CN1086204C - Technology for measuring cytochemistry component content with biological tissue in situ three-D - Google Patents
Technology for measuring cytochemistry component content with biological tissue in situ three-D Download PDFInfo
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- CN1086204C CN1086204C CN97107435A CN97107435A CN1086204C CN 1086204 C CN1086204 C CN 1086204C CN 97107435 A CN97107435 A CN 97107435A CN 97107435 A CN97107435 A CN 97107435A CN 1086204 C CN1086204 C CN 1086204C
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Abstract
The present invention particularly relates to a technical method for measuring the content of biological cell chemical components, which belongs to the technical field of a biological tissue measuring or inspecting method. The technical method comprises the steps that the volume and the size distribution of a target cell are measured, and the size distribution of the average optical speed of the target cell or a cell nucleus are measured, the target cell or the cell nucleus, the volume integral optical density and the size distribution of the volume integral optical density are analyzed and calculated, and the contents of some chemical components among three-dimensional target cell groups are compared. A great amount of statistic and complete cell information can be obtained by adopting the measuring analysis technology, and the accuracy and the speed of the measurement are increased.
Description
The invention belongs to the mensuration or the method for inspection technical field of biological tissue, be specifically related to a kind of method of measuring biomass cells Chemical Composition content.
At present, biological tissue's home position observation intact cell form or measure the content technology of its Chemical Composition, all adopt successive to organize thin section and three-dimensionalreconstruction technology to realize, its typical context file is: " three-dimensional confocal laser scans the cell measurement that thick tissue slice is made the dna ploidy body " (.P.Tekola, J.P.A.Baak, H.A.H.M.Jinkel, et a1.Three-dimensional confocal laserscanning DNA ploidy cytometry in thick histological sections.J Pathology, 1996; 180; 214-222); " laser confocal microscope is made the three dimensional DNA imaging cell measurement of the thick tissue samples of prostatosis " (.T.Irinopoulou, J.Vassy, M.Beil, et al.Three-dimensional DNA image cytometry by confocal scanning lasermicroscopy in thick tissue blocks of prostatic lesions.Cytometry, 1997,27 (2): 99-105), use this two pieces of methods that document relates to, the speed that obtains intact cell form and its Chemical Composition content is slow, be difficult to obtain to have in a large number the result of statistical significance, and required instrument costs an arm and a leg, and is difficult for popularizing.
The present invention is directed to the prior art above shortcomings, purpose is to provide a kind of biological tissue in-situ three-dimensional to measure the method for cytochemistry component content, it can carry out the velocity of sound and detects exactly to the Chemical Composition content of cell in enormous quantities, obtain intact cell in biological tissue's in-situ chemical component content information.
Theoretical basis of the present invention:
1. many provinces of cell chemical composition content should be with the basis that is measured as of its single intact cell.Therefore, have following relationship:
Single intact cell
Total amount=intact cell Chemical Composition concentration * the intact cell volume 1.
Certain Chemical Composition
2. absorbancy (claiming optical density(OD) again) is the tolerance of the degree of light absorbing substance, also can take this to understand the concentration of extinction material.Carry out microscopic image two dimension absorbance measuring and can state following formula as:
A＝Log[Io(x，y)/I(x，y)]＝K(x，y)C(x，y)L(x，y)；②
A: absorbancy Io: emergent light intensity I: incident intensity
K: specific absorbance C: measured matter concentration L: measured matter thickness
(x, y): the locus of measuring pixel in the micro-image 2. formula can be transformed into following formula:
C(x，y)＝A/K(x，y)L(x，y) ③
Be not difficult to find out from 3. formula, if on the tissue slice in the cell specific absorbance and the slice thickness of extinction material do not change, average (average optical) of respectively measuring the absorbancy of pixel in its tangent plane promptly can be used as the concentration of the interior extinction material of this unit slice thickness.As long as the requirement of stochastic sampling is satisfied in the sampling of the cell tangent plane of surveying, the distribution of its average optical promptly is the probability distribution of the extinction substrate concentration in this unit slice thickness.
