WO2002012860A2 - Procede et dispositif de mesure de l'activite d'une substance bioactive dans une preparation histologique - Google Patents

Procede et dispositif de mesure de l'activite d'une substance bioactive dans une preparation histologique Download PDF

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Publication number
WO2002012860A2
WO2002012860A2 PCT/EP2000/007706 EP0007706W WO0212860A2 WO 2002012860 A2 WO2002012860 A2 WO 2002012860A2 EP 0007706 W EP0007706 W EP 0007706W WO 0212860 A2 WO0212860 A2 WO 0212860A2
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WO
WIPO (PCT)
Prior art keywords
preparation
optical property
substance
selected image
change
Prior art date
Application number
PCT/EP2000/007706
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German (de)
English (en)
Other versions
WO2002012860A3 (fr
Inventor
Gerhard Lewandovski
Original Assignee
Gerhard Lewandovski
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 Gerhard Lewandovski filed Critical Gerhard Lewandovski
Priority to PCT/EP2000/007706 priority Critical patent/WO2002012860A2/fr
Priority to AU2000272733A priority patent/AU2000272733A1/en
Publication of WO2002012860A2 publication Critical patent/WO2002012860A2/fr
Publication of WO2002012860A3 publication Critical patent/WO2002012860A3/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/59—Transmissivity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence

