WO2023113098A1

WO2023113098A1 - Microalga image continuous measurement apparatus

Info

Publication number: WO2023113098A1
Application number: PCT/KR2022/001233
Authority: WO
Inventors: 김종락; 장수현; 백지원; 유광태; 백승철
Original assignee: 주식회사 유앤유
Priority date: 2021-12-16
Filing date: 2022-01-24
Publication date: 2023-06-22
Also published as: KR102390074B1

Abstract

The present invention relates to a microalga image continuous measurement apparatus and, more specifically, to a microalga image continuous measurement apparatus which disperses a microalga aggregate through a dilute solvent and an ultrasonic homogenizer when a microalga sample is injected in order to acquire a high-resolution microalga image and allows the dispersed microalga sample to pass through a flow cell, and thus can acquire a high-resolution microalga image, and enables the microalga sample to continuously pass through the flow cell by means of a transfer pump, and thus can continuously acquire a plurality of microalga images.

Description

Microalgae image continuous measuring device

The present invention relates to a microalgae image continuous measuring device, and more particularly, when a microalgae sample is injected to obtain a high-resolution microalgae image, the aggregates of microalgae are dispersed through a dilution solvent and an ultrasonic disperser, and the dispersed By passing the microalgae sample through the flow cell, a high-resolution microalgae image can be obtained, and the microalgae sample can continuously pass through the flow cell by a transfer pump to continuously acquire a plurality of microalgae images , It relates to a microalgae image continuous measuring device.

Recently, the global environment is rapidly deteriorating, and the amount of harmful substances flowing into the water is increasing, and accordingly, there is a social demand for securing safe water resources.

In particular, phytoplankton are photosynthetic microscopic organisms of water quality, and exist everywhere, such as seas, rivers, and lakes, in an environment where the water temperature is higher than the appropriate temperature and light exists. algae, cyanobacteria), as well as various species such as green algae and diatoms, which are progressive organisms. In rivers and lakes, when algae proliferate excessively and cause hydration, a unique odor occurs, causing problems in the water treatment process. In the case of some blue-green algae, microcystin Since it secretes toxic substances harmful to the human body, such as, there is a problem of causing health and environmental problems.

These algae groups sometimes differ in dominant species depending on the region, environment, and season, and it is important to secure a DB for each of these algae groups in order to properly manage and monitor the water quality and environment of a lake or river.

Accordingly, research on various algae monitoring devices and methods is being conducted. Conventional algae monitoring technology mainly uses light having a plurality of wavelengths to a sample containing algae, such as Korean Patent Registration No. 10-1898712, Korean Patent Publication No. 2012-0133974, and Korean Patent Registration No. 10-0917030. There is a method of analyzing the wavelengths generated by algae by irradiation, and there is a method of detecting chromaticity, temperature, and illuminance as in Korean Patent No. 10-1928919, and a method of detecting DNA extracted from algae as in Korean Patent No. 10-1683379. There is a method of analyzing, and a method of photographing algae images with a microscope, such as in Korean Patent Registration No. 10-2100197.

The method of detecting the wavelength, chromaticity, temperature, and illuminance of light has limitations in discriminating species because the main purpose is to analyze the amount of algae generated, and only the level classified by color can be analyzed in classification. For species classification, DNA analysis extracted from algae or algae images magnified through a microscope are classified according to the experimenter's judgment. However, based on the National Institute of Biological Resources’ native species list established in 2015, 1,738 species of diatoms, 1,308 species of freshwater green algae, 686 species of flagella, and 239 species of cyanobacteria were found. There are time and cost issues and the possibility of recognition errors.

On the other hand, recently, research on a technique for learning images and classifying objects using artificial intelligence algorithms has been conducted. Image classification through deep learning has the advantage of being able to analyze a large amount of images with high accuracy, but a sufficient amount of learning is required for this. It is known.

Therefore, in order to secure a DB for each group of algae, there is a need for an invention related to a device capable of acquiring thousands of high-resolution microalgae image files faster than conventional techniques using samples taken from rivers or water sources.

The present invention relates to a microalgae image continuous measuring device, and more particularly, when a microalgae sample is injected to obtain a high-resolution microalgae image, the aggregates of microalgae are dispersed through a dilution solvent and an ultrasonic disperser, and the dispersed By passing the microalgae sample through the flow cell, a high-resolution microalgae image can be obtained, and the microalgae sample can continuously pass through the flow cell by a transfer pump to continuously acquire a plurality of microalgae images aims to

In order to solve the above problems, the present invention is an apparatus for continuously measuring microalgae images, comprising: a filter unit for removing contaminants included in microalgae samples; a dilution unit supplying a solvent to the microalgae sample to control the turbidity of the microalgae; An ultrasonic disperser for dispersing the microalgae by irradiating ultrasonic waves to the microalgae sample with ultrasonic intensity for each type of microalgae for pretreatment of the microalgae sample; a flow cell through which the microalgae sample to which ultrasonic wave is applied by the ultrasonic disperser flows; a microscope for continuously photographing the microalgae sample inside the flow cell; and a transfer pump transferring the microalgae sample from the filter unit to the flow cell.

In one embodiment of the present invention, the dilution unit, a turbidimeter for measuring the turbidity of the microalgae sample; A dilution solvent capable of diluting the microalgae sample based on the turbidity measured by the turbidimeter; and a dilution chamber for diluting the microalgae sample using the dilution solvent, wherein the dilution solvent includes distilled water, and may optionally further include a chemical agent for fixing the microalgae.

In one embodiment of the present invention, the ultrasonic diffuser, a calibration step of deriving a device driving value for the energy unit or the input energy unit corresponding to the input tide; A device driving step of driving the ultrasonic diffuser with the device driving value derived in the calibration step; may be performed.