3. the volume size distribution state of measuring single intact cell volume and colony thereof at tissue in situ that develops into of stereology is laid a good foundation.Adopt stereoscopic frame sampling and nuclear can obtain the individual cells volume and the volume size distribution thereof of isotropy section, terrace cut slice or the upward number weighting of cutting into slices arbitrarily apart from measuring technology.As long as follow the sampling requirement of stereology, the volume size distribution of its individual cells number weighting promptly is the probability distribution of this cell colony volume size.
4. the purpose of measuring single intact cell Chemical Composition content is to understand the content between different groups and the difference of content distribution state.According to the definition of independent probability incident as can be known, 1. concentration in the formula and volume are two independently probability events, therefore, 1. in the formula average of certain Chemical Composition content of single intact cell colony be that the product of the probability distribution of the probability distribution of such cell Chemical Composition concentration in the available tissue and such cell volume is tried to achieve.
5. what of two groups of amount of substances relatively, adopt two kinds of mathematical way to express usually:
A. the absolute value of two groups of amount of substances subtracts each other promptly: C=A amount of substance-B amount of substance
B. the absolute value of two groups of amount of substances is divided by promptly: C=A amount of substance/B amount of substance
When C=0 or C=1, A amount of substance=B amount of substance
When C＞0 or C＞1, A amount of substance＞B amount of substance
When C＜0 or C＞l, A amount of substance＜B amount of substance
What of two groups of amount of substances are the mode that employing is subtracted each other compare, and can draw different test results because of different test durations, different testing tools, different test condition and different personnel operation, are difficult for drawing consistent conclusion.And adopt relatively what of two groups of material relative quantities of the mode (being exponential manner) be divided by, be enough to overcome above-mentioned defective, and the 3. requirement of formula that is content with very little, with the detection of many biological tissues and research need consistent.
The equipment of wanting required for the present invention
1. opticmicroscope
The logical optical filter (extinction characteristic according to the survey Chemical Composition is determined wavelength region) of band or
Light-dividing device
3. micro-image analyzer (must satisfy following performance index):
A. chicken red blood cell nuclear smear, Feulgen dyeing, the CV of its two-dimensional areas observed value less than
10％；
B. same target is 30 integral optical densities that different positions is measured in picture frame is deposited body
CV less than 3%;
C. picture frame is deposited identical integral optical density target group in the body (as chicken red blood cell nuclear, laser
Flow cytometer records the CV of its dna content can be less than 3%) single individuality long-pending
Divide optical density(OD) CV less than 8%;
D. identical integral optical density target group's (as chicken red blood cell nuclear) single individuality and two,
It is linear that the integral optical density value of three, four cluster colonies is;
E. have target is examined apart from the algorithm software of measuring.
Measuring method of the present invention is as follows:
One, measures certain the Chemical Composition content and the content distribution frequency thereof of objective cell colony
1. draw materials: the isotropy section or terrace cut slice or the section (distribution character on target is decided) arbitrarily that cut two different thickness.The thin section of a 3-4 μ m thickness (as far as possible making the thickness homogeneous in the section) is made the chemical ingredients specific staining or is not dyeed (the special spectrographic light-dividing device of this Chemical Composition need be arranged), another than slab (can just contain complete object such as nuclear thickness is advisable, about common 10 μ m) do can clear reflection objective contour dyeing.
