Background Art

[0002] Eutrophication of water bodies leads to the massive reproduction of algae, thus forming algal blooms. They not only destroy aquatic ecosystems, but also produce many secondary toxic metabolites, which seriously threaten other aquatic organisms and even human health. For example, the toxins produced by cyanobacteria can inhibit the synthesis of human proteins and damage various organs of the human body. The toxins produced by Microcystis can promote tumor growth. Among them, Microcystis blooms are one of the most common algal blooms in lake basins. Therefore, in order to effectively prevent and control algal blooms, it is particularly important to monitor Microcystis.

[0003] The traditional method of counting Microcystis is to dye the collected water samples with strong oxidants, let them stand for 72 hours for sedimentation, siphoning, concentration and other pretreatment methods to turn the algae cells into specimens, then take 0.1ml of the sample and put it into the microscope frame, calculate the number of Microcystis cells in 100 fields of view by manual microscopy, and finally convert it into the algae density of the entire sample. The preliminary pretreatment is complicated, the process steps are many, the time span is long, and it can only be performed when all the algae cells are dead. Manual counting is slow, the stability and accuracy are poor, and it is easily affected by human factors. When algae bloom occurs, it is impossible to quickly achieve algae counting of the entire water sample in a short period of time.

[0004] At present, the existing image processing method for identifying and counting Microcystis cannot abandon the complicated and time-consuming pre-processing methods. In addition, a large amount of training is required in the early stage to calculate the area of ​​a Microcystis cell, convert the overall area of ​​the water bloom Microcystis and the area of ​​a single algae cell, and finally obtain the algae density of the entire water sample. Although this counting method is fast, the area of ​​a single algae cell is different, and the phenomenon of stratification and overlap of water bloom Microcystis is ignored, resulting in large accuracy errors. Summary of the invention

[0005] The existing counting methods have complicated pre-treatment processes, too long a time span, overlapping clusters of Microcystis in actual water bodies, and cannot be counted quickly and accurately in the case of live algae. The present invention proposes a method for accurately counting Microcystis in water using live fluorescence. The method collects water samples on site, and reinforces the algae cells in the case that the Microcystis cells are easily broken, disperses the overlapping clusters of Microcystis in water, and maintains the activity of the algae cells. The method uses microfluorescence to perform completely and accurately count the algae cells, and achieves accurate counting of live Microcystis in water at the cellular level, which can be used for on-site online analysis of actual water bodies.

[0006] The technical solution adopted by the present invention is as follows:

[0007] A method for accurately counting Microcystis blooms using live fluorescence comprises the following steps:

[0008] Step 1, collecting water samples of Microcystis bloom;

[0009] Step 2, dissolving the ferrous sulfate heptahydrate crystals;

[0010] Step 3, adding the liquid obtained by dissolving in step 2 to the water sample of Microcystis aquae in step 1;

[0011] Step 4: After standing for 5 minutes, shake the mixed sample evenly;

[0012] Step 5, placing the mixed sample under an ultrasonic cell disruptor for disruption;

[0013] Step 6: Take the solution onto a glass slide, place it under a fluorescence microscope, randomly capture the algae field of view and take photos to obtain an algae fluorescence image;

[0014] Step 7: Calculate the number of Microcystis according to the number of fluorescence points in the algae fluorescence image.

[0015] In the above technical solution, in step 3, the volume ratio of the water sample of Microcystis bloom and the ferrous sulfate solution is 40:1.

[0016] In the above technical solution, in step 5, the ultrasonic cell disruptor is set to have a power of 20-100W, a time of 1-2 minutes, a temperature of 50°C, an intermittent working mode, and works once every 2s.

[0017] In the above technical solution, in step 5, the power of the ultrasonic cell disruptor is set to 100W and the time is 2 minutes.

[0018] In the above technical solution, in step 6, the volume of the solution is 0.1 ml.

[0019] In the above technical solution, in step 7, the fluorescence image is converted from an RGB three-channel color image to a single-channel grayscale image.

[0020] In the above technical solution, in step 7, according to the brightness distribution of the fluorescent spots, the grayscale image value is amplified to the range of 0 to 255 using the range normalization method to enhance the contrast, and the grayscale image is binarized using the adaptive threshold method to highlight the fluorescent area.

[0021] In the above technical solution, in step 7, an elliptical kernel with a diameter of 5 is used to perform an opening operation to separate a very small number of still adherent group cells and remove small noise points.

[0022] In the above technical solution, in step 7, in the denoised binary image, the outermost contour and the chain simple estimation method are used to traverse and search for the contours of all highlight areas to find all independent fluorescent points.

[0023] In the above technical solution, in step 7, all contours are counted and stored in order from the upper left to the lower right, and numbers are drawn above each fluorescent point in the original fluorescent image to intuitively display the detection results.

[0024] Beneficial effects:

[0025] The present invention discloses a method for accurately counting Microcystis blooms by live fluorescence, which can break up the Microcystis blooms population without destroying the single cell morphology structure, thereby separating the overlapping cells of Volvox, and then using a digital image processing algorithm to count single fluorescent spots. The present invention selects the fluorescence effect of water samples of Microcystis blooms in the Chaohu Lake Basin at different powers. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The present invention is a flow chart of a method for accurately counting Microcystis blooms using live fluorescence.

[0027] Figure 2 Adding Fe to Microcystis aquatica treated with 20W ultrasonic disruptor 2+ Fluorescence image of .

[0028] Figure 3A Adding Fe to Microcystis aquatica treated with 70W ultrasonic disruptor 2+ Fluorescence image of .

[0029] Figure 3B This is a fluorescence image of the Microcystis aquae stock solution processed by an ultrasonic disruptor at 70W.

