CN1793862A - Optical detection method for membrane protein molecule interaction - Google Patents
Optical detection method for membrane protein molecule interaction Download PDFInfo
- Publication number
- CN1793862A CN1793862A CNA2005101115944A CN200510111594A CN1793862A CN 1793862 A CN1793862 A CN 1793862A CN A2005101115944 A CNA2005101115944 A CN A2005101115944A CN 200510111594 A CN200510111594 A CN 200510111594A CN 1793862 A CN1793862 A CN 1793862A
- Authority
- CN
- China
- Prior art keywords
- molecule
- matrix
- fluorescence intensity
- image
- cancellation
- Prior art date
- Legal status (The legal status 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 status listed.)
- Granted
Links
- 108010052285 Membrane Proteins Proteins 0.000 title claims abstract description 26
- 102000018697 Membrane Proteins Human genes 0.000 title claims abstract description 26
- 230000003287 optical effect Effects 0.000 title claims description 16
- 238000001514 detection method Methods 0.000 title claims description 15
- 230000003993 interaction Effects 0.000 title abstract description 9
- 238000000034 method Methods 0.000 claims abstract description 42
- 238000012545 processing Methods 0.000 claims abstract description 16
- 210000000170 cell membrane Anatomy 0.000 claims abstract description 9
- 238000006116 polymerization reaction Methods 0.000 claims abstract description 8
- 239000011159 matrix material Substances 0.000 claims description 83
- 108090000623 proteins and genes Proteins 0.000 claims description 28
- 102000004169 proteins and genes Human genes 0.000 claims description 27
- KZMAWJRXKGLWGS-UHFFFAOYSA-N 2-chloro-n-[4-(4-methoxyphenyl)-1,3-thiazol-2-yl]-n-(3-methoxypropyl)acetamide Chemical compound S1C(N(C(=O)CCl)CCCOC)=NC(C=2C=CC(OC)=CC=2)=C1 KZMAWJRXKGLWGS-UHFFFAOYSA-N 0.000 claims description 25
- 210000004027 cell Anatomy 0.000 claims description 15
- 239000004744 fabric Substances 0.000 claims description 11
- 238000003384 imaging method Methods 0.000 claims description 11
- 238000009826 distribution Methods 0.000 claims description 10
- 230000008569 process Effects 0.000 claims description 9
- 238000010586 diagram Methods 0.000 claims description 8
- 230000008859 change Effects 0.000 claims description 6
- 238000006471 dimerization reaction Methods 0.000 claims description 5
- 238000012632 fluorescent imaging Methods 0.000 claims description 5
- 239000007850 fluorescent dye Substances 0.000 claims description 4
- 238000001215 fluorescent labelling Methods 0.000 claims description 4
- 238000003672 processing method Methods 0.000 claims description 4
- 238000004458 analytical method Methods 0.000 claims description 3
- 239000002120 nanofilm Substances 0.000 claims description 3
- 238000001917 fluorescence detection Methods 0.000 claims description 2
- 238000010791 quenching Methods 0.000 abstract description 5
- 230000000171 quenching effect Effects 0.000 abstract description 5
- 238000000799 fluorescence microscopy Methods 0.000 abstract 1
- 238000002866 fluorescence resonance energy transfer Methods 0.000 description 9
- 230000006916 protein interaction Effects 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 230000003915 cell function Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- MURGITYSBWUQTI-UHFFFAOYSA-N fluorescin Chemical compound OC(=O)C1=CC=CC=C1C1C2=CC=C(O)C=C2OC2=CC(O)=CC=C21 MURGITYSBWUQTI-UHFFFAOYSA-N 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 241001522296 Erithacus rubecula Species 0.000 description 1
- 206010038743 Restlessness Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000010382 chemical cross-linking Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000000749 co-immunoprecipitation Methods 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000000879 optical micrograph Methods 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000004557 single molecule detection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000001086 yeast two-hybrid system Methods 0.000 description 1
Images
Landscapes
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
A method for optically detecting an interaction between membrane protein molecules, the method comprising the steps of: detecting a cell membrane by adopting a total internal reflection fluorescence imaging system, and acquiring a plurality of groups of images of target membrane protein molecules in different areas of the cell membrane, wherein each group of images corresponds to one area of the cell membrane and comprises a plurality of sequence pictures of the membrane protein molecules; performing data processing by using a computer, drawing a relation curve graph of fluorescence intensity and time, and judging the number of molecules of the polymerized membrane protein contained in the image by using the quenching times of the relation curve of the fluorescence intensity and the time; determining the position of each molecule in the polymerized membrane protein by adopting a center positioning method, thereby determining the space between the molecules of the membrane protein; and repeating the second step and the third step to carry out data processing on each group of images one by one, and drawing a quenching step statistical chart and a molecular distance statistical chart to obtain a statistical result of the polymerization degree and the molecular distance of the membrane protein.
Description
Technical field
The present invention relates to biology protein, interactional optical detection method between particularly a kind of membrane protein molecule.