In one embodiment of the present invention, the calibration step may include: a first measuring step of driving the ultrasonic diffuser with a first device driving value, applying ultrasonic waves to a reference medium, and measuring first change information of the temperature of the reference medium; a second measurement step of driving the ultrasonic diffuser with a second device driving value, applying ultrasonic waves to the reference medium, and measuring second change information of the temperature of the reference medium; a first derivation step of deriving a first energy unit for a first device driving value based on the first change information; a second derivation step of deriving a second energy unit for a second device drive value based on the second change information; and deriving relationship information between the device drive value and the energy unit based on the regression information including the first device drive value, the second device drive value, the first energy unit, and the second energy unit. , It is possible to derive the ultrasonic intensity for each type of microalgae based on the relational information between the device driving value and the energy unit.

In one embodiment of the present invention, in the device driving step, the ultrasonic wave is irradiated to the microalgae sample for a predetermined time with the ultrasonic intensity derived in the calibration step to disperse flocs, which are microalgal aggregates.

In one embodiment of the present invention, the flow cell includes one or more flow passages, and each of the one or more flow passages includes an inlet passage formed in a vertical direction, an intermediate passage extending horizontally from the inlet passage, and an intermediate passage extending from the inlet passage. It includes an outlet passage extending in a vertical direction, and an inner surface of the intermediate passage is coated with nanomaterials to alleviate the problem of residual algae attachment.

In one embodiment of the present invention, the intermediate flow passage, the inlet side intermediate flow passage having a first width; a middle-side intermediate passage extending from the inlet-side intermediate passage and having a second width; and an outlet-side intermediate passage extending from the middle-side intermediate passage and having a third width, wherein the second width may be greater than the first width and the third width.

In one embodiment of the present invention, the microscope, the lighting unit; a stage unit disposed below the lighting unit and having a through hole therein; and a lens unit located below the stage unit and movable up and down, wherein the flow cell is seated on an upper surface of the stage unit, and the lens unit can be inserted into the through hole.

According to one embodiment of the present invention, microalgal floes can be minimized through sample dilution and cell fixation automation based on the turbidity of the microalgal sample, thereby achieving an effect of stably measuring the image.

According to one embodiment of the present invention, by giving a change in width to a flow cell flow path having a conventional straight line shape, it is possible to achieve an effect of facilitating the flow of algae and measuring stable images.

According to one embodiment of the present invention, the possibility of algae contacting the flow path can be reduced by nano-coating the inner surface of the flow cell flow path, thereby alleviating the problem of remaining algae adhesion and preventing contamination by long-term monitoring. can

According to one embodiment of the present invention, by performing a pre-processing process and a photographing process through an automated system, it is possible to exert an effect of increasing efficiency in acquiring microalgae images.

According to one embodiment of the present invention, through the pretreatment method through the ultrasonic disperser, it is possible to exhibit the effect of increasing the single cell recovery rate and solving the efficiency and time problems compared to the conventional pretreatment method using chemicals and heat treatment.

According to an embodiment of the present invention, the intensity of the ultrasonic diffuser can be standardized through the calibration step, and through this, cross-validation between laboratories can be implemented.

According to an embodiment of the present invention, the health of the aquatic ecosystem can be secured through real-time monitoring of streams, etc., and the quality of purified water can be improved by monitoring the intake source, thereby contributing to securing water quality stability.

According to one embodiment of the present invention, it is possible to analyze the species of microalgae by water area, and it is possible to detect or prevent the occurrence of algae in advance through real-time monitoring.

1 schematically shows an apparatus for continuously measuring microalgae images according to an embodiment of the present invention.

2 shows an image of microalgae according to a pretreatment method according to an embodiment of the present invention.

Figure 3 shows microalgae images according to the intensity of the ultrasonic disperser for irradiating ultrasonic waves to microalgae samples according to an embodiment of the present invention.

4 schematically illustrates steps performed in a calibration step according to an embodiment of the present invention.

5 shows a graph for calculating relationship information between a device drive value and an energy unit in a calibration step according to an embodiment of the present invention.

6 shows an actual photograph and a schematic diagram of a flow cell according to an embodiment of the present invention.

7 schematically shows a structural diagram of a flow path in a flow cell according to an embodiment of the present invention.

8 shows images of microalgae according to the height of the flow cell passage according to an embodiment of the present invention.

9 shows images of microalgae according to the shape of a flow path in a flow cell according to an embodiment of the present invention.

10 schematically shows the structure of an inverted microscope according to an embodiment of the present invention.

In the following, various embodiments and/or aspects are disclosed with reference now to the drawings. In the following description, for purposes of explanation, numerous specific details are set forth in order to facilitate a general understanding of one or more aspects. However, it will also be appreciated by those skilled in the art that such aspect(s) may be practiced without these specific details. The following description and accompanying drawings describe in detail certain illustrative aspects of one or more aspects. However, these aspects are exemplary and some of the various methods in principle of the various aspects may be used, and the described descriptions are intended to include all such aspects and their equivalents.

Moreover, various aspects and features will be presented by a system that may include a number of devices, components and/or modules, and the like. It should also be noted that various systems may include additional devices, components and/or modules, and/or may not include all of the devices, components, modules, etc. discussed in connection with the figures. It must be understood and recognized.

"Example", "example", "aspect", "exemplary", etc., used herein should not be construed as preferring or advantageous to any aspect or design being described over other aspects or designs. . The terms '~unit', 'component', 'module', 'system', 'interface', etc. used below generally mean a computer-related entity, and for example, hardware, hardware It may mean a combination of and software, software.

Also, the terms "comprises" and/or "comprising" mean that the feature and/or element is present, but excludes the presence or addition of one or more other features, elements and/or groups thereof. It should be understood that it does not.