[0030] Figure 4A Adding Fe to Microcystis aquatica treated with 100W ultrasonic disruptor 2+ Fluorescence image of .

[0031] Figure 4B This is a fluorescence image of the Microcystis aquae stock solution processed by an ultrasonic disruptor at 100W.

[0032] Figure 5 The algal cell counting diagram does not show the counting box.

[0033] Figure 6 Figure 2 shows the algal cell count graph showing the counting frame. DETAILED DESCRIPTION

[0034] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. However, the following embodiments are limited to explaining the present invention, and the protection scope of the present invention should include the entire contents of the claims, and through the description of the following embodiments, those skilled in the art can fully implement the entire contents of the claims of the present invention.

[0035] Example

[0036] like Figure 1 As shown, a method for accurately counting Microcystis blooms by living fluorescence comprises the following steps:

[0037] Step 1. Fe 2+ It is very unstable in the environment and is easily oxidized to exist in the form of trivalent iron ions, so it is stored in the form of ferrous sulfate heptahydrate crystals. Dissolve 95% ferrous sulfate crystals, use an electronic analytical balance to accurately weigh 20g of crystals and place them in a beaker, add 80ml of deionized water, stir continuously at a water temperature (25-32℃), and prepare a 20% ferrous sulfate solution;

[0038] Step 2: Mix the algae collected in the wild with Fe 2+ Mix according to the volume ratio of 40:1, shake well and let it stand for 3 minutes to allow the algae cells to fully fuse with the iron ions;

[0039] Step 3. Place the mixed algae liquid under an ultrasonic cell disruptor with a frequency of 20-25KHz, set the temperature to 50°C, and the real-time temperature to 22.3°C; the working mode is intermittent, working once every 2s, and the total time is preferably 1-2min, more preferably 2min; the power is preferably 20-100w, more preferably 100w;

[0040] Step 4. Place the prepared glass slide flat on the stage of the fluorescence microscope, set the fluorescence microscope objective to 20X, open the phytoplankton microfluorescence imaging intelligent analysis software independently developed by the Anhui Institute of Optics and Precision Mechanics, Chinese Academy of Sciences, obtain the autofluorescence image of algae chlorophyll a, click the digital device (camera) in the taskbar to collect, and then project the microscope field of view onto the computer. Adjust the coarse / fine focus knob and brightness, and adjust the white balance on the software at the same time to find the clearest field of view, click to take a photo, and the photo is captured to obtain a clear fluorescent photo.

[0041] In step 3, multiple powers were used in parallel to disrupt the cells. The lowest power of 20w was preferred, and the working time was 1min and 2min respectively. A large number of clustered algal cells could be seen under fluorescence, which were not completely separated. Figure 2 As shown; when the power is increased to 70w, a small amount of algae cells can still be seen, such as Figure 3A , Figure 3BAs shown; finally, the power was increased to 100w, and the working time was 2min. Under the microscope, it can be seen that the red fluorescence produced by the algae cells has a clear and bright outline, and no obvious red fluorescence is found outside the cells. The red fluorescent spots are scattered alone without clustering, as shown in the figure. Figure 4A , Figure 4B As shown; at the same time, the algae stock solution was placed in 20w, and the algae cells were broken in large quantities. Under 100w for 2min, the algae cells were completely broken.

[0042] Step 5. Convert the fluorescence image from RGB three-channel color image to a single-channel grayscale image. According to the brightness distribution of the fluorescence points, use the range normalization method to enlarge the grayscale image value to the range of 0 to 255 to enhance the contrast. Use the adaptive threshold method to binarize the grayscale image, highlight the fluorescent area, and then use an elliptical kernel with a diameter of 5 to perform an opening operation to separate the very few still adherent group cells and remove small noise points. In the denoised binary image, use the outermost contour and chain simple estimation method to traverse and find the contours of all highlighted areas, find all independent fluorescent points, sort and count all contours from upper left to lower right, and draw numbers above each fluorescent point in the original fluorescence image to intuitively display the detection results, such as Figure 5 and Figure 6 shown.

[0043] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

Claims

1. A method for accurately counting Microcystis blooms using live fluorescence, characterized in that: The steps include: Step 1, collecting water samples of Microcystis bloom; Step 2, dissolving the ferrous sulfate heptahydrate crystals into a ferrous sulfate solution with a mass concentration of 20%; Step 3, adding the ferrous sulfate solution obtained by dissolving in step 2 into the water sample of Microcystis aquaeum in step 1 at a volume ratio of 1:40; Step 4, placing the mixed sample in an ultrasonic cell disruptor, and treating it for 2 minutes at a power of 100 W, a temperature of 50° C., and an intermittent working mode, wherein the work is performed once every 2 seconds; Step 5, take 0.1 ml of the treated solution onto a glass slide, and use a fluorescence microscope to capture the chlorophyll a autofluorescence image; Step 6: Perform grayscale conversion, range normalization, and adaptive threshold binarization on the fluorescence image. After denoising by elliptical opening operation, the number of independent fluorescence points is counted by the outermost contour and chain simple estimation method to achieve single-cell counting of living Microcystis.

2. The method for accurately counting Microcystis blooms by living fluorescence according to claim 1, characterized in that: In step 5, the ultrasonic cell disruptor is set to have a power of 100 W, a duration of 2 minutes, and an intermittent working mode of working once every 2 seconds.

3. The method for accurately counting Microcystis blooms by living fluorescence according to claim 1, characterized in that: The image processing in step 6 includes: (a) After converting the RGB image to a grayscale image, the grayscale value is mapped to the range of 0-255 using the range normalization method; (b) Adaptive thresholding was used for binarization, and an elliptical kernel with a diameter of 5 pixels was used for opening to separate adherent cells; (c) Counting of independent fluorescent spots by outermost contour detection and chained simple estimation algorithm.

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