Definitions

  • the invention relates to a method for measuring the activity of a biologically active substance, such as an enzyme or an enzyme substrate in a histological preparation, in particular for metabolic and / or tumor diagnosis, and a device for carrying out the method.
  • a biologically active substance such as an enzyme or an enzyme substrate in a histological preparation, in particular for metabolic and / or tumor diagnosis
  • DE 196 40 121 discloses a method for determining a time-dependent changing variable, the maximum value being determined in a range with a linear reaction course and the time and the size of the reaction time window being variable with the linear range and the type of reaction and is dependent on the reaction conditions.
  • the concentration of the substance to be measured in the solution is measured and they do not allow direct measurement on a tissue preparation itself.
  • V-max the maximum approximately linear reaction rate of the conversion
  • DE 44 11 661 finally shows a video-technical analysis method for the simultaneous quantitative hydrogeological determination of a large number of chemical parameters of a liquid or gaseous medium, such as groundwater, in matrix-like reagent fields on the basis of color intensities.
  • the object of the invention is to provide a method and a device with which the activity of a specific biologically active substance can be at least approximately quantitatively measured directly on a histological preparation.
  • the preparation - such as a histological section or a tissue sample - together with the reagents, which are due to the activity of the measuring substance change the respective optical property of the preparation, arranged in front of the image recording device.
  • the substance to be measured causes a change in the optical property in that area of the preparation in which the substance to be measured is present. If you then take several images of the specimen in succession, you can see a change in the optical property in the area of the specimen over time.
  • the rate of change in particular the maximum rate of change of the optical property, is in a previously known proportional relationship to the concentration of the substance.
  • a measure of the substance concentration is obtained from the change over time, specifically in association with the respectively selected image area of the preparation, to distinguish between those image areas in which the substance is active and those in which it is not active or to a different extent , A visual representation of substance concentrations is thus obtained.
  • a particularly precise measurement of the substance concentration is possible if the concentration of the substance is determined from the greatest temporal change in the optical property in the selected image area and if the concentration of the substance in a region of approximately linear temporal change in the optical property, in particular the change in extinction, is determined in the selected image area.
  • the optical property can include the brightness or / and the color or / and the color intensity or / and the increase in a dye concentration or / and the fluorescence of the preparation.
  • At least one reference image can be taken from the specimen before the multiple images are taken. Then, data values of the reference image relating to the optical property can be offset against, in particular subtracting from, data values of the images taken in succession relating to the optical property. Then the change in the optical property of the selected Image area can be determined by comparing the interference-free, successively recorded images.
  • the reference image can be used to determine the zero value, and not the absolute value of the concentration of the substance, but the difference in the optical property between the reference image and the later images can be used to determine the activity of the substance to be measured.
  • the reference image can reproduce the same image section of the specimen as the later measurement images, the reference image preferably being the first image of the images taken in succession.
  • a reference value can be used which is recorded in a tissue-free area of the preparation in which no activity of the substance to be measured is to be expected, this reference value being offset against image data of the at least one selected image area, in particular being subtracted therefrom.
  • the biologically active substance can be an enzyme substrate or an enzyme whose activity is measured on the basis of the change in concentration over time of the assigned enzyme substrate or the assigned enzyme product.
  • the temperature, the pH value and / or the ion concentration in the preparation or / and the intensity of the illumination can be kept constant during the measurement.
  • a photo camera, a film camera or, particularly preferably, a video camera can be used as the image recording device, the change in the optical property in the selected image area being measured and evaluated pixel by pixel.
  • a device for measuring the activity of a biologically active substance in a histological preparation comprising: an image recording device for successively taking multiple images of the preparation, which has been treated with reagents, which by activity of the substance has at least one optical one Change property of the specimen, a device for determining a temporal change in the optical property in at least one selected image area by comparing the images taken in succession; and a device for determining the concentration of the substance in the selected image area from the temporal change in the optical property in the selected image area.
  • Fig. 1 shows a diagram of an apparatus for performing the method
  • Figure 2 shows a timing diagram of a typical enzyme-enzyme substrate reaction
  • FIG. 3 shows a measurement example with gray values
  • FIG. 4 shows the measurement example from FIG. 3 after the gray values have been converted into extinction values.
  • Fig. 1 shows an apparatus for performing the method according to the invention.
  • a microscope 1 here a transmitted light microscope, for viewing a specimen 3 to be examined is equipped with a color or black-and-white video camera 5 for taking an image of the specimen 3.
  • the microscope 1 and the camera 5 form an image recording device which is arranged in an air-conditioning and darkening chamber 7, which is here air-conditioned to ⁇ 0.5 ° C.
  • the camera 5 is connected via a video recorder 9 to a computer 11, in which the images supplied by the camera 1 or the video recorder 9 can be evaluated mathematically.
  • the climate chamber 7 is air-conditioned by an external air conditioner 13, which is supplied with cooling water by a cyrostat water bath 15. Both the air conditioner 13 and the illumination of the microscope 1 are supplied with current, in this case direct current, with a power supply unit 17.
  • the video camera 5 is connected to a control power supply 19 arranged outside the climatic chamber 7.
  • the video recorder 9 is connected to a video monitor 21, and the computer 11 to a computer monitor 23.
  • a temperature sensor 25 is arranged near the specimen 3, the output of which is connected via a temperature measuring device 27 to a channel of a two-channel voltage-time recorder 29.
  • the other channel of the recorder 29 receives a signal from a lux measuring probe 31, which is fixed on an edge area of the screen of the computer monitor 23, in order to monitor any fluctuations in the basic brightness of the entire recording system of microscope illumination, camera 5, computer 11, for example as a result of fluctuations in the mains voltage.