In addition, terms including ordinal numbers, such as first and second, may be used to describe various components, but the components are not limited by the terms. These terms are only used for the purpose of distinguishing one component from another. For example, a first element may be termed a second element, and similarly, a second element may be termed a first element, without departing from the scope of the present invention. The terms and/or include any combination of a plurality of related recited items or any of a plurality of related recited items.

In addition, in the embodiments of the present invention, unless otherwise defined, all terms used herein, including technical or scientific terms, are generally understood by those of ordinary skill in the art to which the present invention belongs. has the same meaning as Terms such as those defined in commonly used dictionaries are to be interpreted as having a meaning consistent with the meaning in the context of the related art.

It should be, unless it is clearly defined in the embodiments of the present invention, it is not to be interpreted in an ideal or excessively formal meaning.

As shown in FIG. 1, an apparatus for continuously measuring microalgae images, comprising: a filter unit 100 for removing contaminants included in microalgae samples; A dilution unit 110 for supplying a solvent to the microalgae sample to adjust the turbidity of the microalgae; An ultrasonic disperser 120 for dispersing the microalgae by irradiating ultrasonic waves to the microalgae sample at ultrasonic intensity for each type of microalgae for pretreatment of the microalgae sample; a flow cell 130 through which the microalgae sample to which ultrasonic wave is applied by the ultrasonic disperser 120 flows; a microscope for continuously photographing the microalgae sample inside the flow cell 130; and a transfer pump 140 for transferring the microalgae sample from the filter unit 100 to the flow cell 130.

On the other hand, the dilution unit 110, a turbidimeter for measuring the turbidity of the microalgae sample; A dilution solvent capable of diluting the microalgae sample based on the turbidity measured by the turbidimeter; and a dilution chamber for diluting the microalgae sample using the dilution solvent, wherein the dilution solvent includes distilled water, and may optionally further include a chemical agent for fixing the microalgae.

Specifically, in the filter unit 100, the first step of the pretreatment steps for dispersing flocs, which are microalgae aggregates present in the microalgae sample, is performed, and the microalgae sample is filtered using a microsieve filter. The microsieve filter is a mesh filter having a pore size of several tens of μm to several thousand μm, and removes contaminants present in the microalgae sample. Unlike algae cultures, samples obtained from water sources require a process of removing contaminants and dilution. The microsieve filter may be used by removing foreign substances according to the characteristics of the algae or by adjusting the pore size so that only algae having a target size or smaller may be introduced into the flow cell 130 .

According to one embodiment of the present invention, a microsieve filter with a pore size of 1000 μm can be used to prevent foreign substances from entering the flow cell 130, and a microsieve filter with a pore size of 50 μm can prevent microalgae colonies. Can be used to disperse into entities. According to another embodiment of the present invention, even if a microsieve filter having a small pore size is used, the object dispersion effect may not appear clearly, and an additional pretreatment step may be performed to compensate for this. The additional pretreatment step includes a dilution step and an ultrasonic dispersion step, which will be described in detail later.

In the dilution unit 110, the second step of the pretreatment step for dispersing the floc in the microalgae sample is performed, and after measuring the turbidity of the microalgae sample, a dilution solvent is used based on the measured turbidity to reduce the microalgae Samples can be diluted.

The turbidity of the microalgae sample may be measured by a turbidity meter provided in the dilution unit 110. Specifically, the turbidimeter can measure the turbidity of the microalgae sample by irradiating the microalgae sample with light and comparing the degree of reflection or scattering of the light transmitted through the sample by the dispersed particles with a standard solution.

Based on the turbidity of the microalgae sample measured through the turbidimeter, the dilution unit 110 may adjust the amount of the dilution solvent injected into the dilution chamber provided. As an embodiment of the present invention, it is preferable to inject the dilution solvent into the dilution chamber so that the turbidity of the microalgae sample is less than 200 NTU, and distilled water is used as the dilution solvent.

Meanwhile, as an embodiment of the present invention, a chemical agent may be selectively included in the dilution solvent to fix the algae in the microalgae sample. Formaldehyde and the like may be used as the chemical agent, and as an embodiment of the present invention, a chemical agent such as KOH (potassium hydroxide) or NaOH (sodium hydroxide) may be additionally used to disperse the flocs in the microalgae sample. .

As another embodiment of the present invention, the dilution solvent may be automatically injected into the dilution chamber through an automated system without operator intervention. As shown in FIG. 1 , the dilution solvent is contained in a separate dilution solvent storage tank and may be injected into the dilution chamber through the transfer pump 140 . Through the automated system as described above, contamination of the microalgae sample can be prevented, and the effect of increasing the efficiency of the measurement method can be expected.

The ultrasonic disperser 120 is a device used in the last step of the pretreatment step of dispersing the flocs in the microalgal sample by irradiating the microalgal sample with ultrasonic waves.

Algae dispersal pretreatment techniques include chemical treatment techniques and heat treatment techniques in addition to techniques using ultrasonic waves. However, in the present invention, an ultrasonic technique that showed excellent results in terms of dispersal time and single cell recovery rate was adopted in the single cell recovery rate experiment conducted by the present invention. . A detailed comparison between the pretreatment technique using ultrasonic waves and the pretreatment technique without using ultrasonic waves will be described later.

On the other hand, by performing a calibration step in the ultrasonic diffuser 120, it is possible to derive an energy unit corresponding to the injected microalgae sample or a device driving value for the input energy unit. That is, the optimal ultrasonic intensity corresponding to the microalgae sample can be derived to maximize the dispersing effect of microalgae, and since the energy unit is a standardized value, cross-verification with other laboratories can be implemented. Details of the calibration step will be described later.

