WO1998005942A1 - Procede servant a accroitre la precision d'images microscopiques et appareil associe - Google Patents

Procede servant a accroitre la precision d'images microscopiques et appareil associe Download PDF

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Publication number
WO1998005942A1
WO1998005942A1 PCT/CN1997/000079 CN9700079W WO9805942A1 WO 1998005942 A1 WO1998005942 A1 WO 1998005942A1 CN 9700079 W CN9700079 W CN 9700079W WO 9805942 A1 WO9805942 A1 WO 9805942A1
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WO
WIPO (PCT)
Prior art keywords
microscope
image
ccd camera
value
cells
Prior art date
Application number
PCT/CN1997/000079
Other languages
English (en)
Chinese (zh)
Inventor
Kangting Gu
Maqi Shi
Original Assignee
Kangting Gu
Maqi Shi
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kangting Gu, Maqi Shi filed Critical Kangting Gu
Publication of WO1998005942A1 publication Critical patent/WO1998005942A1/fr

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Classifications

    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G02B21/365—Control or image processing arrangements for digital or video microscopes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G02B21/08—Condensers
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G02B21/362—Mechanical details, e.g. mountings for the camera or image sensor, housings

Definitions

  • the present invention relates to a system for improving the quantitative analysis of microscopic images of biological standard ice and accurate analysis of microscopic images. Specifically, the invention relates to a method for calculating the microscopic images captured by mx to freeze, store and manage, Carry out qualitative and quantitative analysis of molecular and cell-level features for each image, making it suitable for scientific research, education, and a microscopic image analysis system that serves daily pathological work.
  • the microscope uses the qualitative Kohler illumination method, the uniformity of the light intensity in the field of view in the space cannot be guaranteed.
  • the CAS-200 uses a computer to prepare the image.
  • the pixel-by-point correction software makes the picture selected by the tested cells only occupy about 60% of the imaging area of 3 ⁇ 4 ⁇ . And the correction software takes up a lot of memory space.
  • the purpose of the present invention is to overcome the shortcomings of the prior art, improve the accuracy of microscopic image analysis, repeatability, easy-to-operate quantitative analysis, and a microscopic image analysis system.
  • the present invention also achieves:
  • the method of the present invention includes establishing a 256-level grayscale illumination for a microscope in a microscopic image analysis system and establishing a DA value of 7. 18Pg (7.18X 10-12 grams) reference weight.
  • Step the present invention establishes a 256-level grayscale illumination method, which consists of a system including a microscope, a gain CCD camera, a computer system including an image acquisition card, a high-rate imager, and a software 256-degree test card Use the following: ⁇ Complete 256 3 ⁇ 4 3 ⁇ 4 quantity illumination, standard disturbance state for quantitative analysis of biological cells, according to the order of colors: 1. Select the luminous source: tungsten halogen lamp, the geometry of the filament is «JI"" Or W-shaped; 2. Keep the power supply with high stability: The output voltage is one thousandth of the rated voltage, and the stability of the output voltage is equal to or less than one-thousandth of the rated voltage. 3. Adjust
  • Level 180 ⁇ 6. Determine the standard theorem illumination value: The grayscale quantitative value of the display is 100 ⁇ 130; 7. Use the imager to check the parts that do not meet the standards: Correct the interior of the microscope that does not meet the standards ⁇ I, including the tube of the video interface Wall reflection flare phenomenon; 8. ⁇ E for standard quantitative lighting value: Measured with an area value of 20 ⁇ 50 ⁇ m 2 , so that ⁇ 3 ⁇ 4 is not greater than 2%. Moreover, the number of turns of the ⁇ 3 ⁇ 40 1 ⁇ 2 ⁇ 3 ⁇ 4 filament 3 ⁇ 43 ⁇ 4 is 7-9 turns.
  • the microscopic image analysis system of the present invention includes a biological microscope, a CO camera, a high-performance power supply, a PC, a host, a true-color image processing card, an image display, and a printer.
  • the microscope is equipped with a binocular binocular lens barrel, and a black and white CCD camera and a color CCD camera are respectively installed on the lens barrel.