Background technology
Protein is the main executive of cell function, the realization of cell function comes down in the cell or the result of iuntercellular range protein interaction of molecules, therefore the interaction of studying between the protein is a key of understanding chemical machinery running in the cell activities process, is the key subjects in the field of biology.Especially after the human genome examining order is finished, life science has entered the back genomics epoch, interacting between research protein and the protein on the molecular level, disclosing biological vital movement process under the body state becomes the opportunities and challenges that life science, information science face jointly.
But up to now, interactional means are very limited between the research protein molecule.Known Measurement for Biochemistry, as co-immunoprecipitation (Robin.L, J.Virology, Vol.71,9803～9807,1997), chemical crosslinking (Jean.D.G, PNAS, Vol.86,4007～4011,1989), fragment chromatography (Pedro.C, PNAS, Vol.61,636～643,1968) etc. method is in conjunction with the analysis that genetic manipulation produces protein mutant and protein structures, screened the interaction of many key proteins and its architecture basics is had gained some understanding, and brought abundant information to us.Yet these Biochemical Information all are the results of stripped (in vitro) research, can not react the situation under the physiological situation, can't answer interactional dynamics problem between living cells internal protein molecule.
Yeast-two hybrid technique is the method for studying protein interaction under body (in vivo) condition that extensively adopts at present, and its energy fast detecting protein interaction comprises corresponding structure territory and gene code (Fields S, Nature, Vol.340,245～246,1989; Chien C.T, PNAS, Vol.88,9578～9582,1991; Bai C, Methods Enzymol, Vol.283,141～156,1996).But this method also has intrinsic defective: 1. its screening system just can not be directly connects protein interaction and specific cell function; 2. and it can only detect endonuclear protein interaction, to research memebrane protein interaction then very difficult; 3. can cause false-positive error message (Rossi F, PNAS, Vol.94,8405～8410,1997) in some cases.
The optical microphotograph formation method can be realized surveying at body of protein interaction, but the spatial resolution of single imaging is subjected to the restriction of diffraction limit, can't break through 200nm, can't on more deep level, annotate detailed information (the Georgiou G.N of vital movement, FEBS.Lett, Vol.250,487-492,1989).Though the near field optic formation method can be realized the spatial resolution of nm level, because the restriction of its intrinsic detection principle is difficult to be implemented in body and surveys (Shen Yumin, Zhu Xing, physics, Vol.29,13,2000).
The FRET (fluorescence resonance energy transfer) technology (hereinafter to be referred as FRET) that grow up the nineties in last century, promoted Study of Interaction between the biomolecule greatly with the brand-new principle that is different from traditional means, its appearance makes us survey the interaction in 1～10nm scope becomes possibility, and can be used at body environment (Truong K, Curr.Opin.Struct.Biol, Vol.11,573～578,2001; Sorkin.A, Curr.Biol, Vol.10,1395～1398,2000).But the FRET method still is faced with a lot of problems, and for example it needs suitable D-A molecule to (Pollok B A, Trends.Cell.Biol, Vol.9,57～60,1999; Sekar R B, J.Cell.Biol, Vol.160,629～633,2003); And to from FRET efficient, obtain the range information of molecule, also must obtain molecule between R
0(Forster radius, the efficient of FRET are 50% o'clock donor, the distance between the acceptor) and molecular dipole orientation factor κ
2Authentic communication, otherwise just can't utilize FRET efficient to carry out accurate distance calculation (Siegel R M, Sci STKE, Vol.38, PL1,2000; Dietrich A, Biotechnology, Vol.82,211～231,2002).Because most at present FRET technology still is the level that is applied in whole cell, and that obtain is the result of ensemble average, this has covered from the right unique information of individual molecule on the other hand.Therefore, development of life science presses for the ways and means that provides new, solves interactional detection problem between the molecule.
Summary of the invention
The present invention is directed to the deficiency of above-mentioned technology formerly to interaction detection aspect between the molecule, especially the deficiency of under concrete conditions in the establishment of a specific crime, surveying, propose a kind of in living cells about interactional optical detection method between the membrane protein molecule, to obtain the interactional situation of membrane protein molecule.
Technical solution of the present invention is as follows:
Interactional optical detection method between a kind of membrane protein molecule is characterized in that this method comprises the following steps:
The first step adopts the total internal reflection fluorescent imaging system to survey cell membrane, and the zones of different of target film albumen is obtained the images of organizing memebrane proteins more, a zone of every group of corresponding cell membrane of image, and every group of image comprises several molecular sequences pictures;
Second step utilized computing machine to carry out data processing, drew fluorescence intensity and time relation curve map, and the cancellation number of times of employing fluorescence intensity and time relation curve is judged the number of contained membrane protein molecule in the image;
The 3rd step adopted the centralized positioning method to determine the position of each molecule in the memebrane protein;
The 4th step repeated for second step and the 3rd step was carried out data processing one by one to every group of image, drew cancellation step statistical graph and intermolecular distance statistical graph, with the extent of polymerization of acquisition memebrane protein and the statistics of intermolecular distance.