The flow cell 130 includes a plurality of thin flow passages, and the microalgae sample that has undergone the pretreatment process can flow through the flow passages. Microalgae images can be obtained for algae samples. As an embodiment of the present invention, the width of the channel is preferably set to 50 μm to 1000 μm in order to capture images of microalgae of various sizes. Details of the flow cell 130 will be described later.

The transfer pump 140 is positioned between the filter unit 100 and the dilution unit 110 and between the dilution solvent and the dilution chamber to help transfer the microalgae sample or the dilution solvent. A micro pump may be used as the transfer pump 140 to precisely control the amount of the microalgal sample flowing into the flow cell 130. The flow rate and flow rate of the microalgal sample transferred through the micropump can be adjusted according to the size and shape of the passage in the flow cell 130. The micro pump refers to a fluid device capable of transferring a minute flow rate of 1 µl or less per minute.

In addition, the operating state of the transfer pump 140 may be controlled through pump control software. Through the pump control software, it is possible to control the flowing amount of the microalgae sample or dilution solvent, control the operation of the syringe, and control the continuous operation of the pump. Once the transfer pump 140 is set through the pump control software, it can automatically transfer the microalgae sample and the dilution solvent to the dilution chamber, the ultrasonic disperser 120, and the flow cell 130 by the set amount, In measuring the microalgae image, it is possible to exert an effect of increasing efficiency.

As described above, as an embodiment of the present invention, the microalgae image continuous measuring device automatically performs a preprocessing step when only the microalgal sample is initially injected, and then the sample is transferred to the flow cell 130, and the An image of the microalgae sample may be automatically measured in the flow cell 130. An embodiment of the microalgae image continuous measuring device that is automatically performed is as follows.

When the microalgae sample is injected into the microalgae image continuous measuring device, the microalgae sample is transferred to the filter unit 100 by the transfer pump 140, and contaminants and foreign substances present in the microalgae sample are removed. .

The microalgal sample that has passed through the filter unit 100 is automatically transferred to the dilution chamber of the dilution unit 110 by the transfer pump 140, and the turbidimeter automatically measures the microalgal sample in the dilution chamber. Measure the turbidity. The dilution unit 110 automatically calculates the amount of dilution solvent required to dilute the microalgae sample based on the measured turbidity, and based on the calculated amount of the dilution solvent, the microalgae sample is pre-set Dilute below turbidity.

The microalgae sample that has passed through the dilution unit 110 is transferred to the ultrasonic disperser 120 by the transfer pump 140, and in the ultrasonic disperser 120, based on the preset ultrasonic intensity and ultrasonic irradiation time, Ultrasound is irradiated to the microalgae sample transferred to the ultrasonic disperser 120.

The microalgal sample that has completed the pretreatment step in the ultrasonic disperser 120 is transferred to the flow cell 130 by the transfer pump 140, and the microalgal sample is continuously imaged in the flow cell 130 can be filmed

The movement of the microalgae sample by the transfer pump 140 is automatically operated according to a predetermined value.

Schematically, FIG. 2 (a) shows a microalgae image of a microalgae sample using a chemical treatment technique among algal dispersal pretreatment techniques, and FIG. 2 (b) shows microalgae microalgae using an ultrasonic irradiation technique among algal dispersal pretreatment techniques An image of the microalgae of the sample is shown.

Specifically, algae spraying pretreatment techniques can be largely divided into single pretreatment techniques and complex pretreatment techniques. The single pretreatment technique includes a chemical treatment technique, a heat treatment technique, and an ultrasonic technique, and the complex pretreatment technique is a technique in which two or more of the single pretreatment techniques are mixed. In general, in the case of a complex pretreatment technique, the dispersion effect is increased compared to a single pretreatment technique, but time and cost may also increase.

A plurality of experiments were performed to derive the optimal pretreatment technique for the present invention, and in FIG. 2, as an embodiment of the present invention, the single cell recovery rate of the pretreatment technique using ultrasound and the pretreatment technique without ultrasound Show an image showing the difference. The alga used in the experiment corresponding to FIG. 2 is Chlorella vulgaris, and in the plurality of experiments, a plurality of algae including Chlorella vulgaris were conducted, but this is not shown separately.

Figure 2 (a) is an embodiment of the present invention, a microscopic image of Chlorella vulgaris using a chemical treatment technique, and a 0.01M sodium hydroxide solution was used in the experiment. As shown in (a) of FIG. 2, although it seems to be relatively well dispersed in the 6th and 8th regions, there are flocs that are not properly dispersed, as indicated by the red circles in the 19th and 21st regions. can confirm that

Figure 2 (b) is an embodiment of the present invention, a microscopic image of Chlorella vulgaris using an ultrasonic technique, and in the experiment, ultrasonic waves were irradiated for 20 seconds at an ultrasonic intensity of 200 kJ / L. As shown in (b) of FIG. 2, it can be confirmed that the algae are evenly dispersed in the entire area as a result of using the ultrasonic technique.

Based on the foregoing, in the present invention, an ultrasonic treatment technique having a high dispersion effect is used. However, according to the type of microalgae, the intensity of the ultrasonic wave and the ultrasonic irradiation time at which the dispersion effect is best for the microalgae are different, and to compensate for this, in the present invention, the optimal ultrasonic intensity according to the type of microalgae through the calibration step And the ultrasonic irradiation time can be derived.

Figure 3 shows microalgae images according to the intensity of the ultrasonic disperser 120 for irradiating ultrasonic waves to microalgae samples according to an embodiment of the present invention.

Schematically, (a) of FIG. 3 shows an image of microalgae when the microalgae sample is irradiated with an ultrasonic intensity of 0.004 W/ml. Figure 3 (b) shows a microalgae image when the microalgae sample is irradiated with an ultrasonic intensity of 0.08 W / ml. Figure 3 (c) shows a microalgae image when the microalgae sample is irradiated with an ultrasonic intensity of 0.3 W / ml.