  • the interface of the color CCD camera is equipped with a 1: 0.5 scale video connector to form a microscope camera system, of which color
  • the CCD camera and the black-and-white CCD camera are respectively connected to the host of the PC, and the output ends of the host are respectively connected to the color imager and the printer.
  • the 256-level gray-scale illumination method of the microscopic image is used to ensure the uniform light intensity in the field of view in space.
  • the background background color is not greater than the percentage of 256 levels: t £, that is, not greater than 13 sensitivity, therefore, no pixel-by-pixel correction software is required, and the measured cell selection screen occupies more than 90% of the imaging area.
  • the difference between different batches of staining is corrected by using the ratio of the cell integrated optical density value to the reference optical density value, thereby eliminating the need to make special animal cell standard slides and corrected staining differences that are large and the standard accuracy is not very high.
  • the optical density color scale is made, so that the measurement is more convenient and the testing cost is lower.
  • Eyepiece field of view 1 1 biological reproduction, can realize teaching reading and disease ⁇ reading.
  • Fig. 2 is a structural diagram of a binocular binocular lens barrel of a CCD camera.
  • FIG. 4 is a schematic structural diagram of a condenser lens assembly according to the present invention.
  • FIG. 5 is a schematic structural diagram of another light accumulation medullary component of the present invention.
  • FIG. 6 is an imaging linear Y-coordinate diagram of the microscopic image analysis system of the present invention.
  • Attached Table 1 is the imaging relationship of the microscopic image analysis system of the present invention ⁇ 3 ⁇ 4 degree relationship table ⁇ detailed description of the preferred embodiment
  • the invention takes a microscope with It light source ⁇ 3 ⁇ 4 as an example, equipped with a 40x achromatic flat field objective lens, 1: 1 »interface and equipped with a 1 / 2-inch gain CCD camera and a computer system, including an image acquisition card And high-resolution image display, in which the computer software has a 256-level grayscale test card.
  • the test card has a measurement sensitivity of less than 1 level and a sampling time of not more than 1 second. It can measure the gray on the needle surface and each point of the image display. Degree instant male.
  • Preferred luminous source 6V, 20W or 6V 30W halogen lamps, the geometry of the filament is "or" W "shape and thickness, the number of turns of 3 ⁇ 4E is 7 ⁇ 9 turns, preferably 8 turns.
  • the second step requires high stability for the power supply of the luminous body.
  • the ripple of the output voltage is 1% of the rated voltage.
  • the stability of the output voltage is not greater than 1.5 of the rated voltage.
  • the third step requires the microscope: 1.
  • the center of the microluminous body ⁇ S adjustment function Adjust the part of the fixed light on the right and back right side to adjust the filament, focus the filament on the slide, and then adjust the lamp with 1 or 4 objective lens
  • the center of the lamp is in the center of the field of view of the microscope, and the center of the filament is monitored with an image S / monitor. It is better to use an objective lens of 40 ⁇ . »The geometric center of the imager is consistent with the center of the lamp, that is, the filament's geometry. Consistent with Zi Na, at this time, the number of horizontal filaments of the paste must be 8 and the reciprocal turning point of the filament must be outside the vertical plane of the display. 2.
  • Microscope installation product This product is shown in Figure 4 and Figure ⁇ .
  • Figure 4 is a combination of a 51 lens external light source 54 and a 51 mm frosted glass 56 and Imra frosted glass 55. Spacers 52 are provided between the semi-convex lens 53, the two frosted glass and the semi-convex lens 53.
  • FIG 5 is a frosted glass 56 and lmra 3mm mill _ 3 ⁇ 4g projections 57 and the field optical fiber 58 between the columns 55 of glass sand, this structure can be both functional lighting, and adjustment of field diaphragm.
  • ⁇ ⁇ Two types of integrated filament blurring can be adjusted according to the level of blurring, the distance between glass sheets, the particle size of the matte surface, and one or both sides. 3.
  • the gray-scale quantitative value of the display is 180.
