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CELL OBSERVATION DEVICE, CELL OBSERVATION METHOD AND PROGRAM THEREOF meetings

Foreign code F150008129
File No. AF30-01WO
Posted date Feb 12, 2015
Country WIPO
International application number 2013JP081894
International publication number WO 2014084255
Date of international filing Nov 27, 2013
Date of international publication Jun 5, 2014
Priority data
  • P2012-259880 (Nov 28, 2012) JP
Title CELL OBSERVATION DEVICE, CELL OBSERVATION METHOD AND PROGRAM THEREOF meetings
Abstract A cell observation device comprising: an outline extracting section for extracting edge pixels from a cell image in a captured image of single layered cells and synthesizing an edge image consisting of the edge pixels thus extracted; a plastid region extracting section for extracting pixels of plastid of the cell image in the captured image and synthesizing a plastid image consisting of the plastid pixels thus extracted; and an image synthesizing section for, in a synthesized image obtained by overlapping the edge image and the plastid image together, detecting a cell image region and a background image region in the captured image on the basis of the pixel luminance value distribution and thus detecting the cell image region in the captured image.
Outline of related art and contending technology BACKGROUND ART
In recent years, when detecting the status of a cell, a live state cells can be observed by a polarization microscope over a long period of experimental apparatus has been put to practical use. By using such an experimental apparatus, such as cell growth or cell division can be observed in real time the course. Then, the progress of the change of these cells to capture image data of the resulting cells by analyzing the time series, and detail analysis changes occurring in the cell becomes possible.
For example, fermentation was performed using a unicellular yeast, beer, alcoholic beverages having a variety of shochu can be manufactured. Is for maintaining the quality of an alcoholic beverage, the yeast used for fermentation such physiological states determined before fermentation, it is possible to predict the effect of subsequent to the fermentation is performed (for example, see Patent Document 2). That is, yeast fermentation, brewing substance production and the like, from which the production of a biological condition of contemplated for use in yeast cells is ascertained in advance, foreseeing the success or failure of the fermentation, and stabilized products having high quality is required in order to obtain.
Microalgae, mainly refers to unicellular photosynthetic organisms. This microalgae is, by photosynthesis, light energy is converted into chemical energy, self-energy conversion of the figure is used for survival and growth. In addition, the microalgae is, depending on the type, the hydrocarbon and the essential unsaturated fatty acid as a useful component (for example DHA (Docosa Hexaenoic Acid), such as EPA (Eicosa Pentaenoic Acid)), starch, and is combined with the raw pigment and the like, function in the biosynthesis of these industrial application has been expected.
The microalgae using a highly efficient production of a useful component in future, the physiological state of the microalgae cells it is important to properly grasp. That is, the physiological state of the microalgae cells, media composition, carbon dioxide concentration, light intensity, culture temperature, cell density of the surrounding environment such as changes greatly depending on the growth conditions. Then, the microalgae cells, the production of a useful component in accordance with the physiological conditions and the stored quantity also changes in some cases.
Therefore, a useful component by cells of microalgae for the production of a high-efficiency, optimization of the conditions of the growth of cells in the course of, a useful component in the course of the production by the cells also also, microalgae in culture to grasp the state of the metabolic, useful component is necessary to know the amount of production. In addition, by culturing the cell of the organism are mixed into the medium of the other type of microalgae influence the physiological state of cells is. Other types of physiological conditions due to the intrusion of the influence on the organism, a useful component by the cells are not uncommon problem in the production run. Therefore, the above-described culture into the culture medium for the detection of other types may be mixed, with the cells of the microalgae is a highly efficient for the production of a useful component becomes important.
As an example of the microalgae cells, and a kind of microalgae Haematococcus pluvialis. Is this Haematococcus pluvialis, is also used as a health food red anti-oxidant astaxanthin biosynthesis can be industrially very useful. Is Haematococcus pluvialis, reflects the physiological state of cells according to the various cell lines. The amount of accumulation of astaxanthin, varies depending on the culture conditions (for example, see non-patent document 1).
To this end the production of astaxanthin by Haematococcus pluvialis, the use of high-producing strain or culture condition optimization has been carried out (for example, see Patent Document 1). However, in the present situation is as not considered to have a high, a further improvement in productivity is expected. Further, in recent years, other types of parasitic Haematococcus pluvialis as an example of a fungal organism Paraphysoderma sedebokerensis is found. Haematococcus pluvialis Paraphysoderma sedobokerensis is infected, a dark brown color change from green cells, leading to death (for example, see non-patent document 2).
Haematococcus pluvialis have been identified and a plurality of the culture, a plurality of types of ordered from all over the world, organism other than the mixed Haematococcus pluvialis was examined. As a result, it has been surprisingly found, industrially used mixed in culture and contains all of the strains was confirmed. Therefore, the physiological state of cell Haematococcus pluvialis, a useful component of astaxanthin accumulation amount, to a mixing rate of the organism from other species can be grasped, a critical issue in industrial use.
As a method to grasp the state of the metabolic, for budding yeast microorganism, such as the evaluation method of discrimination techniques of killing by methylene blue method (for example, see non-patent document 3) have been known. However, in the method according to this Non-Patent Document 3, the physiological state of cells cannot be determined various aspects. In addition, the patent document 2, the quantitative value of the cell morphology of a physiological condition of yeast used evaluation method is shown. Specifically, the contour of the object of the yeast cell, nucleus, and fluorescent staining of the actin cytoskeleton and the image is subjected to image analysis, yeast cell morphological feature set form in advance based on the parameter, a value obtained by quantitative analysis of cell morphology, a database prepared in advance the value by comparing the, for the purpose of assessing a biological condition of yeast.
However, in this method cells fixed, coloring treatment and which require observation by fluorescence microscopy, such as in a production site for field and is not suitable for real-time of a physiological condition of grasp. The microalgae cells to incorporate not suggest no consideration. In addition, as a method for detecting an accumulation amount of the useful component, in patent document 1, the amount of astaxanthin Haematococcus pluvialis, extracted from the cells by the plastid dimethyl sulfoxide, 750 nm 492 nm and quantitated by measuring the absorbance of the method is shown. However, a large number of microalgae cells can be measured with the extraction of the color bodies from the required, time is required for the extraction of the plastid.
In addition, for the detection of other types, diatoms microalgae cells in the parasitic fungus to the pot of fungi, a component of the pot cell walls of fungi calcofluor white chitin binding specifically to a method of coloring is shown (non-patent document 4). In addition, according to another prior study, the capsular bag of a plant parasitic Haematococcus pluvialis Paraphysoderma sedebokerensis and stained with FITC-WGA(non-patent document 5).
However, any of these, and the processing and staining of the cells, cells in culture is not directly determined. Also, such as Patent Document 2 and non-patent document 5 in the method, the need for a fluorescence microscope, such as for field and is not suitable for the determination at a production site. Or more as mentioned above, the growing condition of the cells of the microalgae, the microalgae cells in the culture medium of other contaminating species such as biological (for example flora) mixed state, the amount of production of microalgae cell by the cells of each of the useful substance to monitor in real time is not a simple method.
Scope of claims (In Japanese)請求の範囲 [請求項1]
 一層の細胞を撮像した撮像画像から、当該撮像画像内の細胞の画像のエッジの画素を抽出し、抽出したエッジの画素からなるエッジ画像を生成する輪郭抽出部と、
 前記撮像画像における細胞の画像の色素体の画素を抽出し、この抽出した色素体の画素からなる色素体画像を生成する色素体領域抽出部と、
 前記エッジ画像と前記色素体画像とを重ね合わせて合成された合成画像において、前記撮像画像における細胞の画像領域と背景の画像領域とを、前記画素の輝度値の分散により検出し、前記撮像画像における細胞の画像領域を検出する画像合成部と
 を備えることを特徴とする細胞観察装置。