Specifically, referring to FIG. 3 as an embodiment of the present invention, it can be confirmed that the algae dispersion effect appears well as the ultrasonic wave is irradiated to the microalgae sample at a strong ultrasonic intensity, and in the case of FIG. 3 (c), a single The cell recovery rate was determined to be about 90%. As another embodiment of the present invention, in the case of using the complex pretreatment technique using both the chemical treatment technique and the ultrasonic treatment technique, the single cell recovery rate can be measured higher than the ultrasonic treatment technique, but the single cell recovery rate is high even when only the ultrasonic treatment technique is used. Since this has been measured, in the present invention, considering cost and time issues, a single pretreatment technique of only sonication is used.

As shown in FIG. 4, the ultrasonic diffuser 120 includes a calibration step of deriving an energy unit corresponding to the input tidal current or a device driving value for the input energy unit; A device driving step of driving the ultrasonic diffuser 120 with the device driving value derived in the calibration step; may be performed, and the calibration step drives the ultrasonic diffuser 120 with the first device driving value, a first measurement step (S100.1) of applying ultrasonic waves to and measuring first change information of the temperature of the reference medium; A second measuring step (S100.2) of driving the ultrasonic diffuser 120 with the second device driving value, applying ultrasonic waves to the reference medium, and measuring second change information of the reference medium temperature; a first derivation step (S110.1) of deriving a first energy unit for a first device driving value based on the first change information; a second derivation step of deriving a second energy unit for a second device drive value based on the second change information (S110.2); and the first device driving value and the second device driving value. Based on the regression information including the first energy unit and the second energy unit, deriving relationship information between the device drive value and the energy unit; including, based on the relationship information between the device drive value and the energy unit The ultrasonic intensity for each type of microalgae can be derived.

Specifically, the calibration step is a step of standardizing the device driving value of the ultrasonic diffuser 120 through a calorimetric method. In the method using the conventional ultrasonic disperser 120, even if the ultrasonic disperser 120 is operated with the same device driving value, the result value is different as the laboratory environment, equipment used, and processing container are changed, and thus the same Even if the sample was conducted in the same experiment method, the result value was different, resulting in a problem of low reliability of the experiment. In order to solve this problem, in the present invention, by performing a calibration step, a more rigorous comparison is possible through standardized power no matter which laboratory conducts the experiment, and the reliability of the experiment is improved through cross-laboratory cross-validation (Round Robin Test). It can exert an effect that can be improved. In addition, if the intensity of the ultrasonic wave applied to the microalgae sample is too strong, since the algae cells may be destroyed, by performing the calibration step, according to the type of microalgae, the dispersion effect of the microalgae can be expected to be maximized Device drive value can be found. The reference medium, preferably as an embodiment of the present invention, may be a microalgal sample diluted by the dilution unit 110.

As shown in FIG. 5, the ultrasonic diffuser 120 performs a device driving step of driving the ultrasonic diffuser 120 with the device driving value derived in the calibration step, and in the device driving step, the calibration Depending on the device driving value derived in the step, ultrasonic waves may be irradiated to the microalgae sample for a predetermined time with ultrasonic intensity to disperse flocs, which are aggregates of microalgae.

Specifically, referring to FIG. 4, in the calibration step, a measurement step (S100.1 to S100.N or less, S100) and a derivation step (S110.1 to S110.N or less, S110) are performed, and the measurement step ( S100) is sequentially performed from the first measuring step (S100.1) to the Nth measuring step (S100.N), and the derivation step (S110) is performed corresponding to the measuring step. In the measuring step, a device drive value is set and the ultrasonic diffuser 120 is driven with the device drive value, the ultrasonic diffuser 120 applies ultrasonic waves to a reference medium, and according to the irradiation time of the reference medium to which the ultrasonic waves are applied, the ultrasonic diffuser 120 is driven. Record the change information about the changed temperature of the reference medium.

As an embodiment of the present invention, (a) and (b) of FIG. 5 show graphs for the measurement step (S100), and (a) and (b) of FIG. 5 are results performed in different laboratories, respectively. is shown graphically.

Referring to (a) of FIG. 5, after setting the first device drive value to 10%, the temperature change according to the ultrasonic irradiation time was recorded. After the first measurement step (S100.1) was completed, the second device drive value was set to 20% and the second measurement step (S100.2) was performed, and the sixth device drive value was sequentially set to 100%. Up to 6 measurement steps (S100.6) were performed.

In the case of (b) of FIG. 5, the temperature change according to the ultrasonic irradiation time was recorded by experimenting twice with one device driving value. In (b) of FIG. 5, the first change information was obtained by setting the first device drive value to 20%, and sequentially in the fifth measurement step (S100.5), the fifth device drive value was set to 100%. , fifth change information is acquired. After obtaining the fifth change information, using the same reference medium as the reference medium used in the first measurement step (S100.1) to the fifth measurement step (S100.5), the sixth device driving value is set to 20% again Then, the 6th change information is obtained, and the 10th change information is obtained after sequentially setting the 10th device drive value to 100% in the 10th measurement step (S100.10).

In the deriving step (S110), the change information obtained through the measuring step (S100); and _mass and specific heat information of the reference medium according to the initial conditions of the experiment. In the deriving step (S110), the energy unit applied to the reference medium can be obtained by substituting the three conditions information into the following equation (1).

- formula (1)

Equation (1) above can be derived by transforming the relational expression between heat quantity and specific heat. Since the unit of heat quantity is J (Joule), and dividing J by time (s) is W (Watt), both sides of the relational expression between heat quantity and specific heat are divided by unit time to obtain Equation (1).