The concrete steps of the described first step are as follows:
1. in order to carry out fluorescence detection, target film protein molecular to be measured is done fluorescence labeling;
The living cells that 2. will have fluorescently-labeled target film protein molecular places the total internal reflection fluorescent imaging system, take the image of many group membrane protein molecules with the zones of different of high-sensitive CCD pair cell, every group of corresponding zone of image, every group of image comprises several molecular sequences pictures;
The process of the data processing in described second step is as follows:
1. each width of cloth picture in the set of diagrams picture is measured, obtained fluorescence intensity maximal value or fluorescence intensity mean value, draw fluorescence intensity and time relation curve map, be called for short fluorescence intensity figure;
2. analysis of fluorescence intensity map, fluorescence intensity among this figure relation curve in time presents stair-stepping decline, and the sudden change that descends the first time of fluorescence intensity is called first step cancellation, and the sudden change that descends for the second time is called the second step cancellation, the sudden change that descends for the third time is called the 3rd step cancellation, so analogizes; In this fluorescence intensity curves, before the first step cancellation the zone be called a interval, be called the b interval between the first step cancellation and the second step cancellation, the second step cancellation and the 3rd goes on foot between the cancellation and is called the c interval, Judge the extent of polymerization of target film albuminous cell according to the cancellation number of times of fluorescence intensity figure: the polymerization of albumen does not take place in step cancellation explanation, contain two molecules in the two step cancellation representative pictures, the dimerization that is called albumen, three step cancellation show that three molecules are arranged, the dimerization of albumen has taken place, by that analogy.
Described centralized positioning method is a kind of data processing method, and is as follows to the data handling procedure of bimolecular memebrane protein:
1. a certain single picture that is in the b interval with a certain group of target film albumen image reads in computing machine, promptly obtains one 10 * 10 two-dimentional intensity matrix, is called original matrix I
1, this matrix I
1The fluorescence intensity that includes molecule 1 distributes;
2. to original matrix I
1Do statistic histogram, the fluorescence intensity cut off value is set at the background value B of image among this figure;
3. to original matrix I
1The fluorescence intensity subtracting background value B row interpolation of going forward side by side handle, form 100 * 100 new matrix I
1';
4. be curvilinear equation with following formula (1), I
1' in matrix element be data point, utilize least square method that image is carried out match, the central point that obtains image is the position coordinates (x of molecule 1
1, y
1),
In the formula: (x
1, y
1) center of promptly putting object; N
Xy, N
00Be respectively that (x y) locates and center (x pixel
1, y
1) the imaging intensity level located; S is the full width at half maximum of point spread function;
5. a certain picture with a interval reads in computing machine, promptly obtains one 10 * 10 two-dimentional intensity matrix, is called original matrix I
2, this matrix I
2The fluorescence intensity that includes molecule 1 and molecule 2 distributes;
6. with matrix I
2Subtract matrix I
1, the fluorescence intensity distribution matrix that obtains molecule 2 row interpolation of going forward side by side is handled, 100 * 100 fluorescence intensities that the form molecule 2 new matrix I that distributes
2';
7. 4. carry out data processing by above-mentioned steps, obtain the position coordinates (x of molecule 2
2, y
2);
8. calculate the distance between molecule 1 and the molecule 2:
d(2，1)
x＝|x
2-x
1|；d(2，1)
y＝|y
2-y
1|。
Described centralized positioning method is a kind of data processing method, and is as follows to the data handling procedure of three molecular film albumen:
1. a certain single picture that is in the c interval with a certain group of target film albumen image reads in computing machine, promptly obtains one 10 * 10 two-dimentional intensity matrix, is called original matrix I
1, this matrix I
1The fluorescence intensity that includes molecule 1 distributes;
2. to original matrix I
1Do statistic histogram, the fluorescence intensity cut off value is set at the background value B of image among this figure;
3. to original matrix I
1Fluorescence intensity carry out interpolation processing, form 100 * 100 new matrix I
1';
4. be curvilinear equation with following formula (1), I
1' in matrix element be data point, utilize least square method that image is carried out match, the central point that obtains image is the position coordinates (x of molecule 1
1, y
1),
In the formula: (x
1, y
1) center of promptly putting object; N
Xy, N
00Be respectively that (x y) locates and center (x pixel
1, y
1) the imaging intensity level located; S is the full width at half maximum of point spread function;
5. a certain picture with the b interval reads in computing machine, promptly obtains one 10 * 10 two-dimentional intensity matrix, is called original matrix I
2, this matrix I
2The fluorescence intensity that includes molecule 1 and molecule 2 distributes;
6. with matrix I
2Subtract matrix I
1, the fluorescence intensity distribution matrix that obtains molecule 2 row interpolation of going forward side by side is handled, 100 * 100 fluorescence intensities that the form molecule 2 new matrix I that distributes
2';
7. 4. carry out data processing by above-mentioned steps, obtain the position coordinates (x of molecule 2
2, y
2);
8. a certain picture with a interval reads in computing machine, promptly obtains one 10 * 10 two-dimentional intensity matrix, is called original matrix I
3, this matrix I
3The fluorescence intensity that includes molecule 1, molecule 2 and molecule 3 distributes; From original matrix I
3Deduct matrix I
2, the fluorescence intensity distribution matrix that obtains molecule 3 row interpolation of going forward side by side is handled, 100 * 100 fluorescence intensities that the form molecule 3 new matrix I that distributes
3' 4. carry out data processing by above-mentioned steps, obtain the position coordinates (x of molecule 3
3, y
3);
9. calculate the distance between molecule 1, molecule 2 and the molecule 3:
Distance between molecule 1, the molecule 2:
d(2，1)
x＝|x
2-x
1|
d(2，1)
y＝|y
2-y
1|；
Distance between molecule 1, the molecule 3:
d(3，1)
x＝|x
3-x
1|
d(3，1)
y＝|y
3-y
1|；
Distance between molecule 2, the molecule 3:
d(3，2)
x＝|x
3-x
2|
d(3，2)
y＝|y
3-y
2|。
About the unexpected variation in time of monomolecular fluorescence intensity, available statistical theory is explained.For molecule, the optical quenching incident is a Poisson process, and it defers to exponential distribution
In the formula, P (t) is the probability in the cancellation of moment t fluorescence molecule; τ is the time constant of optical quenching, it depend on molecule absorption cross section, be optical physics parameter and external environment conditions such as a transit time.If two or more molecular aggregatess are together, because the optical quenching time constant difference of different molecular will show the phenomenon that fluorescence intensity repeatedly changes suddenly, i.e. multistep cancellation, the step number of cancellation equals to assemble the number of molecule.
