CN101025397A - Cell-growth automatic detector and method - Google Patents
Cell-growth automatic detector and method Download PDFInfo
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- CN101025397A CN101025397A CN 200610030824 CN200610030824A CN101025397A CN 101025397 A CN101025397 A CN 101025397A CN 200610030824 CN200610030824 CN 200610030824 CN 200610030824 A CN200610030824 A CN 200610030824A CN 101025397 A CN101025397 A CN 101025397A
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Abstract
The invention relates to an automatic detector of cell growth and method. The invented detector includes machine platform to hang cell culture table and light microscope to aim at the machine platform, the light microscope connect a camera, the output of camera input the computer per the image collect card, the one output of the computer is display, the another output of the computer is printer, and the another output controls the movement of machine platform per a controller. The method of invention operates on two manner of successive (automation) scanning pattern and flying sopt (selected) scanning pattern in. Under the successive (automation) scanning pattern, it can acquire the image of the all cell on the cell culture table of the platform; under the flying sopt (selected) scanning pattern, it can acquire the image of specific selected cell on the cell culture of the platform. Under the two patterns, each sighting port has the complete image display, and stored in the computer image file form, it supplies for off lining analysis. The invention of the detector and the method has the advantages of fast and automatic, can automatic batch detect the cell growth.
Description
Technical field
The present invention relates to a kind of porous cell culture plate cell growth condition be observed automatic observational record instrument and method with image recording.Can also can under flying spot (selecting) scan pattern, carry out controlled position observation and record putting in order plate continuous hole observation and record to the porous cell culture plate under (automatically) scan pattern continuously to arbitrarily independent hole.Under two kinds of patterns, each viewport all has complete image to show, and with the storage of computer picture document form, can supply off-line analysis.
Background technology
Along with the enforcement and the propelling of the Human Genome Project, life science has entered the genome times afterwards comprehensively.In these epoch, the main research object of life science is a functional genomics, comprises research of structural gene group and proteome research etc.Although the genome of existing a plurality of species is checked order now, from the genome to protein, there are transcriptional level, translation skill and three kinds of regulatory mechanisms of translation back level, the information that obtains in the genomics can not be represented protein expression level comprehensively.Protein is the executor of physiological function, is the direct agent of biological phenomena, will directly illustrate the change mechanism of life under physiology or pathological conditions to the research of protein structure and function.The existence form of protein itself and mechanics as problems such as posttranslational modification, protein-protein interaction and protein conformations, still depend on direct research to protein and solve.Though special natures such as the changeability of protein and diversity have caused the protein research technology more complicated and much more difficult than nucleic acid technology far away, these changeabilities and diversity participate in and affect whole life process just.
Oneself can't satisfy the requirement of genome times afterwards comprehensively traditional mode that single protein is studied.This be because: the generation of (1) biological phenomena is multifactor impact often, must relate to a plurality of protein.(2) participation of a plurality of protein is woven into network, or parallel generation, or is the cascade cause and effect.(3) performance of protein is various, dynamic when carrying out physiological function, and basic fixed is constant unlike the base group.Therefore to comprehensive and deep understanding be arranged to the complicated activity of life, must on the level of whole, dynamic, network, study protein.Therefore in the mid-90 in last century, produced new branch of science one proteomics in the world, it be with all protein in the cell exist and manner is a research object.We can say that carrying out of proteome research be not only the milestone that life science enters the genome times afterwards comprehensively, also is one of core content of genome times afterwards comprehensively life science.
Antibody be research protein properties and function and with the important tool of the relation of disease.Antibody is the ideal tools of analysis of cells protein group, specificity height not only, and can separate the relative abundance of reading protein in the cell pyrolysis liquid sample very delicately, use various antibody, by quantitatively monitoring the variation of protein expression profiles at different cell proteins; Utilize antibody chip or the same tissue of different tissues and different situations is carried out parllel screening, can determine differentially expressed protein with same antibody, or even the albumen of new agnoprotein or disease association.The antibody engineering technology is constantly perfect along with the development of modern biotechnology, and is the main force of biotechnology industry.
At present in the MONOCLONAL ANTIBODIES SPECIFIC FOR process, traditional method is to use albumen or antigen immune mouse, determines antibody by the ELISA method and produces many hybridomas, clones and builds strain, further the monoclonal antibody of purifying is identified then, detected its epitope again as needs.In real work, often obtain the hybridoma of a large amount of generation antibody when fusion rate is high, in each cell clone screening process, cell detection needs the experimenter to carry out cell observation at microscopically for a long time.The staff faces a large amount of workloads, and existing Observations Means becomes the rate-limiting step of work, and work efficiency can't be protected.For example,, need observe one by one whole double dish, interested cell is carried out record by the professional who possesses professional knowledge at the cell that detects 96 porocyte double dish.Whole testing process need spend considerable time, and intensity is quite big.Therefore, the testing staff is tired easily, thus the result that influence detects.Owing to spend considerable time, the growth of pair cell is unfavorable in addition.The present invention proposes a cover and can repeat pair cell and cultivate and to carry out autoscan observation and pick-up unit and method, can satisfy the automatic collection that makes things convenient for cell image, can effectively address the above problem.