As an embodiment of the present invention, (c) and (d) of FIG. 5 show graphs for the derivation step (S110) corresponding to (a) and (b) of FIG. 5, respectively.

5 (c) and (d), each blue point is an energy unit derived based on the change information when the ultrasonic diffuser 120 is driven with the device drive value as the y value is recorded as the x value.

A relational expression between the device driving value and the energy unit may be derived through the relational information between the device driving value and the energy unit. Specifically, referring to FIG. 4 , the regression information may correspond to a relational expression between the device drive value and energy, and the accuracy of the regression information may be determined through a coefficient of determination (R ² ) for the relational expression. .

As an embodiment of the present invention, referring to FIG. 5 , it can be further confirmed that the coefficient of determination increases when the number of measurement steps (S100) is increased in the calibration step. That is, as the value of N in FIG. 4 increases, an accurate correlation between the device driving value and the energy unit can be obtained. Specifically, as shown in (b) and (d) of FIG. 5, after subdividing the level of the device driving value into n, the value of N may be increased through repeated measurement. Here, 'N = n x number of iterations'. As another embodiment of the present invention, the value of N may be increased by further subdividing the level of the device driving value, that is, by increasing the value of n. Here, 'N = n'.

However, in the case of (c) of FIG. 5, the coefficient of determination is 0.9981 when the N value is 5, and in the case of (d) of FIG. 5, the result of the coefficient of determination is 0.9997 when the N value is 10. In (S100), it is preferable to set the value of n to 4 or more, and it is more preferable to set the value of n to 4 to 6 in consideration of efficiency.

In this way, when the experiment is performed by converting the ultrasonic intensity applied to the sample into an energy level through the calibration step, the experiment can be conducted and recorded at a standardized energy level without affecting the laboratory environment.

In addition, when the actual algae are pretreated, the energy transmitted to the microalgae sample can be quantified and standardized, so that the optimum ultrasonic intensity that can maximize the dispersion effect according to the type of microalgae or the source of the microalgae sample can be obtained. It can be derived and by applying the optimal ultrasonic intensity derived from the laboratory environment to the microalgae image continuous measurement device, it is possible to exert an effect of stably obtaining a high-resolution microalgae image.

On the other hand, as an embodiment of the present invention, additional characteristics of microalgae can be found as data information on the optimal ultrasonic intensity for each laboratory is accumulated.

6 shows an actual photograph and a blueprint of a flow cell 130 according to an embodiment of the present invention, and FIG. 7 schematically shows a structural diagram of a flow path in the flow cell 130 according to an embodiment of the present invention. .

As shown in FIGS. 6 and 7 , the flow cell 130 includes one or more flow passages, and each of the one or more flow passages includes an inlet passage 131 formed in a vertical direction and a horizontal inlet passage 131. and an outlet passage 133 extending in a vertical direction from the middle passage 132, and the inner surface of the middle passage 132 is provided to alleviate the problem of remaining algae adhesion. coated with nanomaterials.

Schematically, FIG. 6(a) is an actual photograph of an actual flow cell 130 having different channel widths, and FIG. 6(b) is a picture of the flow cell 130 shown in FIG. 6(a). It shows the blueprint. 7 shows the structure of a flow path located inside the flow cell 130.

Specifically, as shown in FIG. 6 , one flow cell 130 includes two or more flow paths. Each flow path has a different width and is characterized by a technical feature that microalgae samples flowing through the flow path can be photographed at different magnifications in one flow cell 130. Images can be taken. In addition, as shown in FIG. 7, when the microalgae sample is introduced through the inlet passage 131 formed in the vertical direction, each flow passage forms an intermediate passage 132 extending horizontally from the inlet passage 131. The microalgae sample flows through and the microalgae sample is discharged through the outlet passage 133 extending in the vertical direction from the intermediate passage 132. The inlet passage 131 and the outlet passage 133 are open in the same direction.

Referring to FIG. 7 , the flow cell 130 uses a PDMS material for the PDMS chip, which is a part including the flow path, and the lower part of the PDMS chip is made of slide glass. The PDMS material is a colorless polymer material similar to silicon and has excellent formability, so it is used in various fields such as biology, medicine, pharmacy, material engineering and mechanical engineering. In order to compensate for the disadvantage of poor durability of the PDMS material, a structure in which a slide glass is attached to the lower end of the PDMS chip may be manufactured. As an embodiment of the present invention, the PDMS chip may be manufactured with a height of 4.5 mm, preferably with a horizontal length of 63 mm and a vertical length of 20.5 mm, and depending on the laboratory environment and the size of the flow path, it may be larger or larger than that. It can also be produced in small shapes.

In addition, the inner surface of the passage is coated with nanomaterials. When the microalgae sample passes through the inside of the flow cell 130 at low speed, microalgae may adhere to the inner surface of the flow path and block the flow path. As described above, the remaining algae are coated by coating the inner surface of the flow path It alleviates the adhesion problem and allows the microalgae sample to pass through the flow cell 130 well.

Specifically, the height of the passage means the height of the intermediate passage 132 with reference to FIG. 7 . The height of the flow path is an important setting factor in microalgae image capture. As shown in FIG. 8, when the height of the flow path is higher than a certain height, the amount of light transmitted to the microalgae sample is reduced, and the microscope focuses difficulties may arise. However, if the height of the flow path is lower than a certain height, the microalgae sample may not flow smoothly in the flow path and the flow path may be clogged. To solve the problem, if the flow rate is increased in the flow cell 130 The resolution of the microalgae image may be lowered.