Monomolecular accurate location is based on such fact: though because the restriction of diffraction limit, imaging system can't be differentiated the size of object, but, can carry out unlimited accurate recovery, so-called centralized positioning method that Here it is to the center of object according to the hot spot that object became.M.K Cheezum (Biophys.J.Vol.81,2378-2388,2001) and R.E Thompson (Biophys.J.Vol.82,2775-2783,2002) etc. people's research is pointed out, for the low signal-to-noise ratio imaging, the centralized positioning method adopts two-dimensional Gaussian function that the diffraction limit light spot image of object is carried out match, can obtain best positioning result.
The expression formula of two-dimensional Gaussian function is
In the formula, (x
0, y
0) center of promptly putting object; N
Xy, N
00Be respectively that (x y) locates and center (x pixel
0, y
0) the imaging intensity level located; S is the full width at half maximum of point spread function; B is a background, and it comprises readout noise, dark current and the photon scattering noise of CCD.
The degree of accuracy that the method for centralized positioning simultaneously can reach and the relating to parameters of imaging system, the formula of embodying is
N is a signal strength values sum in the image; A is the Pixel Dimensions of detector; B is the standard deviation of noise.
Obviously, characteristics of the present invention are quantitatively to obtain the relative distance between two or more molecules, and can obtain relative distance over time.This method has remedied the shortcoming that the FRET technology can only be surveyed the intermolecular distance of 1～10nm, simultaneously also having broken through the restriction of optical microscope (far field) resolution greater than 200nm, is a kind of optics new method to interacting and survey between the membrane protein molecule.
The invention has the advantages that:
1, at the interactional lossless detection of body memebrane protein.
2, the diffraction limit resolution of big quantum jump far-field optics imaging.
3, the accurate of distance determined between the interactional molecule of generation.
Description of drawings
Fig. 1 is three step cancellation synoptic diagram of molecular fluorescence intensity and time relation curve map
Fig. 2 is one group of membrane protein molecule sequence of pictures of the embodiment of the invention
Fig. 3 is two step cancellation synoptic diagram of embodiment of the invention membrane protein molecule fluorescence intensity and time relation curve map
Fig. 4 is the 25th width of cloth molecule picture among Fig. 2
Fig. 5 is that Fig. 4 Molecular Graphs is as original matrix I
1Statistic histogram
Fig. 6 is the Flame Image Process process flow diagram
Fig. 7 is the three-dimensional fluorescence intensity displayed map of Fig. 4 molecule picture match
Fig. 8 is the 10th width of cloth molecule picture among Fig. 2
Fig. 9 is the three-dimensional fluorescence intensity displayed map of the 10th width of cloth molecule picture match
Figure 10 is the statistical graph of embodiment of the invention cancellation step
Figure 11 is the statistical graph of embodiment of the invention intermolecular distance
Embodiment
The invention will be further described below in conjunction with embodiment and accompanying drawing, but should not limit protection scope of the present invention with this.
See also Fig. 2,3,4,5,6,7,8,9 and Figure 10, Fig. 2 to Figure 10 is the main synoptic diagram in each stage of the interactional optical detection method of memebrane protein of the present invention embodiment, and the process of present embodiment is as follows:
＜1〉the target film albumen in the living cells to be measured is done fluorescence labeling.Make up the fusion plasmid of memebrane protein and fluorescin in the present embodiment earlier, be transfected into then in the cell, finish the fluorescence labeling of testing sample.Owing to be single-molecule detection, must control and make after the transfection concentration of protein expression very low.
＜2〉in the total internal reflection fluorescent imaging system, take the unicellular image of many group target film albumen with high-sensitive CCD in the zones of different of cell membrane, take 100 groups altogether, every group of corresponding zone, every group contains 60 unicellular sequence of pictures, sees Fig. 2.The present invention adopts concrete parameter to be: object lens NA=1.45, enlargement ratio be 100 *; λ=570nm, CCD are of a size of 0.06 μ m/pixel (pixel is a pixel), and the physical distance of the corresponding imaging space of each pixel of CCD is 60nm like this; The size in each zone is 10 * 10 pixels; The time shutter of every width of cloth picture is 200ms in one group, and be 0 the shooting time delay between the picture, and every group of T.T. of taking is 12s.
