• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
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  • 优先权
    • 专利摘要:
    The present invention consists of a method for estimating the microalgal growth of a culture in a precise, fast and inexpensive way, avoiding problems derived from conventional counting systems (time consumed, need of the same person for visual counts or expensive equipment). The methodology object of the invention is based on the capture and subsequent treatment of images for the determination of the cell density or number of cells of a culture for the industrial production of microalgae, which comprises the steps: a) Capture and acquire digital images of microalgae culture. b) Process the digital images in order to extract the measurable parameters median, media and fashion of each image. c) Correlate the modal, average and average values extracted from the different color models, with the cell density or canteo obtained experimentally. d) Selection of the representative function of algal growth.
    公开号:ES2685220A2
    申请号:ES201700447
    申请日:2017-03-30
    公开日:2018-10-05
    发明作者:Ángel Manuel SÁNCHEZ BERMÚDEZ；Bran TOURIÑO ANDRADE；María Ángeles CANCELA CARRAL；José Luis SALGUEIRO FERNÁNDEZ；Leticia PÉREZ RIAL
    申请人:Universidade de Vigo；
    IPC主号:
    专利说明:

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    DESCRIPTION
    METHOD FOR ESTIMATING MICROALGAL GROWTH
    SECTOR OF THE TECHNIQUE
    The present invention relates to a methodology to be followed for the estimation of microalgal growth, in terms of cell number or cell density, by taking digital photographs of cultures and their subsequent treatment. The invention is applicable to any laboratory, research center or company that carries out microalgae cultures and that seeks to control their growth in an economical, simple, accurate and fast way. By using this technique, the phase in which a crop is found can be more accurately and agilely known.
    BACKGROUND OF THE INVENTION
    Currently there are two conventional systems that are the most used to carry out the cell count: the hematocytometer or Neubauer chamber and the Coulter counter.
    The hematocytometer or Neubauer chamber is based on the visual count, being a thick glass slide-shaped plate, about 30x70 mm and about 4 mm thick which is divided into 3 parts. In most cases, the central part of the plate has 2 counting zones (double chamber), one upper and one lower than the longitudinal axis of the chamber. To make a count with this system it is necessary to take a sample of the culture (approximately 10 pL) and deposit it on top of the chamber covering with a coverslip. This assembly is taken under a microscope and the visual count is carried out. The great disadvantages of this system is that the counts must always be carried out by the same person (otherwise there would be errors in the measurement) and that it is a technique that consumes a lot of time.
    As for Coulter counters, they are microchannel-based devices that connect two chambers filled with an electrolyte solution on which a difference is applied.
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    of potential The solution is forced to flow from one chamber to another, dragging along the particles that are in suspension. The meter detects changes in electrical resistance and based on this determines the number of cells present in a defined volume of fluid. The main problem is associated with the high prices of these equipment.
    In recent years, other methodologies have also been developed whose purpose is to provide more reliable, faster and cheaper systems than the traditional methods cited.
    Document CN103955937 (A) collects an automatic counting method based on digital image processing. It carries out the realization of photographs through a microscope that are later enlarged to be treated and converted to grayscale, then improving the contrast of the images and filtering through the median using statistical methods based on the variance
    Document FR3002355 (A1) contains a method for the determination of the cell density of photosynthetic microorganisms according to a chrominance and luminance component by means of photographs taken to the cultures and subsequent treatment and calculation of the derivative of the temporal evolution of the component color over time.
    Document CN101694724 (A) focuses on a method for locating microalgae only with a circular structure from the capture of micro-images thanks to a micrograph capture device and the creation of a database. However, if the algae present in the micrograph has a different shape than the circular one, it would not be taken into account by the proposed system.
    Document CN101604330 (A) collects a method for the determination of only circular microalgae based on their surface texture by microscopic imaging. Like the previous document, this method would only be valid to identify circular microalgae.
    In view of the cited documents, the present invention aims at estimating
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    microalgal growth in terms of cell density or the number of cells existing only by taking photographs and subsequent treatment of them thanks to the relationship between the color of the culture and the cell density of the same, not influencing the shape of the microalgae present and not necessary equipment such as a microscope.
    EXPLANATION OF THE INVENTION
    In the present invention, "counting" is understood as the method used to estimate the number of microalgal cells present in a given culture volume and is usually expressed as cell / pL. In order to perform said determination, the aforementioned conventional systems are used (chamber of Neubauer or Coulter counter.) On the other hand, "cell density" means the amount of algal biomass present in a given crop volume and is usually expressed as g / L. In order to make this determination, a certain volume of culture is extracted from which the biomass is separated (by filtration, centrifugation, etc.) and subsequently dried to constant weight.