[請求項2]
 前記合成画像において色素体の存在する細胞の画像領域を対象細胞画像とし、色素体の存在しない細胞の画像領域を非対象細胞画像として検出し、前記合成画像における全細胞の画像における当該非対象細胞画像の比率を求める細胞形態検出部
 をさらに備えることを特徴とする請求項1に記載の細胞観察装置。

[請求項3]
 前記細胞の画像領域における色素体の輝度値から前記色素体の量である色素体量を算出する色素値抽出部
 をさらに備えることを特徴とする請求項1または請求項2に記載の細胞観察装置。

[請求項4]
 前記細胞の画像領域から色素体の平均輝度値を求めておき、前記撮像画像を撮像した当該細胞から色素体を抽出して色素体量を求め、平均輝度値と細胞当たりの色素体量との回帰式を予め求めて記憶部に記憶させておき、
 前記色素値抽出部が前記細胞画像における平均輝度値を求め、前記回帰式から細胞当たりの色素体量を求めることを特徴とする請求項3に記載の細胞観察装置。

[請求項5]
 輪郭抽出部が、一層の細胞を撮像した撮像画像から、当該撮像画像内の細胞の画像のエッジの画素を抽出し、抽出したエッジの画素からなるエッジ画像を生成する輪郭抽出過程と、
 色素体領域抽出部が、前記撮像画像における細胞の画像の色素体の画素を抽出し、この抽出した色素体の画素からなる色素体画像を生成する色素体領域抽出過程と、
 画像合成部が、前記エッジ画像と前記色素体画像とを重ね合わせて合成された合成画像において、前記撮像画像における細胞の画像領域と背景の画像領域とを、前記画素の輝度値の分散により検出し、前記撮像画像における細胞の画像領域を検出する画像合成過程と
 を含むことを特徴とする細胞観察方法。

[請求項6]
 細胞の形状を観察する細胞観察装置の動作をコンピュータに実行させるプログラムであり、
 コンピュータを、
 一層の細胞を撮像した撮像画像から、当該撮像画像内の細胞の画像のエッジの画素を抽出し、抽出したエッジの画素からなるエッジ画像を生成する輪郭抽出手段、
 前記撮像画像における細胞の画像の色素体の画素を抽出し、この抽出した色素体の画素からなる色素体画像を生成する色素体領域抽出手段、
 前記エッジ画像と前記色素体画像とを重ね合わせて合成された合成画像において、前記撮像画像における細胞の画像領域と背景の画像領域とを、前記画素の輝度値の分散により検出し、前記撮像画像における細胞の画像領域を検出する画像合成手段
 として機能させるためのプログラム。

  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
  • Inventor
  • OHYA Yoshikazu
  • KAWANO Shigeyuki
  • NOGAMI Satoru
  • OHNUKI Shinsuke
  • OTA Shuhei
  • WATANABE Koichi
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JP KE KG KN KP KR KZ LA LC LK LR LS LT LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN TD TG
Reference ( R and D project ) CREST Creation of Basic Technology for Improved Bioenergy Production through Functional Analysis and Regulation of Algae and Other Aquatic Microorganisms AREA
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