CN1632520A - Fluorescent in situ hybridization counting slide and counting method thereof - Google Patents
Fluorescent in situ hybridization counting slide and counting method thereof Download PDFInfo
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- CN1632520A CN1632520A CN 200510002789 CN200510002789A CN1632520A CN 1632520 A CN1632520 A CN 1632520A CN 200510002789 CN200510002789 CN 200510002789 CN 200510002789 A CN200510002789 A CN 200510002789A CN 1632520 A CN1632520 A CN 1632520A
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Abstract
This invention relates to fluorescence home position cross counting plate and its method, which belongs to fluorescence home cross test technique field. The counting plate adopts the wave-pad composed of marking area and color covering area. The method is as the following: to take some sample to coat on the covered wave-pad cross area to make fluorescence home position cross; to record and store the images within n pieces of small square with image process software; to measure the fluorescence intensity of the images without overlapped cells to get the single cell fluorescence intensity; to separately measure other images fluorescence intensity and sum to get the total fluorescence intensity of the n pieces of images.
Description
Technical field
The invention belongs to the fluorescence in situ hybridization detection technical field, particularly the FISH design and the method for counting thereof of hybridization tally.
Background technology
Along with the development of present FISH (fluorescence in situ hybridization, fluorescence in situ hybridization) technology, the FISH technology has been widely used in monitoring biological community structure, function and dynamic dispatching aspect.But at present the cell quantity in the test sample do not had a good method of counting, come only can for a relative populations or relative scale, can not satisfy the requirement of present research, the structure of FISH microslide commonly used as shown in Figure 6 now, it is of a size of: long 5.75cm, wide 1.9cm, thick 0.1cm, nearly 1/4 colourless part on the microslide left side is a ground glass 1, be used for numbering, mark etc., 3/4 remaining part has then covered one deck dyestuff, in the zone 2 that dyestuff covers, there are several circles not cover the zone of dyestuff, for hybridizing zone 61.
This microslide does not have the function of counting, in most cases, can only provide a relative scale according to the number of each cell in the same visual field, and can not draw absolute quantity.Though, can calculate a relative dilution ratio according to the sample size, regional area and the visual field area of hybridization that spread upon in the hybridization zone, and ratio calculates the quantity of cells in sample thus, but may not guarantee that in operating process sample evenly spreads upon in the hybridization zone, so just can not guarantee that resulting dilution ratio is an entirely accurate, just can not be called and counted more accurately and calculate cell quantity in the sample by such ratio; As if the method for taking all visuals field in the border circular areas are counted one by one, owing to can't guarantee in counting process, not have the omission or the repetition in the visual field, so also can't guarantee the accurate counting of pair cell.Up to the present, when utilizing FISH technology for detection sample, also do not have a kind ofly can carry out the comparatively method of accurate counting to the absolute quantity of cells in sample.
Summary of the invention
The objective of the invention is at present can't be to this shortcoming of cells in sample quantity accurate counting in the FISH technology for overcoming, design a kind of novel fluorescence in situ hybridization tally, propose a kind of this tally that adopts simultaneously the cell in the sample is carried out the comparatively method of accurate counting.The present invention can compare counting accurately to the cell quantity in the sample.
A kind of fluorescence in situ hybridization tally that the present invention proposes adopts the microslide of being made up of marked region and dyestuff overlay area, is distributed with a plurality of hybridization zones that do not cover dyestuff in this dyestuff overlay area; It is characterized in that, in each hybridization zone, be divided into n little square,
R is microscopical diameter, when
The hybridization zone is a square during for integer; When
When being not integer, then hybridizing the zone and be one and longly be
Widely be
Rectangle, wherein a * b=n and a, b are integer.