    In order to achieve the objectives and avoid the inconveniences mentioned in the previous sections, the invention proposes a methodology for the determination of the cell density or number of cells existing in a culture for the industrial production of microalgae, thanks to the capture of images digital color and subsequent treatment of them. It is a simple and economical method that includes the following stages:
    a) Capture and acquire digital images of microalgae culture;
    b) Process the digital images in order to extract the median, average and fashion measurable parameters of each image that allow the color to be correlated with the cell density or count values measured directly from the microalgae culture;
    c) Correlate the modal, average and average values extracted from the different color models, with the cell density or count obtained experimentally, in order to obtain the slope of the function that relates to both.
    d) Obtaining the representative function of algal growth such as that
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    function that best fits the experimental values obtained, specifically the estimation of algal growth will correspond to the function that presents a higher coefficient of determination (R2) and a lower error, among all the functions obtained for the mean, mode and median in the decomposition of images.
    A preferred embodiment, the capture of color images should be performed under the same conditions throughout the days of cultivation (same position, inclination, lighting and distance). The images taken must comprise a minimum resolution of 4608x2592 pixels.
    Another preferred embodiment, step b) of image processing in turn comprises the following steps:
    i) Upload and crop images obtained in stage a);
    ii) Decompose the images into their primary colors (RGB);
    iii) Transform the images to grayscale;
    iv) Convert the RGB decomposition values (red, green and blue) to decomposition values in the CMYK printing model (cyan, magenta, yellow and black);
    where stages ii), iii) and iv) an extraction of median values of median, medium and mode is performed for each image decomposition.
    Another preferred embodiment is that extraction of measurable values of median, average and mode for each image decomposition of stages ii) and iii) above must be between 0 and 255.
    With this invention, the usual errors that occur when a visual count is performed using a microscope are avoided, and it is not necessary for the same person to take the photographs as in the visual count. Its main use is that it serves to count all types of mlcroalgae regardless of their shape or size. In addition, the time required to carry out a count or determination of cell density is reduced, the immediate treatment of the images not being necessary. On the other hand, the use of the present invention would result in savings with respect to any of the methods mentioned above since only a device for taking images and a
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    computer.
    BRIEF DESCRIPTION OF THE DRAWINGS
    To complement the description that is being made and in order to help a better understanding of the characteristics of the invention, a set of drawings is attached as an integral part of said description, where illustrative and non-limiting nature has been represented. next:
    Figure 1.- Shows a diagram that illustrates the steps that must be followed to carry out the methodology to be patented.
    Figure 2.- Shows an example illustration of the average obtained from an image that has decomposed to RGB with their respective coefficients of determination for each channel.
    Figure 3.- Shows an example illustration of the median obtained from an image that has decomposed to RGB with their respective coefficients of determination.
    Figure 4.- Shows an example illustration of the fashion obtained from an image that has decomposed to RGB with their respective coefficients of determination.
    Figure 5.- Shows an example illustration of the average obtained from an image that has decomposed to CMYK with their respective coefficients of determination.
    Figure 6.- Shows an example illustration of the median obtained from an image that has decomposed to CMYK with their respective coefficients of determination.
    Figure 7.- Shows an example illustration of the fashion obtained from an image that has decomposed to CMYK with their respective coefficients of determination.
    Figure 8.- Shows an example illustration of the mean, median and mode
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    obtained from an image that has decomposed to grayscale, and presents its respective coefficients of determination.
    PREFERRED EMBODIMENT OF THE INVENTION
    As indicated, the method for estimating the microalgal growth object of the invention, in its preferred embodiment, comprises the following steps:
    1. CAPTURE AND ACQUISITION OF DIGITAL IMAGES.
    The taking of images is carried out by means of a digital camera (1) which must remain fixed or always be placed in the same position so as to allow the taking of images in the same conditions throughout the days of cultivation (same position, inclination, lighting and distance). The images taken must be made with a minimum resolution of 4608x2592 pixels.
    2. PROCESSING IMAGES
    For the processing of the digital images taken, the Matlab ver software tool can be used. R2016a. From this software, the following functions are performed:
    - Load the images taken by the camera.
    - Crop images in regions of interest (2).
    - Decomposition of images in their primary RGB colors (3).
    - Transformation of grayscale images (3).
    - Conversion of the RGB model to the CMYK model (3).
    - Extraction of measurable values of decompositions (5).
    • Upload images taken by the camera:
    The Image Processing toolbox is activated and the code is written to load the images directory. Matlab considers the images as matrices in which each point of the matrix is composed of each pixel of the image and therefore, the matrix will have a size of 4608x2592.