  • Use a grayscale test card to select and measure any point in the middle part of the image display or any beam matrix point.
  • select a power supply voltage adjust the focusing column, make the numerical aperture of the illumination match the numerical aperture of the objective lens, and make the CCD
  • the camera amplifier gain is at 1: l ⁇ g or the automatic gain control is turned off. Adjust the TO- to adjust the test to the maximum, and then adjust the power of the halogen lamp to make the test 200 levels, that is, 1 to 255 levels.
  • the ftg of the lifting wheel at this point basically makes the platform close to the maximum displacement of the bottom product, and the lifting wheel dH is moved up and down at noon, and the test value will be significantly reduced and changed. Adjust the lifting wheel to move the lifting wheel from level 200 to level 180.
  • the thickness of the glass slide used is 1.2 mm. Because the thickness of the glass slide is constant, fine adjustment is to use the thickness error.
  • the gray level of the background color is unchanged, and the ship is a quantitative value of about 10% that is reduced by 200 levels after fine adjustment. 4.
  • the grayscale quantitative value of its display is 100 ⁇ 130
  • the black and white OCD camera can be between 100 and 110
  • the color CCD camera can be higher, that is, 120 ⁇ 130 ⁇ 1
  • quantitative inspection at this time the gray value error between single points or single beam matrix is less than 2 levels
  • 90% of each point on the image display surface is less than 10 levels.
  • hit fOCD gain to make the system background quantitative 3 ⁇ 43 ⁇ 4200 ⁇ 230 levels.
  • the lighting standard quantitative value slide glass with a thickness of 1.2, artificially produced 20 ⁇ 50 ⁇ m 2 Pat known value of the area, within which a uniform gray area, and the background gray level difference of 15 ⁇ 20, while the above-described
  • the known area value is measured through the system, and the error is preferably less than 2%.
  • the standard background light of 256-level gray-scale illumination is used.
  • the intensity of the measured structure is less than 2 % of m 2 , and the uniformity of the large-area illumination is 90%.
  • the background background color body is less than 13 degrees, so that the quantitative analysis of the brain object ice image cell has a reference interface, as long as the system Not only is the cage correct and reliable, but it can also be made. Therefore, the knowledge of the measured morphological analysis software can be directly used as a clinical leg or even legal judgment.
  • the microscopic product can be composed of two pieces of frosted glass of different thicknesses and semi-convex pieces, as shown in FIG. 4, and the convexity can also be set between two pieces of glass with different thicknesses as shown in FIG. 5 Field light bar composition. It is also suitable for the use of rear 3 ⁇ 4a micromirrors.
  • the invention can be combined with a CCD camera and a binary image processing system, and can distinguish 256 levels of gray, which cannot be achieved by the "critical lighting” and “Kuler lighting” brigades.
  • the average value of the measured integrated optical density of monocytes I0D (called IOD "2) is equal to or greater than the average value of basal white blood cell density IOI, and the average value of integrated optical density IOD ⁇ 2 , At 3 ⁇ 43 ⁇ 480 ⁇
  • the transitional cells are mainly white blood cells in human normal blood. «That is to say, the ratio of the density value of the product ⁇ 3 ⁇ 4 is used as the correction value for the poor staining. It is caused by the transition tissue and the basic tissue. As long as it is technically guaranteed, such Correction
  • Chicken blood cells can also be used for the selection of cells.
  • the principle is the same, but except for AJfiL cell tissue, any type of cell tissue must be used as the benchmark value.
  • the present invention uses a 256 «degree illumination biological microscope 5 Bfifi binocular binocular tube 3 (as shown in Figure 2), or equipped with a binocular lens tube 16 (as shown in Figure 3
  • the black and white CCD camera 4 and the color CCD camera 1 are respectively installed on the lens barrel.
  • the color CCD camera 1 interface is equipped with a 1: 0.5 scale video connector 2 to form a micro rattan image system, in which the color CCD Camera 1 and black and white CCD camera 4 are connected to host 6, respectively.
  • the X-axis represents the attenuation film attenuation » and ⁇ « represents the actual test value of the ma grayscale measurement device. Any point or any beam can be selected for sampling.