＜3〉first group of image analyzed, wherein contained 60 width of cloth pictures.Obtain maximum of intensity or average strength from every unicellular picture, present embodiment is got the fluorescence intensity maximal value through measurement, totally 60 data, and data are as shown in table 1 below:
Table 1
| The picture numbering | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Maximum of intensity (arbitrary unit) | 300 | 202 | 206 | 205 | 209 | 208 | 205 | 200 | 208 | 204 |
| The picture numbering | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| Maximum of intensity (arbitrary unit) | 206 | 208 | 209 | 207 | 202 | 204 | 209 | 209 | 204 | 209 |
| The picture numbering | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 |
| Maximum of intensity (arbitrary unit) | 102 | 102 | 106 | 103 | 102 | 100 | 107 | 104 | 109 | 105 |
| The picture numbering | 31 | 32 | 33 | 34 | 35 | 36 | 37 | 38 | 39 | 40 |
| Maximum of intensity (arbitrary unit) | 104 | 108 | 105 | 102 | 107 | 108 | 100 | 107 | 104 | 108 |
| The picture numbering | 41 | 42 | 43 | 44 | 45 | 46 | 47 | 48 | 49 | 50 |
| Maximum of intensity (arbitrary unit) | 2 | 7 | 3 | 5 | 2 | 7 | 4 | 9 | 9 | 6 |
| The picture numbering | 51 | 52 | 53 | 55 | 55 | 56 | 57 | 58 | 59 | 60 |
| Maximum of intensity (arbitrary unit) | 5 | 9 | 8 | 6 | 8 | 7 | 3 | 3 | 4 | 5 |
Data according to table 1 are drawn fluorescence intensity maximal value and time relation curve map, are called for short fluorescence intensity figure, see Fig. 3.As we can see from the figure, the fluorescence intensity of molecule point presents two step cancellation, contains two molecules in the key diagram picture.
＜5〉select a width of cloth molecule picture arbitrarily in the interval b of Fig. 3, present embodiment is chosen the 25th width of cloth picture, sees Fig. 4.Picture is imported computing machine handle, process flow diagram is seen Fig. 6, and concrete treatment step is as follows:
1. read in image, image is shown as a two-dimentional fluorescence intensity matrix I in computing machine
1, see Table 2.This matrix is called original matrix, and size is 10 * 10, and unit is an arbitrary unit:
2. to original matrix I
1Do statistic histogram, see Fig. 5.Intensity cut off value in the histogram is the cut off value of ground unrest and flashlight, and this cut off value is set at the background value of image, is the B value in the formula (1), B=20.
3. to two-dimentional fluorescence intensity matrix I
1Fluorescence intensity subtracting background value B value do interpolation processing, obtain a new matrix I
1', its size is 100 * 100;
4. be curvilinear equation with formula (1), I
1In matrix element be data point, carry out the curve fitting of least square method.Fitting result is seen Fig. 7.
5. obtain the position coordinates (x of molecule 1
1, y
1)=(5.4309,5.2499) pixel.
Table 2
| 15.291 | 16.747 | 16.019 | 16.747 | 20.388 | 13.106 | 22.572 | 26.941 | 13.835 | 15.291 |
| 21.844 | 18.203 | 24.028 | 12.378 | 18.203 | 24.756 | 11.65 | 13.835 | 18.203 | 18.203 |
| 25.485 | 19.66 | 17.475 | 21.116 | 17.475 | 16.019 | 19.66 | 16.747 | 20.388 | 18.931 |
| 14.563 | 21.844 | 21.116 | 56.604 | 87.242 | 82.079 | 48.565 | 17.475 | 15.291 | 17.475 |
| 17.475 | 24.028 | 17.475 | 63.979 | 96.46 | 95.467 | 51.254 | 16.019 | 19.66 | 21.116 |
| 24.028 | 24.756 | 22.572 | 65.159 | 97.677 | 102 | 66.342 | 21.116 | 19.66 | 24.028 |
| 18.931 | 18.203 | 23.3 | 33.352 | 44.577 | 42.364 | 30.233 | 12.378 | 18.931 | 24.028 |
| 18.203 | 18.931 | 14.563 | 19.66 | 17.475 | 16.747 | 18.931 | 18.203 | 15.291 | 21.844 |
| 21.844 | 18.931 | 20.388 | 14.563 | 19.66 | 21.116 | 21.844 | 21.116 | 12.378 | 16.019 |
| 16.747 | 21.116 | 16.019 | 20.388 | 12.378 | 17.475 | 14.563 | 18.931 | 18.203 | 11.65 |
Select a width of cloth molecule picture in a interval of Fig. 3 arbitrarily, present embodiment is chosen the 10th width of cloth picture, sees Fig. 8, and the input computing machine is handled, and treatment scheme is as follows:
6. read in this picture, obtain another two-dimentional intensity matrix, be called original matrix I
2The fluorescence intensity that comprises molecule 1 and molecule 2 in this matrix distributes;
7. with matrix I
2With matrix I
1Subtract each other, promptly obtain the intensity distributions of molecule 2;
8. repeat the 4. step of above-mentioned treatment scheme, fitting result is seen Fig. 9, obtains the position coordinates (x of molecule 2
2, y
2)=(5.4287,5.4577) pixel.