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    It is advisable to load the images in .jpeg format in the RGB model.
    • Crop images in regions of interest (2):
    Each image taken is trimmed so that the area of interest is reduced to a rectangular region (ROI) of 120 rows and 60 columns, constituting a total of 7200 pixels of RGB that covered the section of the equipment where the crop under measurement is located .
    • Decomposition of images in their primary RGB colors (3):
    Once the ROI region is defined and for the decomposition process to be more
    Fast accuracy of images is reduced. Subsequently, the
    following commands to obtain the reduced R, G and B planes of the images and
    in turn, the matrix of each image is transformed into a vector:
    red {k} = single (reshape (crop (k} (:, 1), 1, [])).
    green {k) = s¡ngle (reshape (crop {k) (:.: .2), 1, [])). bluejk] = s¡ngle (reshape (crop (k) (:, 3), 1, [])).
    Where "k" is represented as the variable that defines the set n images of the
    species studied in its growth process.
    • Conversion of the RGB model to the CMYK model.
    For the conversion of the RGB model to the CMYK model it is necessary to use an intermediate CMY value since the scales of both models are different. While in the RGB model its scale varies from 0 to 255 in the CMYK model its scale varies from 0 to 1. To do this, the steps to follow are as follows:
    First, the scale of the values of the decompositions R (red), G (green) and B (blue) obtained in Matlab is varied. For this it is necessary to divide the median, average and mode values collected by the maximum value of the scale (255) and thus obtain values in scale from 0 to 1:
    R '=
    R
    image 1
    B '=
    B
    255
    255
    255
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    Where R represents the red component of a color image, G represents the green component of a color image and B represents the blue component of a color image. R ', G' and B 'represent the new values of the decompositions R, G, B passed to scale of the CMYK model.
    The black component of a color image Identified as K, of the CMYK model, is equivalent to the difference between the maximum possible value on the working scale (1) minus the maximum value of the vector formed by the values R ', G', B 'previously calculated:
    K = 1 - max (R ', G', B ')
    Once this "K" value is obtained, the conversion of the CMY values of this new model can be carried out as follows:
    (l-R'-K) (l-G'-K) (1 - B '- K)
    C "(i - fQ (í-ff) (i -«)
    Where C represents the cyan component of a color image, M represents the magenta component of a color image, Y represents the yellow component of a color Image, K represents the black component of a color image and R ', G' and B 'represent the new values of the decompositions R, G, B passed to the scale of the CMYK model.
    • Transformation of grayscale images:
    The following command is entered:
    gray {k} = s i ngIe (reshape (rgb2gray (crop {k}), 1, [])).
    • Extraction of medial values of decompositions (5):
    It is necessary to extract measurable parameters that allow correlating the images taken with the cell density or counting values measured directly from the culture. The parameters used in this design are: medium, medium and mode of each image.
    The commands used to obtain the values of the decomposition mean
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    RGB, grayscale and CMYK are:
    meanredlk} = mean (red {k)): meangreenfk] = mean (green {k}); meanblue {k) = mean (blue {k}); meangray {kl = mean (gray {k)); meancyan {k) = mean (cyan {k}); meanmagenta {k} = mean (magentaík)); meanye11ow | k} = mean (ye 11ow {k}): meanblack (k) = mean (black {k}):
    The commands to be used to obtain the median values are:
    medianredlk] = median (red {k}). mediangreenfkl = median (green (k}). med i anbIue | k} = med i an (bIue {k}). mediangray {k} = median (gray {k}). mediancyan (k) = median (cyan {k }). medium-sized {k) = medium-sized (magenta {k |). med i anye 11ow | k} = med i an (ye 11ow Ikl). med i anbIack {k] = med i an (bIack {k}).
    Finally, the commands used to obtain the value corresponding to fashion
    They are as follows:
    modered (k) = mode (red {k}); modegreen (k) = mode (green (k)): modebluelk) = mode (blue (k}): modegraylk) = mode (gray (k (): modecyan (k} = mode (cyan (k)): modemagenta { k} = mode (magenta (k}): modeye11ow (k} = mode (ye 11ow {k}): modebIack {k} = mode (bIack (k));
    Matlab returns a single median, average and mode value for each image decomposition.
    3. CORRELATION OF MICROALGAL GROWTH
    Once all the digital image processing has been carried out, a comparison is made between the cell count or density values measured directly from the culture and the modal, average and average values of each decomposition of the images obtained, so that by correlating them we obtain the slopes of a series of functions, as can be seen in figure 2, figure 3, figure 4,
    Figure 5, Figure 6, Figure 7 and Figure 8, which are provided by way of demonstrative example.