The present invention propose to the Cytometric method in the sample, may further comprise the steps:
1) adopts a microslide of forming by marked region and dyestuff overlay area as the fluorescence in situ hybridization tally, be distributed with a plurality of hybridization zones that do not cover dyestuff in the dyestuff overlay area of this tally; In each hybridization zone, all be divided into n little square,
R is microscopical diameter, is numbered on each little square, with convenient counting;
2) getting a certain amount of testing sample evenly is applied in the hybridization zone of the microslide that the gelatin bag crossed and is carried out fluorescence in situ hybridization;
3) with fluorescent microscope a hybridization zone is observed, write down and store n the image in the little square simultaneously with imgae processing software;
4) in n image, find out the non-overlapping image of some width of cloth cells, respectively these visual fluorescence intensities are measured, obtain the total fluorescence intensity of these images, simultaneously the cell number in these images is counted, obtain the total cell number in these images, the two is divided by and obtains the fluorescence intensity of individual cells;
5) measure the fluorescence intensity and the addition of other image more respectively, obtain the total fluorescence intensity of n image,, promptly draw and be applied in the interior cell quantity in hybridization zone with the fluorescence intensity of this total fluorescence intensity divided by resulting individual cells;
6) according to the sample size that is applied in the hybridization zone, obtain the cell quantity in the testing sample.
Characteristics of the present invention and good effect
The present invention is by improveing with microslide original FISH hybridization, both: in several hybridization zones on the used microslide of FISH, all engrave n little square of calculating the length of side in each zone with certain relation, and deciding the shape and the size in hybridization zone by the little foursquare length of side and number, this novel FISH hybridization tally can be counted comparatively accurately to the cell in the sample.Method of counting is simply accurate, has greatly expanded FISH The Application of Technology scope, will make the FISH technology can be applied to aspects such as industrial microorganism detection, health quarantine, medical diagnosis on disease, food and water quality detection.
Description of drawings
Fig. 1 is a FISH hybridization tally structural representation of the present invention.
Fig. 2 is the another kind of structural representation of FISH hybridization tally of the present invention.
Fig. 3 is the square hybridization regional enlarged drawing synoptic diagram among Fig. 1.
Fig. 4 is the rectangle hybridization regional enlarged drawing synoptic diagram among Fig. 2.
The synoptic diagram that Fig. 5 examines under a microscope for hybridization of the present invention zone.
Fig. 6 is traditional F ISH hybridization slide configurations synoptic diagram.
Embodiment
FISH hybridization tally design that the present invention proposes and method of counting reach embodiment in conjunction with the accompanying drawings and are described further.
A kind of novel fluorescence in situ hybridization tally that the present invention proposes, be primarily characterized in that, the fluorescence in situ hybridization tally, for traditional microslide size both: long 5.75cm, wide 1.9cm, thick 0.1cm, nearly 1/4 colourless part of on the left side is a ground glass 1, be used for numbering, mark etc., 3/4 remaining part has then covered one deck dyestuff, in the zone 2 that dyestuff covers, several squares 11 (as shown in Figure 1) or rectangle uncovered area 21 (as shown in Figure 2) are arranged, then be the hybridization zone, all engrave n little square in each hybridization zone, this hybridization regional enlarged drawing is respectively as Fig. 3, shown in 4; Its medium and small square number of 111,211 is n, when
The hybridization zone is a square during for integer; When
When being not integer, then hybridizing the zone and be one and longly be
Widely be
Rectangle, wherein a * b=n and a, b are integer, r is microscopical diameter; A, b have many groups to separate, and can observe what visuals field according to user's needs and determine and choose optimal one group and separate and form rectangle.And each little foursquare length of side, by employed microscopical diameter r determine (both:
), just can see at microscopically like this: field of microscope just comprises a square 111, and the little square in the visual field is external in the circle 112 in the visual field, as shown in Figure 5.
The present invention propose to the Cytometric method in the sample, may further comprise the steps:
1) with a microslide of forming by marked region and dyestuff overlay area as the fluorescence in situ hybridization tally, be distributed with a plurality of hybridization zones that do not cover dyestuff in the dyestuff overlay area of this tally; In each hybridization zone, all be divided into n little square,
R is microscopical diameter, is numbered on each little square, with convenient counting;
2) getting a certain amount of testing sample evenly is applied in the microslide hybridization zone that the gelatin bag crossed and is carried out fluorescence in situ hybridization;
3) with fluorescent microscope a hybridization zone is observed, write down and store n the image in the little square simultaneously with imgae processing software;
4) in n image, find out the non-overlapping image of some width of cloth cells, respectively these visual fluorescence intensities are measured, obtain the total fluorescence intensity of these images, simultaneously the cell number in these images is counted, obtain the total cell number in these images, the two is divided by and obtains the fluorescence intensity of individual cells;
5) measure the fluorescence intensity and the addition of other image more respectively, obtain the total fluorescence intensity of n image,, promptly draw and be applied in the interior cell quantity in hybridization zone with the fluorescence intensity of this total fluorescence intensity divided by resulting individual cells;
6) according to the sample size that is applied in the hybridization zone, obtain the cell quantity in the testing sample.