Referring to FIG. 8, as an embodiment of the present invention, an image taken by passing a microalgae sample through a channel having a height of 20 μm, 50 μm, and 80 μm is shown, and the height of the channel is 20 μm. Recognizable level It can be seen that the focus of Based on the plurality of images of microalgae shown in FIG. 8, the height of the passage is preferably set to less than 50 μm, and more preferably, the height of the passage may be set to 20 to 30 μm. The above-mentioned problem that the microalgae sample does not flow smoothly inside the flow cell due to the low height of the passage can be solved by adjusting the width of the passage.

9 shows images of microalgae according to the shape of a flow path in the flow cell 130 according to an embodiment of the present invention.

As shown in FIG. 9 , the middle passage 132 includes an inlet-side middle passage 132.1 having a first width; a middle-side intermediate passage 132.2 extending from the inlet-side intermediate passage 132.1 and having a second width; and an outlet-side intermediate passage 132.3 extending from the middle-side intermediate passage 132.2 and having a third width, and the second width may be greater than the first and third widths.

Schematically, (a) of FIG. 9 is an embodiment of the present invention, which is a line-shaped intermediate flow path 132 used in a conventional experiment; And a microscope image when the intermediate flow path 132 is used; FIG. 9 (b) is an embodiment of the present invention having a first to third width and a form used in the present invention a middle flow path (132); and a microscope image when the intermediate flow path 132 is used.

Specifically, the middle flow passage 132 is a part for photographing the microalgae sample flowing in the flow cell 130 through a microscope. As shown in (a) of FIG. 9 , the straight intermediate flow path 132 used in the conventional experiment is easy to manufacture and has low cost, and has advantages in terms of maintenance and is widely used. However, in order to secure a high-resolution microalgae image, it is preferable that the microalgae sample flow at a low speed in the intermediate flow path 132 in the flow cell 130, and to secure the focus, the height of the intermediate flow path 132 is preferably low do. On the other hand, in the straight-line intermediate flow path 132 shown in (a) of FIG. 9, when the height of the flow path is low and the microalgae sample flows at a low speed, the flow of algae is not smooth, and the flow of algae is smooth. When the microalgae sample is flowed at high speed or the flow cell 130 having a flow path height higher than a certain level is used for this purpose, it is difficult to stably obtain a high-resolution image with reference to FIG. 8 .

In order to solve the problem as described above, in the present invention, the intermediate flow path 132 of the form shown in FIG. 9 (b) was manufactured. Korean Patent Registration No. 10-2100197 also used a flow cell 130 in the form of varying the width of the flow path of the part to be photographed, but in the above registered patent, by performing the process of injecting chemicals into the flow cell 130 , The size of the flow cell 130 is large and the structure is complicated compared to the present invention. Due to this feature, the function of taking images of microalgae at various magnifications through two or more flow channels having various widths in one flow cell 130, which is a technical feature of the present invention, is implemented in the registered patent has a hard point. In addition, the flow cell 130 of the registered patent has a complicated structure and is expensive to manufacture, unsuitable for obtaining high-resolution images, and difficult to maintain. As shown in (b) of FIG. The flow cell 130 having a flow path used in the present invention has improved the above-mentioned problems, and through this, it is possible to take images of microalgae efficiently.

10 schematically shows the structure of a microscope 200 according to an embodiment of the present invention.

As shown in Figure 10, in one embodiment of the present invention, the microscope 200, the lighting unit 210; a stage unit 220 disposed below the lighting unit 210 and having a through hole therein; and a lens unit 230 located below the stage unit 220 and movable up and down, wherein the flow cell 130 is seated on the upper surface of the stage unit 220, and the lens unit 230 may be introduced into the through hole.

Specifically, as shown in FIG. 10, an inverted microscope is used in the present invention. The microscope can be largely classified into an upright microscope and an inverted microscope. The upright microscope has an objective lens positioned above the stage 220, and the inverted microscope has an objective lens positioned below the stage 220. has a positioning form.

The upright microscope has an advantage in observing a slide sample because the lighting unit 210 is located at the bottom of the stage unit 220 and the objective lens is located at the top of the stage unit 220, and the inverted microscope has the lighting unit 210 is located at the top of the stage unit 220, and the objective lens is located at the bottom of the stage unit 220, so that the lower end of the sample is photographed, so it is suitable for photographing the sample contained in the culture medium.

On the other hand, it is preferable to use a high-magnification and high-resolution objective lens in the lens unit 230, and in the present invention, an objective lens with a maximum magnification of 40 is used. Through this, it is possible to take an image of microalgae flowing in the flow path inside the flow cell 130 seated on the stage unit 220, and as an embodiment of the present invention, by using the objective lens, an image of microalgae less than 10 μm in size can also be measured.

The lighting unit 210 is located on top of the stage unit 220 and emits light downward from the top of the inverted microscope. When light emitted from the lighting unit 210 passes through the flow cell 130 , the lens unit 230 photographs the inside of the flow cell 130 receiving light from the lighting unit 210 . In detail, the lens unit 230 can capture the intermediate flow path 132 located inside the flow cell 130, and more specifically, referring to FIG. 9, the second width of the intermediate flow path 132 A sample of microalgae flowing in the middle side intermediate passage 132.2 is photographed.

On the other hand, as the magnification of the objective lens used in the lens unit 230 increases, the working distance becomes shorter, which may cause difficulty in focusing. That is, when the upright microscope is used in the present invention, since the flow path is located at the bottom of the PDMS chip of the flow cell 130, the thickness of the flow cell 130 can reduce the working distance when a high-magnification lens is used. Since the problem of exceeding occurs, the present invention uses the inverted microscope in consideration of structural and functional features. The working distance means the vertical distance between the surface of the objective lens and the slide glass when focus is achieved. Therefore, by using the inverted microscope, a higher magnification objective lens can be used than when using an upright microscope, and through this, microalgae images can be stably captured.