9. calculate molecule 1 and molecule 2 between distance be d
x=0.2nm, d
y=12.5nm.
10. 99 groups of images of remainder are repeated abovely to analyze and take statistics, obtain the statistical graph of cancellation step, see Figure 10.The fluorescence intensity of most of molecules (55/100) presents two step cancellation, illustrates that the interaction between the target film albumen shows as dimerization.From 55 groups of images that show as dimerization, calculate 55 groups of intermolecular distances, make the statistical graph of intermolecular distance, see Figure 11, the range distribution between the molecule presents the normal distribution that central value is 12nm, further specify protein molecular and be not random collision together, but interaction has taken place.
Claims (5)
1, interactional optical detection method between a kind of membrane protein molecule is characterized in that this method comprises the following steps:
The first step adopts the total internal reflection fluorescent imaging system to survey cell membrane, obtains the images of organizing the target film protein moleculars in the zones of different of cell membrane more, a zone of every group of corresponding cell membrane of image, and every group of image comprises the sequence of pictures of several membrane protein molecules;
Second step utilized computing machine to carry out data processing, drew fluorescence intensity and time relation curve map, and the cancellation number of times of employing fluorescence intensity and time relation curve is judged the number of contained membrane protein molecule in the image;
The 3rd step adopted the centralized positioning method to determine the position of each molecule in the memebrane protein of polymerization, determine thus memebrane protein between spacing;
The 4th step repeated for second step and the 3rd step was carried out data processing one by one to every group of image, drew cancellation step statistical graph and intermolecular distance statistical graph, with the extent of polymerization of acquisition memebrane protein and the statistics of intermolecular distance.
2, interactional optical detection method between the membrane protein molecule according to claim 1 is characterized in that the concrete steps of the described first step are as follows:
1. in order to carry out fluorescence detection, the living cells of target film protein molecular to be measured is done fluorescence labeling;
The living cells that 2. will have fluorescently-labeled target film protein molecular places the total internal reflection fluorescent imaging system, take the image of many group membrane protein molecules with the zones of different of high-sensitive CCD pair cell, every group of corresponding zone of image, every group of image comprises several molecular sequences pictures.
3, interactional optical detection method between the membrane protein molecule according to claim 1 is characterized in that the process of described second data processing that goes on foot is as follows:
1. each width of cloth picture in the set of diagrams picture is measured, obtained fluorescence intensity maximal value or fluorescence intensity mean value, draw fluorescence intensity and time relation curve map, be called for short fluorescence intensity figure;
2. analysis of fluorescence intensity map, fluorescence intensity among this figure relation curve in time presents ladder and descends, and the sudden change that descends the first time of fluorescence intensity is called first step cancellation, and the sudden change that descends for the second time is called the second step cancellation, the sudden change that descends for the third time is called the 3rd step cancellation, so analogizes; In this fluorescence intensity curves, before the first step cancellation the zone be called a interval, be called the b interval between the first step cancellation and the second step cancellation, the 3rd step cancellation and second goes on foot between the cancellation and is called the c interval, Judge the extent of polymerization of target film albumen according to the cancellation number of times of fluorescence intensity figure: the polymerization of albumen does not take place in step cancellation explanation, contain two molecules in the two step cancellation representative pictures, the dimerization that is called albumen, three step cancellation show that three molecules are arranged, the trimerizing of albumen has taken place, by that analogy.