2. measurement target cell volume and volume size distribution thereof:
(1) sampling: adopt isotropy section or the terrace cut slice or the section (distribution character on target is decided) arbitrarily of thicker (about common 10 μ m); Use the micro objective of large-numerical aperture, the fine setting of adjusting microscope, image outline cell in the tissue slice changes from small to big, again from large to small or the cell image profile be sampling and measuring object by constant individual cells that diminishes when big or nucleus largest contours image, behind cell of every sampling or the nucleus, change pick up camera and microscopic fields of view plane mutual angle (definite at random before the sampling) on parallel direction, have nuclear as imager and examine apart from measuring scale angle function, can economize this operation approximately apart from the automatic rotation of method of masurement requirement.The individual cells largest contours of being sampled can be stored in the disk, concentrate and measure;
(2) measure: adopt the calculation formula of stereology nuclear apart from method of masurement, intersection point in length and breadth with "+" font cursor on the artificial mobile imager of mouse is located in the core (being center or center of gravity) of individual cells largest contours ash image, move four the short end lines of "+" word cursor and the coincident of cell largest contours again, to determine four nuclear distances through core, imager calculates the volume of this cell automatically; Or imager is determined the core (being center or center of gravity) of individual cells largest contours bianry image automatically and through four nuclear distances of core, and calculates this cell or nuclear volume automatically.Change video camera imaging plane and microscopic fields of view plane mutual angle sampling cell largest contours image on parallel direction as not adopting, behind cell of then every measurement, imager need by in advance artificially or the angle of determining at random rotate nuclear automatically apart from the direction of just measuring, measure another cell largest contours image.This measuring method mainly uses mutually perpendicular four of " ten " font 90 degree to examine carpenters square measurement target cell volumes on cell largest contours image, comprises that also rightabout two nuclear carpenters squares of employing 180 degree and 120 degree intervals three examine carpenters square and 60 degree, six nuclear carpenters squares and more multidirectional nuclear carpenters squares at interval.
The volume of the cell of sampling as stated above and measuring and size distribution thereof are the probability distribution of such cell colony volume.Aforesaid method also is applicable to the measurement to particle volume in the abiotic tissue slice.
3. measurement target cell section average optical and size distribution thereof:
(1) measuring system setting: use micro objective (common 20 * object lens), adjust the setting of this system by the operational requirement of the micro image analysis system that satisfies measuring light density performance index than small value aperture; Select the illumination light of the spectral filter or the specific wavelength of suitable wavelength for use according to the spectral response curve of survey cell chemical composition;
(2) sampling: adopt isotropy section or the terrace cut slice or the section (distribution character on target is decided) arbitrarily of thin (3-4 μ m usually), make the cell chemical composition specific staining or do not dye (light with specific wavelength is made illumination light), evenly randomly draw the microscopic fields of view shooting;
(3) press the operational requirement of micro-image analyzer, average optical and intragroup size distribution thereof in more accurate measurement target cell or the nucleus profile.This distribution is the probability distribution of the concentration of certain Chemical Composition in target cell or the nucleus.
4. analytical calculation target cell or nucleus volume integral optical density and size distribution thereof:
With above-mentioned such cell that obtains respectively or the probability distribution of nucleus volume and average optical,, obtain the optical density(OD) average and the big or small frequency distribution thereof of target cell or nucleus volume integration by integration after the multiplied by weight of each individuality in probability distribution.The optical density(OD) average of this volume integral and big or small frequency distribution thereof promptly are the content average of certain Chemical Composition of this cell colony and the distribution (containing the information of tested cell chemical composition specific absorbance and the information of slice thickness unit) of different content individual difference thereof.As long as obtained 2 and 3 original measurement quantity; No matter who finishes measurement earlier, has the micro-image analyzer of aforementioned calculation method program or the dedicated analysis software of computer and all can promptly finish this analytical calculation.