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  • Engineering & Computer Science (AREA)
  • Multimedia (AREA)
  • Physics & Mathematics (AREA)
  • Computer Vision & Pattern Recognition (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Microscoopes, Condenser (AREA)

Abstract

On décrit un procédé servant à accroître la précision analytique d'images microscopiques, ainsi qu'un appareil associé, ce procédé étant caractérisé en ce qu'il consiste à établir un standard d'éclairage à échelle de gris de niveau 256 pour le microscope du système et à établir également le poids standard de 7,18 Pg DNA. L'appareil d'analyse d'images microscopiques est caractérisé en ce qu'il comprend un microscope biologique, une caméra à CCD, une alimentation électrique, un processeur principal d'ordinateur personnel, un écran de contrôle et une imprimante, en ce que le microscope biologique binoculaire reçoit un éclairage à échelle de gris de niveau 256, en ce que l'on a placé respectivement sur le cylindre une caméra à CCD en noir et blanc et une en couleurs, en ce que l'on a couplé un coupleur vidéo 1:0,5 à l'interface de la caméra en couleurs afin de former le système de caméra microscopique dans lequel la caméra en couleurs et la caméra en noir et blanc sont respectivement reliées au processeur principal d'un ordinateur personnel, et en ce que la sortie du processeur est reliée respectivement à l'écran de contrôle en couleurs et à l'imprimante.
PCT/CN1997/000079 1996-08-05 1997-08-05 Procede servant a accroitre la precision d'images microscopiques et appareil associe WO1998005942A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN 96116450 CN1172960A (zh) 1996-08-05 1996-08-05 显微镜用的256级灰度照明方法
CN96116450.6 1996-08-05

Publications (1)

Publication Number Publication Date
WO1998005942A1 true WO1998005942A1 (fr) 1998-02-12

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4741043A (en) * 1985-11-04 1988-04-26 Cell Analysis Systems, Inc. Method of and an apparatus for image analyses of biological specimens
JPH01167648A (ja) * 1987-12-23 1989-07-03 Hitachi Software Eng Co Ltd Ｄｎａパターン読取り装置
EP0334152A1 (fr) * 1988-03-21 1989-09-27 Siemens Aktiengesellschaft Procédé d'évaluation de l'information de l'image dans une inspection de la surface au moyen d'un scanner
US4881818A (en) * 1986-04-22 1989-11-21 The University Of New Mexico Differential imaging device
WO1994011841A1 (fr) * 1992-11-06 1994-05-26 Quatro Biosystems Limited Procede et appareil pour l'analyse d'images
US5351307A (en) * 1991-11-15 1994-09-27 Societe Nationale D'etude Et De Construction De Moteurs D'aviation "S.N.E.C.M.A." Process and apparatus for the acquisition and processing of screen images
EP0677823A1 (fr) * 1994-04-15 1995-10-18 Fuji Photo Film Co., Ltd. Appareil pour l'analyse d'images

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4741043A (en) * 1985-11-04 1988-04-26 Cell Analysis Systems, Inc. Method of and an apparatus for image analyses of biological specimens
US4741043B1 (en) * 1985-11-04 1994-08-09 Cell Analysis Systems Inc Method of and apparatus for image analyses of biological specimens
US4881818A (en) * 1986-04-22 1989-11-21 The University Of New Mexico Differential imaging device
JPH01167648A (ja) * 1987-12-23 1989-07-03 Hitachi Software Eng Co Ltd Ｄｎａパターン読取り装置
EP0334152A1 (fr) * 1988-03-21 1989-09-27 Siemens Aktiengesellschaft Procédé d'évaluation de l'information de l'image dans une inspection de la surface au moyen d'un scanner
US5351307A (en) * 1991-11-15 1994-09-27 Societe Nationale D'etude Et De Construction De Moteurs D'aviation "S.N.E.C.M.A." Process and apparatus for the acquisition and processing of screen images
WO1994011841A1 (fr) * 1992-11-06 1994-05-26 Quatro Biosystems Limited Procede et appareil pour l'analyse d'images
EP0677823A1 (fr) * 1994-04-15 1995-10-18 Fuji Photo Film Co., Ltd. Appareil pour l'analyse d'images

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Publication number Publication date
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