4, interactional optical detection method between the membrane protein molecule according to claim 1 is characterized in that described centralized positioning method is a kind of data processing method, and is as follows to the data handling procedure of two molecular film albumen:
1. a certain single picture that is in the b interval with a certain group of target film albumen image reads in computing machine, promptly obtains the two-dimentional intensity matrix of a 10x10, is called original matrix I
1, this matrix I
1The fluorescence intensity that includes molecule 1 distributes;
2. to original matrix I
1Do statistic histogram, the fluorescence intensity cut off value is set at the background value B of image among this figure;
3. to original matrix I
1The fluorescence intensity subtracting background value B row interpolation of going forward side by side handle, form the new matrix I of 100x100
1';
4. be curvilinear equation with following formula (1), I
1' in matrix element be data point, utilize least square method that image is carried out match, obtain the central point of image, be the position coordinates (x of molecule 1
1, y
1),
In the formula: (x
1, y
1) center of promptly putting object; N
Xy, N
00Be respectively that (x y) locates and center (x pixel
1, y
1) the imaging intensity level located; S is the full width at half maximum of point spread function;
5. a certain picture with a interval reads in computing machine, promptly obtains the two-dimentional intensity matrix of a 10x10, is called original matrix I
2, this matrix I
2The fluorescence intensity that includes molecule 1 and molecule 2 distributes;
6. with matrix I
2Subtract matrix I
1, the fluorescence intensity distribution matrix that obtains molecule 2 row interpolation of going forward side by side is handled, the 100x100 fluorescence intensity that the forms molecule 2 new matrix I that distributes
2';
7. 4. carry out data processing by above-mentioned steps, obtain the position coordinates (x of molecule 2
2, y
2);
8. calculate the distance between molecule 1 and the molecule 2:
d(2，1)x＝|x
2-x
1|； d(2，1)
y＝|y
2-y
1|。
5, interactional optical detection method between the membrane protein molecule according to claim 1 is characterized in that described centralized positioning method is a kind of data processing method, and is as follows to the data handling procedure of three molecular film albumen:
1. a certain single picture that is in the c interval with a certain group of target film albumen image reads in computing machine, obtains the two-dimentional intensity matrix of a 10x10, is called original matrix I
1, this matrix I
1The fluorescence intensity that includes molecule 1 distributes;
2. to original matrix I
1Do statistic histogram, the fluorescence intensity cut off value is set at the background value B of image among this figure;
3. with original matrix I
1The fluorescence intensity subtracting background value B row interpolation of going forward side by side handle, form the new matrix I of 100x100
1';
4. be curvilinear equation with following formula (1), I
1' in matrix element be data point, utilize least square method that image is carried out match, the central point that obtains image is the position coordinates (x of molecule 1
1, y
1),
In the formula: (x
1, y
1) center of promptly putting object; N
Xy, N
00Be respectively that (x y) locates and center (x pixel
1, y
1) the imaging intensity level located; S is the full width at half maximum of point spread function;
5. a certain picture with the b interval reads in computing machine, obtains the two-dimentional intensity matrix of a 10x10, is called original matrix I
2, this matrix I
2The fluorescence intensity that includes molecule 1 and molecule 2 distributes;
6. with matrix I
2Subtract matrix I
1, the fluorescence intensity distribution matrix that obtains molecule 2 row interpolation of going forward side by side is handled, the 100x100 fluorescence intensity that the forms molecule 2 new matrix I that distributes
2';
7. 4. carry out data processing by above-mentioned steps, obtain the position coordinates (x of molecule 2
2, y
2);
8. a certain picture with a interval reads in computing machine, obtains the two-dimentional intensity matrix of a 10x10, is called original matrix I
3, this matrix I
3The fluorescence intensity that includes molecule 1, molecule 2 and molecule 3 distributes; From original matrix I
3Deduct matrix I
2, the fluorescence intensity distribution matrix that obtains molecule 3 row interpolation of going forward side by side is handled, the 100x100 fluorescence intensity that the forms molecule 3 new matrix I that distributes
3' 4. carry out data processing by above-mentioned steps, obtain the position coordinates (x of molecule 3
3, y
3);
9. calculate the distance between molecule 1, molecule 2 and the molecule 3:
Distance between molecule 1, the molecule 2:
d(2，1)
x＝|x
2-x
1|
d(2，1)
y＝|y
2-y
1|；
Distance between molecule 1, the molecule 3:
d(3，1)
x＝|x
3-x
1|
d(3，1)
y＝|y
3-y
1|；
Distance between molecule 2, the molecule 3:
d(3，2)
x＝|x
3-x
2|
d(3，2)
y＝|y
3-y
2|。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005101115944A CN100570337C (en) | 2005-12-16 | 2005-12-16 | Optical detection method for membrane protein molecule interaction |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005101115944A CN100570337C (en) | 2005-12-16 | 2005-12-16 | Optical detection method for membrane protein molecule interaction |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1793862A true CN1793862A (en) | 2006-06-28 |
| CN100570337C CN100570337C (en) | 2009-12-16 |
Family
ID=36805430
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2005101115944A Expired - Fee Related CN100570337C (en) | 2005-12-16 | 2005-12-16 | Optical detection method for membrane protein molecule interaction |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100570337C (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103983555A (en) * | 2014-05-28 | 2014-08-13 | 国家纳米科学中心 | Method for detecting interaction of biomolecules |