Exactly analytical calculation is carried out in the volume integrated optical density average and the distribution thereof of the cell colony that has multiple volume or average optical for meticulousr, before finishing above-mentioned analytical calculation, adopt following method to determine the volume of each cell subsets and the corresponding relation of its average optical more exactly, and then analysis meter is calculated the measurement average and the distribution thereof of this colony: when 1. using nuclear to measure cell volume apart from method of masurement (comprising other method), measure the area and the diameter of individual cells largest contours image; When 2. measuring on the two-dimensional slice image individual cells average optical, measure their area, area integral optical density(OD), optical density(OD) deviation, major-minor axis ratio, shape-dependent constant, fractal dimension, parameters such as texture; 3. respectively each above-mentioned parameter is made histogram analysis, understand each parameter distribution state of whole colony; 4. the volume in getting is 1. made two-dimentional scatter diagram with area or diameter respectively; Average optical in getting 2. respectively with 2. in all the other each parameters make two-dimentional scatter diagram; 5. with reference to the analytical results of each parameter histogram distribution, determine the volume and the corresponding distributed areas of each parameter of average optical and other in two dimension is loose figure of each cell subsets respectively, respectively the probability distribution of the take off data in the corresponding distributed areas of two scatter diagrams is carried out probability multiplication, obtain the volume integrated optical density average and the distribution thereof of each subgroup, synthesize the volume integrated optical density average and the distribution thereof of whole colony by the volume integrated optical density calculated value of each subgroup; Also the probability distribution of corresponding distributed areas is carried out probability multiplication, the volume integrated optical density average and the distribution thereof of acquisition different volumes subgroup in the two-dimentional scatter diagram of each the subgroup probability distribution that can be calculated by volume histogram and average optical and area.
Two, the comparison of certain Chemical Composition content between the objective cell colony
From 3. formula as can be known, adopt present technique on tissue slice, obtain the objective cell colony certain Chemical Composition content, compare with the identical chemical component content of another objective cell colony, satisfy following condition:
1. the specific absorbance that is used to the thin tissue section of the cell chemical composition average optical measured should be consistent, and this can realize with consistent sample preparation methods by constant measuring condition.
2. the thickness of thin tissue section that is used to the cell chemical composition average optical measured is identical, this can bury piece section (thickness in the common same section is the comparison homogeneous) by the tissue of two class cell colonys of desire comparison being done same comprising: or accurately measure the two class cell colonys thickness (thickness between section is difficult to accurate unanimity) of place section separately, when analytical calculation cell or nucleus volume integral optical density and size distribution thereof, slice thickness value input computer is made normalized, obtain to have the measuring result of comparability.
After having obtained to satisfy two groups or more objective cell colony Chemical Composition content value of above-mentioned condition, wherein to make denominator with reference to the volume integrated optical density average of certain Chemical Composition of cell colony group or intermediate value or peak value (its CV value should be as far as possible little), probability distribution with the volume integrated optical density of target cell colony is a molecule, is divided by to obtain the objective cell colony and distribute with density index with reference to cell colony Chemical Composition content difference.This distribution value be the objective cell colony with reference to the different ratio of cell colony Chemical Composition real content value difference, the specific absorbance of having eliminated certain Chemical Composition and slice thickness the contribution of being divided by of molecule denominator to observed value, if can adopt the definite absolute value of other method, can determine the distribution of target cell colony Chemical Composition real content absolute value with reference to cell colony Chemical Composition real content.
The inventive method is by being applied to meet in the cytological image analyses instrument of aforesaid device condition of the present invention, be used to measure the testis tissue section and go up the androgone dna content, the dna content that obtains primary spermatocyte is the twice of spermatogonium dna content, be four times of spermatid dna content, the dna content of spermatogonium is the twice of spermatid dna content, and this ratio is consistent with the result who draws with other research method.
Present technique also is applicable to the Chemical Composition Determination on content of certain class particle in abiotic the sheet.
Adopt this Measurement and analysis method, can measure biomass cells in large quantity, obtain a large amount of statistics, and can obtain complete cell information, improved accuracy and the speed measured.Below measure liver cell nuclear with the liver organization in-situ three-dimensional dna content be example, the concrete steps of use this patent method be describeds:
(1) essential equipment
1 opticmicroscope
2 image analyzers or laser confocal microscope
(2) preparation of liver tissue slices
Liver organization is drawn materials, conventional fixing, embedding.Because of the liver cell nuclear almost spherical with liver
Be stochastic distribution in the tissue, make thick, thin two successive hepatic tissue sections of any direction,
Thinly-sliced agreement that contracts a film or TV play to an actor or actress 3～4 μ m, Feulgen dyeing, about the about 10 μ m of thick section, doing can be clear
Analyse the dyeing of reflection liver cell nuclear profile.