  • the preparation 3 is treated with reagents, in particular dyes, which change an optical property, such as the color intensity of the preparation, through the activity of the substance to be measured, in order ultimately to bring about a change in extinction in the preparation.
  • the specimen treated in this way is placed under the objective of the microscope 1.
  • selected image areas Ba shown on the computer monitor 23 in FIG. 1 of the sections are recorded at defined light intensities and constant temperature.
  • An image B is taken every minute for a period of about 15 minutes here.
  • the resulting image data are transmitted to the computer 11 via an image acquisition card (AD converter) and are offset there with an image processing program.
  • AD converter image acquisition card
  • the series of measurements results in a temporal change in the respective optical property due to the activity of the substance to be measured, for example an increasingly intense coloration which can be measured as extinction.
  • the speed, in particular the maximum speed, of the increase is used as a measure of the substance activity.
  • the data of the individual pixels of the first image as a reference image Bref of the image series can be subtracted as zero values from that of the images subsequently recorded. Then you select an area in the histological section that is not to be measured or / and that has no tissue substance (unselected area Bna) and determines a blank value there. This blank value is also subtracted from the data of the pixels of the respective images.
  • This two-stage zero and blank value deduction leads to images B without the unspecific coloring of the dye produced by light, while at the same time changes in the system, for example light fluctuations due to voltage fluctuations in the supply network, are compensated for.
  • This double zero value or blank value subtraction enables the images to be mathematically smoothed, ie freed from noise.
  • Image information is obtained which only indicates the change in the optical property over time.
  • the recording device can be calibrated using a calibrated gray filter. The conversion of the measured absorbance to substance concentration is carried out in a known manner according to the Lambert-Beer law.
  • the rate of conversion of a substrate by an enzyme with a high substrate excess is mainly dependent on the concentration of this enzyme until there is a lack of substrate or inhibition by the product and the rate of turnover decreases.
  • an enzyme moie case takes a certain amount of time to absorb the substrate, to react and to release the product until it reaches the maximum sales rate. If the product concentration rises, the delivery of the product becomes more difficult and the turnover rate decreases.
  • the maximum reaction rate of the conversion (V-max) is specific for an enzyme under defined conditions, such as temperature, pH value, ion concentration. The measurement of the enzyme activities under V-max conditions over time allows a quantification of the enzyme activity. The general course of such an enzyme kinetics is shown schematically in FIG. 2.
  • the extinction E corresponds to the logarithm of the ratio of the intensity l 0 of the incident light to the intensity I of the transmitted light. At the same time, it corresponds to a substance concentration c with a constant irradiated layer thickness s as a function of a substance-specific constant.
  • FIGS. 3 and 4 show the relative change in gray value and the change in extinction of four measurement series calculated therefrom with assigned regression lines in the range of Vmax, which were measured simultaneously on four tissue areas of a specimen.
  • the amount of enzyme in each pixel or in the selected image area Ba can be measured via the assigned substrate or product turnover.
  • the image information can be resolved down to the cellular and subcellular level, e.g. it is possible to measure the activities of enzymes in individual mitrochondria.
  • the absorbance is determined indirectly according to equation 3) for the quantitative detection of enzymatic activity.
  • the change in gray value or change in absorption in a pixel or the selected area Ba is measured over time.
  • the results of this measurement are converted into a system absorbance E '.
  • the correction factor k is determined by calibrating the system with gray filters.
  • the amount of the respective enzyme or substrate is determined by changing the absorbance over time in accordance with the Lambert-Beer law.
  • nematodes of the species Caenorhabditis elegans were incubated under various conditions in tissue embedding agents and frozen and cut to a thickness of 7 ⁇ m in the cyrostat and applied to microscope slide treated with polylysine.
  • the tissue sections were thawed, dried for 10 to 15 minutes and either used immediately or frozen for later use at -70 ° C. Before being introduced into the measuring device, the sections were thawed with the back of the hand and carefully covered with an incubation solution.
  • This incubation solution contained excess substrate, tetrazolium dye, phenazine methosulfate (PMS), cyanide to block the respiratory chain and Tris-HCl buffer to keep the pH constant at 7.4.
  • PMS phenazine methosulfate
  • Tris-HCl buffer Tris-HCl buffer to keep the pH constant at 7.4.
  • the method can also be used in particular for quantifying metabolic enzymes such as SDH (succinate dehydrogenase), MDH (malate dehydrogenase) and LDH (lactate dehydrogenase). Other NAD-dependent metabolic enzymes can also be measured.
  • This method can be used to quantify all enzymes for which a specific staining method is available, such as acid phosphatase, cytochrome oxidase and various proteases.
  • the ⁇ -galactosidase can also be detected via the lacZ -> ⁇ -galactosidase -> Xgal reaction.
  • the lac-Z gene codes in E. coli for the enzyme ⁇ -glactosidase, which hydrolyzes lactose in glucose and galactose, and is used in genetic engineering as a selection marker and reporter gene.
  • the genetic substance to be introduced can also contain the lac-Z gene.
  • ⁇ -galactosidase The presence of the ⁇ -galactosidase is demonstrated with a substrate analog Xgal (5- Bromine-4-chloro-3-indolyl-ß-D-galactoside), which is hydrolyzed in ß-galactose and a blue chromophore, which causes a change in color in the preparation.
  • Xgal 5- Bromine-4-chloro-3-indolyl-ß-D-galactoside
  • a blue chromophore which causes a change in color in the preparation.
  • concentration of the ⁇ -galactosidase and thus the strength of the expression of the introduced sequence can be read from the speed of the change in color.
  • the change in the fluorescence properties of the substrate conversion can also be used as a measurement variable.
  • Suitable dyes are e.g. Methyl-Umbellipheryl substrates. A particularly sensitive camera must be used for this.
  • the change in absorbance can be determined using artificially added dyes, but native substances present in the cell can also be used to measure the enzyme activities.
  • NAD nicotinamide adenine dinucleotide
  • FAD flavin adenine dinucleotide
  • the high sensitivity of the method according to the invention is in this variant especially for processing gel '.
  • a particularly sensitive camera and UV light must be used here.
  • inhibitors of enzymes can also be tested under almost vital conditions without the need for extensive animal experiments.
  • the method is particularly applicable in tumor diagnosis, in particular in the quantification of metabolic enzymes such as SDH and LDH in tumor tissues.