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    4. OBTAINING THE REPRESENTATIVE FUNCTION OF MICROALGAL GROWTH
    Once the different functions of mean, median and mode of the color models are obtained, the coefficients of determination (R2) are analyzed, so that the microalgal growth will be characterized by the function that presents the least error. In the example provided, depending on the coefficient of determination (R2), the microalgal growth is adjusted both to the median function (curve 10) of the G (Green) channel of the RGB color model (figure 3) and to the median (curve 21) of the K (Black) channel of the CMYK color model (figure 6).
    Currently it is not known that any similar methodology is implemented at the state level in any type of industry dedicated to the cultivation of microalgae. This fact has led to an analysis of the existing problem with the current systems of cell count and density determination allowing the development of the methodology object of the invention. The experimental results obtained throughout the research project have allowed the validation of this methodology.
    权利要求:
    Claims (6)
    [1]
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    1. Method for estimating microalgal growth comprising the following stages:
    a) Capture and acquire digital images of microalgae culture;
    b) Process the digital images in order to extract the median, median and fashion medial parameters of each image that allow the color to be correlated with the values of cell density or count measured directly from the microalgae culture;
    c) Correlate the modal, average and average values extracted from the different color models, with the cell density or count obtained experimentally, in order to obtain the slope of the function that relates to both.
    d) Obtaining the representative function of algal growth as that function that best fits the experimental values obtained, specifically the estimation of algal growth will correspond to the function that presents a higher coefficient of determination (R2) and a lower error, of among all the functions obtained for the mean, fashion and median in the decomposition of images.
    [2]
    2. Method according to claim 1, wherein step a) of image capture is characterized in that the captured images must comprise a minimum resolution of 4608x2592 pixels.
    [3]
    3. Method according to claim 1, characterized in that step b) of image processing in turn comprises the following steps:
    v) Upload and crop images obtained in stage a);
    vi) Decompose the images into their primary colors (RGB);
    vii) Transform the images to grayscale;
    viii) Convert the RGB decomposition values (red, green and blue) to decomposition values in the CMYK printing model (cyan, magenta, yellow and black);
    where stages ii), iii) and iv) an extraction of median values of median, medium and mode is performed for each image decomposition.
    [4]
    4. Method according to claim 3, characterized in that the median values,
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    average and mode for each image decomposition of stages ii) and ¡i¡) must be between 0 and 255.
    [5]
    5. Method according to claim 3, characterized in that in step iv) the conversion of the RGB values to CMYK values involves the use of an intermediate CMY value which in turn comprises the following steps:
    - Divide the median, average and mode values collected by the maximum value of the scale (255) and thus obtain values on a scale of 0 to 1 characterized in that it is obtained according to the following equations:
    255
    255
    B '
    B
    255
    Where R represents the red component of a color image, G represents the green component of a color image and B represents the blue component of a color image. R ', G' and B ‘represent the new values of the decompositions R, G, B passed to the CMYK scale.
    - Calculate the black component of a Color Image identified as K, of the CMYK model is characterized by that it is obtained according to the following equation:
    K = 1 - max (R ', G', B ')
    - Conversion of CMY values to the new model characterized by the following equations:
    (l-R'-i O (l-G'-R) (l-B'-K)
    (1- / 0 (1- / 0 “(1 -K)
    Where C represents the cyan component of a color image, M represents the magenta component of a color image, Y represents the yellow component of a color image, K represents the black component of a color image and R ', G' and B 'represent the new values of the decompositions R, G, B passed to the scale of the CMYK model.
    [6]
    6. Use of the method according to any of the preceding claims, characterized in that the count of all types of microalgae is used regardless of their shape or size.
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    同族专利:
    公开号 | 公开日
    ES2685220R1|2018-11-14|
    ES2685220B1|2019-10-21|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    JP2011182731A|2010-03-10|2011-09-22|Incorporated Educational Institution Meisei|Automatic apparatus for measuring growth yield of algae|
    CN102517372B|2011-12-09|2013-05-08|厦门大学|Method for rapidly determining fat content of microalgae cells|
    JP5990127B2|2013-04-08|2016-09-07|日本電信電話株式会社|Method, apparatus and program for determining the concentration of microalgae|
    CN103955937A|2014-05-15|2014-07-30|福州大学|Microalgae automatic counting method based on digital image processing|
    CN105738364B|2015-12-28|2018-08-17|清华大学深圳研究生院|Silastic surface algal grown degree measurement method and device based on image procossing|
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