(3) three-D volumes and the size distribution thereof of measurement liver cell nuclear
According to stereoscopic frame principle, at first determine the center or the center of gravity of the interior liver cell nuclear of the thick section of hepatic tissue (about about 10 μ m), promptly use the micro objective of 100X oil mirror, the fine setting of adjusting microscope, the image outline of choosing liver cell nuclear changes from small to big, and single liver cell nuclear from large to small is as target sample again." ten " font nuclear of determining image analyzer apart from the intersection point in length and breadth of measuring scale in " center or the center of gravity " of liver cell nuclear largest contours image, four short end lines determining this measuring scale again overlap with nucleus largest contours edge of image, and image analyzer calculates the volume of area, diameter and this intact cell nuclear of this nucleus largest contours automatically.After measuring some amount (greater than 50) liver cell nuclear volume, their volume distributed median promptly is the probability distribution of single liver cell nuclear volume in this hepatic tissue.
(4) average optical and the size distribution thereof of liver cell nuclear tangent plane in the measurement hepatic tissue thin section
Randomly draw the microscopic fields of view shooting of hepatic tissue section, the operational requirement of pressing micro-image analyzer, the parameters such as area, shape-dependent constant, area integral optical density(OD), average optical and probability distribution thereof of measurement liver cell nuclear tangent plane.
(5) calculate liver cell nuclear volume integrated optical density and probability distribution thereof in the hepatic tissue section:
The probability distribution of liver cell nuclear volume and the probability distribution of its average optical are multiplied each other volume integrated optical density average and probability distribution thereof that promptly to obtain with single liver cell nuclear volume be unit.The dna content average that this average promptly is is unit with single whole livers nucleus in the liver cell nuclear colony, this probability distribution promptly are the probability distribution of the liver cell nuclear individuality of different dna contents.This computation process can be written as computer software and finish quickly and accurately.
The comparison of certain chemical composition content and the volume of definite each cell mass and the corresponding relation of its average optical between above-mentioned objective cell colony, specific implementation process all can be finished by computer software.
Claims (2)
1. a biological tissue in-situ three-dimensional is measured the method for cytochemistry component content, it is characterized in that it comprises following steps:
(1) certain Chemical Composition content and content thereof of objective cell colony in the measurement tissue slice
Distribution frequency:
1. cut isotropy section or the terrace cut slice or the section arbitrarily of two different thickness, thinly-sliced
Sheet 3-4 μ m is thick, makes the Chemical Composition specific staining or does not dye about 10 μ of thick section
M does the dyeing of the clear reflection objective contour of energy;
2. measure tissue slice internal object cell volume and volume size distribution thereof:
A. sampling: adopt than slab, regulate microscope and focus on fine setting, make tissue slice
Interior cell image profile takes place ascending, descending again or cell image wheel
Wide diminish greatly by constant, with the image of the maximum space diameter that obtains cell to be measured,
And with the largest contours image of this cell as sampling and measuring object, every sampling one
Behind the individual cell, change video camera imaging plane and microscopic fields of view plane parallel side
Mutual angle makes progress; Or utilize that imager has revolve nuclear automatically apart from measuring scale
The angle function is rotated the largest contours figure that the direction of examining carpenters square is got individual cells automatically
Picture, to be measured with storing in the individual cells largest contours image set of being sampled;
B. measure: adopt stereology nuclear apart from method of masurement calculation formula, artificial mobile imager
" ten " font nuclear carpenters square is determined or is determined the individual cells maximum automatically by imager
Four nuclear distances of the core of profile bianry image and process core, and by imager certainly
The moving volume that calculates this cell, the volume and the size distribution thereof of the cell of sampling are
The probability distribution of such cell volume;
3. the average optical of measurement target cell section and size distribution thereof:
A. measuring system setting: use micro objective, survey by satisfying than small value aperture
The micro-image analyzer operational requirement of amount optical density performance index is adjusted this system,
According to the spectral response curve of survey cell chemical composition select for use suitable wavelength spectral filter or
The illumination light of specific wavelength;
B. thin section is evenly randomly drawed the microscopic fields of view shooting, press micro-image analyzer
Operational requirement, the average optical in measurement target cell or the nucleus profile and
The size distribution of colony is the dense of interior certain Chemical Composition of target cell or nucleus
The probability distribution of degree;
4. analytical calculation tissue slice internal object cell volume integral optical density and probability distribution thereof: will
The probability distribution of such cell volume of above-mentioned acquisition and the probability distribution of average optical,
Carry out probability multiplication, obtain target cell volume integrated optical density average and distribution thereof, promptly
Reflect the content of certain Chemical Composition of this cell colony and the distribution of different content individuality thereof.