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  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

La présente invention concerne un procédé et un dispositif permettant la mesure et la représentation sous forme d'image de l'activité d'une substance bioactive, notamment d'une enzyme, dans une préparation histologique. A cet effet, la préparation (3) est traitée avec des réactifs qui, en raison de l'activité de la substance, modifient au moins une propriété optique de la préparation, et la préparation ainsi pré-traitée est disposée devant un dispositif de prise d'image (1, 5). Pour finir, plusieurs images de la préparation sont prises successivement. Puis, la modification temporelle de la propriété optique dans au moins une partie d'image sélectionnée (Ba) est déterminée au moyen d'un ordinateur (11) par comparaison des images (B) prises successivement, et la concentration de la substance dans la partie d'image sélectionnée (Ba) est déterminée à partir de la modification de la propriété optique dans ladite partie d'image sélectionnée.
PCT/EP2000/007706 2000-08-08 2000-08-08 Procede et dispositif de mesure de l'activite d'une substance bioactive dans une preparation histologique WO2002012860A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/EP2000/007706 WO2002012860A2 (fr) 2000-08-08 2000-08-08 Procede et dispositif de mesure de l'activite d'une substance bioactive dans une preparation histologique
AU2000272733A AU2000272733A1 (en) 2000-08-08 2000-08-08 Method and device for measuring the activity of a biologically active substance in a histological preparation

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/EP2000/007706 WO2002012860A2 (fr) 2000-08-08 2000-08-08 Procede et dispositif de mesure de l'activite d'une substance bioactive dans une preparation histologique

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WO2002012860A2 true WO2002012860A2 (fr) 2002-02-14
WO2002012860A3 WO2002012860A3 (fr) 2009-02-19

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5126569A (en) * 1989-03-10 1992-06-30 Massachusetts Institute Of Technology Apparatus for measuring optical properties of materials
DE19527160C1 (de) * 1995-07-25 1997-01-23 Brahms Diagnostica Gmbh Verfahren zur Messung der Konzentration eines Enzyms durch photometrische Absorptionsmessung eines enzymatisch gebildeten Farbstoffs in einer Meßlösung
US5914245A (en) * 1998-04-20 1999-06-22 Kairos Scientific Inc. Solid phase enzyme kinetics screening in microcolonies
EP0997729A1 (fr) * 1998-10-27 2000-05-03 Commissariat A L'energie Atomique Dispositif de détermination de la concentration d'une substance mélangée à un fluorophore et procédé de mise en oeuvre de ce dispositif
DE19919539C1 (de) * 1999-04-29 2001-01-11 Gerhard Lewandovski Verfahren zur Messung der Aktivität einer biologisch wirksamen Substanz in einem histologischen Präparat

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5126569A (en) * 1989-03-10 1992-06-30 Massachusetts Institute Of Technology Apparatus for measuring optical properties of materials
DE19527160C1 (de) * 1995-07-25 1997-01-23 Brahms Diagnostica Gmbh Verfahren zur Messung der Konzentration eines Enzyms durch photometrische Absorptionsmessung eines enzymatisch gebildeten Farbstoffs in einer Meßlösung
US5914245A (en) * 1998-04-20 1999-06-22 Kairos Scientific Inc. Solid phase enzyme kinetics screening in microcolonies
EP0997729A1 (fr) * 1998-10-27 2000-05-03 Commissariat A L'energie Atomique Dispositif de détermination de la concentration d'une substance mélangée à un fluorophore et procédé de mise en oeuvre de ce dispositif
DE19919539C1 (de) * 1999-04-29 2001-01-11 Gerhard Lewandovski Verfahren zur Messung der Aktivität einer biologisch wirksamen Substanz in einem histologischen Präparat

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AU2000272733A8 (en) 2009-03-19
AU2000272733A1 (en) 2002-02-18
WO2002012860A3 (fr) 2009-02-19

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