The stage part 220 is located in the middle of the inverted microscope, that is, between the lighting part 210 and the lens part 230, and is a part for placing the flow cell 130 thereon. As shown in FIG. 10, two flow cells 130 can be placed on the stage unit 220, and a lens unit 230 capable of taking pictures of each flow cell 130 and a camera are provided on the stage unit ( 220) to be installed on the lower part respectively. The lens unit 230 may have objective lenses of different magnifications, each objective lens is connected to the camera, and the camera is connected to the computer, so that the microalgae image captured through the camera is It is collected by an image analysis program installed on the computer. In addition, as an embodiment of the present invention, the two flow cells 130 may include a plurality of passages having different widths therein, and through the plurality of passages, different magnifications may be obtained for one microalgae sample. It is possible to exert the effect of acquiring the image of microalgae.

The stage part 220 includes a through hole therein, and the flow cell 130 is seated on top of the through hole. The slide glass of the flow cell 130 is seated facing the lens unit 230, and the flow cell 130 is mounted on the stage unit (not shown) through a fixing device (not shown) included in the stage unit 220. 220) can be fixed. A small movement of the transfer pump 140 can be controlled through the fixing device, so that a clear image of microalgae can be obtained. As an embodiment of the present invention, preferably, the middle side intermediate flow passage 132.2 inside the flow cell 130 may be installed to be placed in the center of the through hole.

The lens unit 230 can install two or more objective lenses of one microscope 200, and each objective lens can use a different magnification, so that birds having different sizes can be photographed simultaneously, or the same For an algae of the same size, it may be photographed widely through one low magnification objective lens so that many algae cells can come out, or a plurality of algae cells may be photographed in detail one by one using a high magnification objective lens. Also, the lens unit 230 may move up and down to focus, and to focus, the lens unit 230 may be inserted into the through hole.

Although the present invention has been described with reference to the embodiments shown in the drawings, this is only exemplary, and those skilled in the art will understand that various modifications and equivalent other embodiments are possible therefrom. For example, the described techniques may be performed in a different order than the described method, and/or the described devices, structures, devices, circuits, etc. may be combined or combined in a different form than the described method, or other components may be used. Or even if it is replaced or substituted by equivalents, appropriate results can be achieved. Therefore, the true technical protection scope of the present invention should be defined by the technical spirit of the attached claims.

Claims

Microalgae image continuous measuring device,

A filter unit for removing contaminants included in the microalgae sample;

a dilution unit supplying a solvent to the microalgae sample to control the turbidity of the microalgae;

An ultrasonic disperser for dispersing the microalgae by irradiating ultrasonic waves to the microalgae sample with ultrasonic intensity for each type of microalgae for pretreatment of the microalgae sample;

a flow cell through which the microalgae sample to which ultrasonic wave is applied by the ultrasonic disperser flows;

a microscope for continuously photographing the microalgae sample inside the flow cell; and

Microalgae image continuous measurement device comprising a; transfer pump for transferring the microalgae sample from the filter unit to the flow cell.
The method of claim 1,

The dilution part,

A turbidimeter for measuring the turbidity of the microalgae sample;

A dilution solvent capable of diluting the microalgae sample based on the turbidity measured by the turbidimeter; and

A dilution chamber for diluting the microalgae sample using the dilution solvent;

The dilution solvent includes distilled water, and may optionally further include a chemical agent for immobilizing microalgae, microalgae image continuous measuring device.
The method of claim 1,

The ultrasonic disperser,

A calibration step of deriving an energy unit corresponding to the input current or a device driving value for the input energy unit;

Microalgae image continuous measurement device for performing; device driving step of driving the ultrasonic diffuser with the device driving value derived in the calibration step.
The method of claim 3,

In the calibration step,

a first measurement step of driving the ultrasonic diffuser with a first device driving value, applying ultrasonic waves to a reference medium, and measuring first change information of the temperature of the reference medium;

a second measurement step of driving the ultrasonic diffuser with a second device driving value, applying ultrasonic waves to the reference medium, and measuring second change information of the temperature of the reference medium;

a first derivation step of deriving a first energy unit for a first device driving value based on the first change information;

a second derivation step of deriving a second energy unit for a second device drive value based on the second change information; and

Based on the regression information including the first device drive value, the second device drive value, the first energy unit, and the second energy unit, deriving relationship information between the device drive value and the energy unit; Including,

Microalgae image continuous measuring device for deriving the ultrasonic intensity for each type of microalgae based on the relational information between the device drive value and the energy unit.
The method of claim 4,

In the device driving step,

Microalgae image continuous measurement device for dispersing microalgae aggregate floc by irradiating ultrasonic waves to the microalgae sample for a predetermined time with the ultrasonic intensity derived in the calibration step.
The method of claim 1,

The flow cell includes one or more flow paths,

Each of the one or more flow passages,

An inlet passage formed in a vertical direction, a middle passage extending horizontally from the inlet passage, and an outlet passage extending vertically from the middle passage,

Microalgae image continuous measuring device wherein the inner surface of the intermediate flow path is coated with nanomaterials to alleviate the residual algae adhesion problem.
The method of claim 6,

The middle flow path,

an inlet-side intermediate passage having a first width;

a middle-side intermediate passage extending from the inlet-side intermediate passage and having a second width;

An outlet-side intermediate passage extending from the middle-side intermediate passage and having a third width;

The second width is greater than the first width and the third width, microalgae image continuous measuring device.
The method of claim 1,

The microscope,

lighting unit;

a stage unit disposed below the lighting unit and having a through hole therein; and

A lens unit located below the stage unit and movable up and down; includes,

The flow cell is seated on the upper surface of the stage part, and the lens part can be introduced into the through hole, microalgae image continuous measuring device.