(2) comparison of certain Chemical Composition content between the objective cell colony:
Obtaining to satisfy two groups or more objective cell colonyization of above-mentioned condition
Learn after the volume integral density value of component content, with certain chemistry of reference cell colony group
The volume integrated optical density average of composition or intermediate value or peak value are made denominator, with cell population of interest
The volume integrated optical density probability distribution of body is for dividing, and being divided by obtains the objective cell colony
With the distribution value of the different ratio of reference cell colony Chemical Composition real content value difference, logical
Cross the absolute value of determining with reference to cell colony Chemical Composition real content, just can obtain order
The distribution of mark colony Chemical Composition real content absolute value.
2. biological tissue according to claim 1 in-situ three-dimensional is measured the side of cytochemistry component content
Method is characterized in that tissue slice internal object cell volume integral optical density and probability distribution thereof are advanced
Before the row analytical calculation, can adopt following method to determine that the volume of each cell subsets and its average light are close
The corresponding relation of degree, and then analysis meter is calculated the measurement average and the distribution thereof of total group:
1. measure cell volume, measure area and the diameter of individual cells largest contours image;
When 2. measuring on the two-dimensional slice image individual cells average optical, measure their face
Long-pending, area integral optical density(OD), optical density(OD) deviation, major-minor axis ratio, shape-dependent constant, branch dimension
Parameters such as number, texture;
3. respectively each above-mentioned parameter is made histogram analysis, understand each parameter branch of whole colony
The cloth state;
4. the volume in getting is 1. made two-dimentional scatter diagram with area or diameter respectively; Average light in getting 2.
Density respectively with 2. in all the other each parameters make two-dimentional scatter diagram;
5. with reference to the analytical results of each parameter histogram distribution, determine each cell subsets respectively
The corresponding range of distribution of each parameter of volume and average optical and other in two-dimentional scatter diagram
Carry out the probability distribution of the take off data in the corresponding distributed areas of 2 diffusing points generally respectively in the territory
Rate multiplies each other, and obtains the volume integrated optical density average and the distribution thereof of each subgroup, is synthesized the volume integrated optical density average and the distribution thereof of whole colony by the volume integrated optical density calculated value of each subgroup; Also the probability distribution of corresponding distributed areas is carried out probability multiplication, the volume integrated optical density average and the distribution thereof of acquisition different volumes subgroup in the two-dimentional scatter diagram of each the subgroup probability distribution that can be calculated by volume histogram and average optical and area.
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| CN108709786B (en) * | 2018-02-08 | 2021-03-19 | 中国科学院化学研究所 | Method for staining and quantitative analysis of rare earth nanoparticles in biological tissues |
| CN116183331A (en) * | 2023-03-03 | 2023-05-30 | 吉林大学 | A tissue slice detection platform based on a three-dimensional gel electrophoresis device |
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| CN1083212A (en) * | 1993-06-08 | 1994-03-02 | 山西大学 | The easy quick paraffin sections method of human body and animal tissue |
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| CN1083212A (en) * | 1993-06-08 | 1994-03-02 | 山西大学 | The easy quick paraffin sections method of human body and animal tissue |
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