| CN104105964A (en) * | 2011-12-05 | 2014-10-15 | 株式会社医学生物学研究所 | Method for detecting protein-protein interaction |
| CN105243677A (en) * | 2015-09-02 | 2016-01-13 | 华中科技大学 | Real-time single-molecule positioning method guaranteeing precision and system thereof |
| CN106661603A (en) * | 2014-06-03 | 2017-05-10 | 韩国科学技术院 | Method for analyzing activation state of signal path and method for selecting personalized medicine using the same |
| CN108195801A (en) * | 2017-11-17 | 2018-06-22 | 北京林业大学 | Single molecules level observation Stomacal guard cell memebrane protein distribution and dynamic method |
| CN108229097A (en) * | 2016-12-09 | 2018-06-29 | 深圳市瀚海基因生物科技有限公司 | Monomolecular identification, method of counting and device |
| CN108229098A (en) * | 2016-12-09 | 2018-06-29 | 深圳市瀚海基因生物科技有限公司 | Monomolecular identification, method of counting and device |
| CN113008842A (en) * | 2019-12-20 | 2021-06-22 | 中国科学院物理研究所 | Fluorescence imaging method for observing biomacromolecule on surface and nearby cell membrane of living cell |
| CN113049552A (en) * | 2021-03-07 | 2021-06-29 | 天津大学 | MUC1 protein quantitative detection method based on exosome detection and single-molecule fluorescence bleaching technology |
-
2005
- 2005-12-16 CN CNB2005101115944A patent/CN100570337C/en not_active Expired - Fee Related
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104105964A (en) * | 2011-12-05 | 2014-10-15 | 株式会社医学生物学研究所 | Method for detecting protein-protein interaction |
| CN103983555A (en) * | 2014-05-28 | 2014-08-13 | 国家纳米科学中心 | Method for detecting interaction of biomolecules |
| CN103983555B (en) * | 2014-05-28 | 2016-04-20 | 国家纳米科学中心 | A kind of method detecting bio-molecular interaction |
| CN106661603A (en) * | 2014-06-03 | 2017-05-10 | 韩国科学技术院 | Method for analyzing activation state of signal path and method for selecting personalized medicine using the same |
| CN105243677A (en) * | 2015-09-02 | 2016-01-13 | 华中科技大学 | Real-time single-molecule positioning method guaranteeing precision and system thereof |
| CN105243677B (en) * | 2015-09-02 | 2019-10-01 | 华中科技大学 | A kind of real-time unimolecule localization method and system guaranteeing precision |
| CN108229097A (en) * | 2016-12-09 | 2018-06-29 | 深圳市瀚海基因生物科技有限公司 | Monomolecular identification, method of counting and device |
| CN108229098A (en) * | 2016-12-09 | 2018-06-29 | 深圳市瀚海基因生物科技有限公司 | Monomolecular identification, method of counting and device |
| CN108195801A (en) * | 2017-11-17 | 2018-06-22 | 北京林业大学 | Single molecules level observation Stomacal guard cell memebrane protein distribution and dynamic method |
| CN108195801B (en) * | 2017-11-17 | 2020-11-24 | 北京林业大学 | Method for observing pore guard cell membrane protein distribution and dynamics at single molecule level |
| CN113008842A (en) * | 2019-12-20 | 2021-06-22 | 中国科学院物理研究所 | Fluorescence imaging method for observing biomacromolecule on surface and nearby cell membrane of living cell |
| CN113008842B (en) * | 2019-12-20 | 2022-05-24 | 中国科学院物理研究所 | Fluorescence imaging method for observing surface of cell membrane of living cell and nearby biomacromolecules |
| CN113049552A (en) * | 2021-03-07 | 2021-06-29 | 天津大学 | MUC1 protein quantitative detection method based on exosome detection and single-molecule fluorescence bleaching technology |
| CN113049552B (en) * | 2021-03-07 | 2022-08-05 | 天津大学 | MUC1 protein quantitative detection method based on exosome detection and single-molecule fluorescence bleaching technology |
Also Published As
| Publication number | Publication date |
|---|---|
| CN100570337C (en) | 2009-12-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1793862A (en) | Optical detection method for membrane protein molecule interaction | |
| US20220154274A1 (en) | Method and System for Multiplex Genetic Analysis | |
| US12106828B2 (en) | Systems and devices for signal corrections in pixel-based sequencing | |
| EP4394778A2 (en) | Systems and methods for characterization and performance analysis of pixel-based sequencing | |
| US6914229B2 (en) | Signal offset for prevention of data clipping in a molecular array scanner | |
| JP6291842B2 (en) | Detection method, microarray analysis method, and fluorescence reader | |
| Claessen et al. | Single-biomolecule kinetics: the art of studying a single enzyme | |
| CA2825727C (en) | Microarray analysis method and microarray reading device | |
| US20050239113A1 (en) | Methods and devices for microarray image | |
| JP2017504307A (en) | Method and system for digitally counting features on an array | |
| US20220301657A1 (en) | Tile location and/or cycle based weight set selection for base calling | |
| May et al. | Tethered fluorophore motion: studying large DNA conformational changes by single-fluorophore imaging | |
| US7173701B2 (en) | CCD-based biochip reader | |
| Sengar et al. | Deep learning aided small-sized portable fluorescence biochip reader | |
| JP4321716B2 (en) | Fluorescence image correction method, apparatus, and program | |
| Liu et al. | Two-channel, quasi-confocal parallel scan fluorescence imaging for detection of biochips | |
| CN113874712B (en) | Positioning microscopy on chip | |
| US20240100518A1 (en) | Flow cell based motion system calibration and control methods | |
| Schneider | Statistical analysis of biomolecular clustering and oligomerization | |
| WO2024229069A2 (en) | Apparatus and method for computational compensation of under-corrected aberrations | |
| Kalb et al. | Three-dimensional single-molecule tracking in living cells | |
| Steindel et al. | Studying the Dynamics of Chromatin-Binding Proteins in Mammalian Cells Using Single-Molecule Localization Microscopy | |
| CA3183581A1 (en) | Tile location and/or cycle based weight set selection for base calling | |
| CN117859086A (en) | Sequencer focus quality metrics and focus tracking for periodically patterned surfaces | |
| Visalli et al. | Microarrays as a Tool for Gene Expression Profiling: Application in Ocular and kk |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20091216 Termination date: 20111216 |