Embodiment one
1. the diameter of general microscopical 40 times of mirrors is 0.42mm, then can calculate it in to connect the little foursquare length of side be 0.3mm, present embodiment is analyzed 256 visuals field, the microslide of the Cai Yonging square that to be provided with several (being generally 6,8,10) length of sides be 4.8mm hybridization zone then, each big square hybridization zone is divided into 256 little squares again.
2. from sample to be measured, take out a certain amount of gelatin bag that evenly is applied in and carried out fluorescence in situ hybridization in certain big square hybridization zone of the microslide of crossing.
3. use the fluorescent microscope microscopy, write down with imgae processing software and (only write down the image in the medium and small square in each visual field during record hybridizing image in 256 little squares in the zone, or the image in the record whole visual field, later image is handled, only kept the image in the medium and small square in the visual field).
4. in resulting 256 image the insides, find out the non-overlapping image of some width of cloth cells, count the total cell number in these images when their total fluorescence intensity is measured, the two is divided by and can obtains the fluorescence intensity of individual cells,
5. again the fluorescence intensity of 256 images is measured respectively and addition, fluorescence intensity divided by individual cells, just can calculate the cell quantity that is applied in the big square hybridization zone, because it is known being applied in the sample size in big square hybridization zone, just can obtain cell quantity in the testing sample according to ratiometric conversion so.
Embodiment two
1. the diameter of general microscopical 100 times of mirrors is 0.16mm, then can calculate it in to connect the little foursquare length of side be 0.11mm, present embodiment is analyzed 400 visuals field, the microslide of the Cai Yonging square that to be provided with several (being generally 6,8,10) length of sides be 2.2mm hybridization zone then, and each big square hybridization zone is divided into 400 little squares.
2. from sample to be measured, take out a certain amount of gelatin bag that evenly is applied in and carried out fluorescence in situ hybridization in certain big square hybridization zone of the microslide of crossing.
3. use the fluorescent microscope microscopy, write down with imgae processing software and (only write down the image in the medium and small square in each visual field during record hybridizing image in 400 little squares in the zone, or the image in the record whole visual field, later image is handled, only kept the image in the medium and small square in the visual field).
4. in resulting 400 image the insides, find out the non-overlapping image of some width of cloth cells, count the total cell number in these images when their total fluorescence intensity is measured, the two is divided by and can obtains the fluorescence intensity of individual cells,
5. again the fluorescence intensity of 400 images is measured respectively and addition, fluorescence intensity divided by individual cells, just can draw the cell quantity that is applied in the big square hybridization zone, because it is known being applied in the sample size in big square hybridization zone, just can obtain cell quantity in the testing sample according to ratiometric conversion so.
Embodiment three
1. the diameter of general microscopical 100 times of mirrors is 0.16mm, then can calculate it in to connect the little foursquare length of side be 0.11mm, present embodiment is analyzed 600 visuals field, then to be provided with several (being generally 6,8,10) length of sides be 3.3mm to the microslide of Cai Yonging, wide is that (when n=600, ab=n has a to 2.2mm
1=60, b
1=10; a
2=50, b
2=12; a
3=30, b
3Many groups such as=20 grades are separated, and we choose a group of helping mapping most and separate, i.e. a
3=30, b
3=20) rectangle hybridization zone, and each rectangle hybridization zone is divided into 600 little squares.
2. from sample to be measured, take out a certain amount of gelatin bag that evenly is applied in and carried out fluorescence in situ hybridization in certain rectangle hybridization zone of the microslide of crossing.
3. use the fluorescent microscope microscopy, image in 600 little squares in the rectangle write down with imgae processing software (only write down the image in the medium and small square in each visual field during record, or the image in the record whole visual field, later image is handled, only kept the image in the medium and small square in the visual field).
4. in resulting 600 image the insides, find out the non-overlapping image of some width of cloth cells, count the total cell number in these images when their total fluorescence intensity is measured, the two is divided by and can obtains the fluorescence intensity of individual cells,
5. again the fluorescence intensity of 600 images is measured respectively and addition, fluorescence intensity divided by individual cells, just can draw the cell quantity that is applied in the big square hybridization zone, because it is known being applied in the sample size in big square hybridization zone, just can obtain cell quantity in the testing sample according to ratiometric conversion so.
Claims (3)
1. a fluorescence in situ hybridization tally adopts the microslide of being made up of marked region and dyestuff overlay area, is distributed with a plurality of hybridization zones that do not cover dyestuff in this dyestuff overlay area; It is characterized in that, in each hybridization zone, be divided into n little square,
R is microscopical diameter, when
The hybridization zone is a square during for integer; When
When being not integer, then hybridizing the zone and be one and longly be
Widely be
Rectangle, wherein a * b=n and a, b are integer.
2. tally as claimed in claim 1 is characterized in that, each little square in described hybridization zone is provided with numbering.
3. the detection method of cell absolute quantity in the fluorescence in situ hybridization is characterized in that, may further comprise the steps:
1) adopts a microslide of forming by marked region and dyestuff overlay area as the fluorescence in situ hybridization tally, be distributed with a plurality of hybridization zones that do not cover dyestuff in the dyestuff overlay area of this tally; In each hybridization zone, all be divided into n little square,
R is microscopical diameter, is numbered on each little square, with convenient counting;
2) getting a certain amount of testing sample evenly is applied in the hybridization zone of the microslide that the gelatin bag crossed and is carried out fluorescence in situ hybridization;
3) with fluorescent microscope a hybridization zone is observed, write down and store n the image in the little square simultaneously with imgae processing software;
4) in n image, find out the non-overlapping image of some width of cloth cells, respectively these visual fluorescence intensities are measured, obtain the total fluorescence intensity of these images, simultaneously the cell number in these images is counted, obtain the total cell number in these images, the two is divided by and obtains the fluorescence intensity of individual cells;
5) measure the fluorescence intensity and the addition of other image more respectively, obtain the total fluorescence intensity of n image,, promptly draw and be applied in the interior cell quantity in hybridization zone with the fluorescence intensity of this total fluorescence intensity divided by resulting individual cells;
6) according to the sample size that is applied in the hybridization zone, obtain the total cell quantity in the testing sample.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100027895A CN1314955C (en) | 2005-01-28 | 2005-01-28 | Fluorescent in situ hybridization counting slide and counting method thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100027895A CN1314955C (en) | 2005-01-28 | 2005-01-28 | Fluorescent in situ hybridization counting slide and counting method thereof |
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| CN1632520A true CN1632520A (en) | 2005-06-29 |
| CN1314955C CN1314955C (en) | 2007-05-09 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102478489A (en) * | 2010-11-23 | 2012-05-30 | 北京汇丰隆经济技术开发有限公司 | Biological particle detection system based on gelatin detection baseband |
| CN102839208A (en) * | 2012-07-24 | 2012-12-26 | 北京工业大学 | Absolute quantification method for fluorescence in-situ hybrid cell counting |
| CN109313191A (en) * | 2016-05-27 | 2019-02-05 | 珀金埃尔默细胞科技德国公司 | The method for determining the quantity of the infection site of cell culture |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2073604U (en) * | 1990-07-30 | 1991-03-20 | 杜群军 | Blood counting board for the use of multicell |
| CN2377532Y (en) * | 1999-06-10 | 2000-05-10 | 中国人民解放军第二军医大学 | Microfluorecyte counting plate |
| TW587694U (en) * | 2003-03-14 | 2004-05-11 | Mau-Guei Jang | Protruded platform type quantitative cell counter plate |
-
2005
- 2005-01-28 CN CNB2005100027895A patent/CN1314955C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102478489A (en) * | 2010-11-23 | 2012-05-30 | 北京汇丰隆经济技术开发有限公司 | Biological particle detection system based on gelatin detection baseband |
| CN102839208A (en) * | 2012-07-24 | 2012-12-26 | 北京工业大学 | Absolute quantification method for fluorescence in-situ hybrid cell counting |
| CN109313191A (en) * | 2016-05-27 | 2019-02-05 | 珀金埃尔默细胞科技德国公司 | The method for determining the quantity of the infection site of cell culture |
| CN109313191B (en) * | 2016-05-27 | 2022-04-05 | 珀金埃尔默细胞科技德国公司 | Method for determining the number of infection sites of a cell culture |
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| CN1314955C (en) | 2007-05-09 |
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