Summary of the invention
The objective of the invention is to defective, a kind of cell-growth automatic detector and method are provided, can repeat the pair cell cultivation and observe and the automatic cell image of gathering, to replace loaded down with trivial details manually-operated at above-mentioned prior art existence.The experimenter only need be placed on Tissue Culture Plate on the workbench, according to the experiment needs, selects autoscan, perhaps flying-spot scanner, images acquired then.Image is exported with flexible way, and the user can handle collecting image.Adopt detector of the present invention and detection method, can improve experimenter's work efficiency greatly.Both can the pair cell cultivation observe and gather automatically cell image, can read the observation and the image acquisition of analysis by pair cell immune clone enzyme connection spot.
In order to achieve the above object, the present invention adopts following technical proposals:
A kind of cell-growth automatic detector, comprise a mechanical platform and an optical microscope of aiming at mechanical platform of shelving Tissue Culture Plate, optical microscope connects a video camera, the outputting video signal of video camera is through computing machine of image pick-up card input, after computing machine is handled vision signal, one the tunnel exports display showed cell image to, another road exports printer prints to and goes out cell image, also has one tunnel output through a controller, to the mechanical platform input control signal, the control mechanical platform moves.
Above-mentioned controller can employing section day new Q2HB68MG control module.
The structure of above-mentioned mechanical platform can be designed as: fix a y guide rail and a y to sliding shoe slip coupling on a substrate, y is fixedlyed connected with a middle layer platform to sliding shoe, the middle layer platform is fixedlyed connected with a nut, this nut and a y revolve to leading screw and join, and y connects a motor output shaft that is fixed in substrate to the leading screw end and connects; There is a top platform platform top, middle layer, fix an x on the platform of middle layer to guide rail, mate to the sliding shoe slip with an x, x is fixedlyed connected with top platform to sliding shoe, top platform is fixedlyed connected with a nut, this nut and an x revolve to leading screw and join, and by the supporting of the bearing in the supporting base that is fixed in the middle layer platform, x connects a motor output shaft that is fixed in the middle layer platform to the leading screw end and connects x to leading screw; All there are the printing opacity breach in described substrate and middle layer platform central authorities, and there is the breach of configuration Tissue Culture Plate in top platform central authorities.
Above-mentioned Tissue Culture Plate is the porous cell culture plate, comprises 96 porocyte culture plates, a series of Tissue Culture Plates or PCR plate (pcr plate) or elisa plates (enzyme linked immunological adsorption plate) such as 384 porocyte culture plates.
A kind of cell-growth automatic detector method adopts cell-growth automatic detector to detect, and it is characterized in that detecting and adopts automatic continuous sweep pattern, and step is as follows:
1) initialization: the moving step length that mechanical platform is set is the distance (as Figure 12) between the adjacent culture hole of Tissue Culture Plate; The coordinate that system is set according to Tissue Culture Plate is a discrete coordinates system (as Figure 13); The platform initial position is set: x axial coordinate X=0, y axial coordinate Y=0; Platform moving direction parameter d ir_x=1 is set, and (platform moves along x axle positive dirction dir_y=1 during dir_x=1, and platform moves in the other direction along the x axle during dir_x=-1; Platform moves along y axle positive dirction during dir_y=1, and platform moves in the other direction along the y axle during dir_y=-1); Platform is set counts n_x=1 along the step-length that the x axle moves at every turn, platform is counted n_y=1 along the step-length that the y axle moves at every turn.
2) image of seizure current location makes X=X+n_xgdir_x.
3) if x axial coordinate X>11 and the dir_x=-1 of platform, perhaps, X≤0 and dir_x=1 then judge Y: if Y 〉=7, then skip to step 5); If Y<7, then platform makes Y=Y+n_ygdir_y along moving n_y the step-length of y-axis shift.
4) if x axial coordinate 0≤X≤11 and the Y of platform are less than or equal to 7, then Y is carried out odd even and judge, if Y is an odd number, dir_x=-1 then, platform is along the mobile n_x of an x axle step-length; If Y is even number, dir_x=1 then, platform is along the mobile n_x of an x axle step-length; Be back to step 2);
5) mechanical platform resets: n_y=7 is set, and dir_y=-1, platform is along moving n_y the step-length of y-axis shift.
Another kind of cell-growth automatic detector method adopts cell-growth automatic detector to detect equally, it is characterized in that detecting adopting the flying-spot scanner pattern, and step is as follows:
1) initialization: the moving step length that mechanical platform is set is the distance (as Figure 12) between the adjacent culture hole of Tissue Culture Plate; The coordinate that system is set according to Tissue Culture Plate is a discrete coordinates system (as Figure 13); The platform current location is set: x axial coordinate X=0, y axial coordinate Y=0; Platform moving direction parameter d ir_x=1 is set, and (platform moves along x axle positive dirction dir_y=1 during dir_x=1, and platform moves in the other direction along the x axle during dir_x-1; Platform moves along y axle positive dirction during dir_y=1, and platform moves in the other direction along the y axle during dir_y=-1); Platform is set counts n_x=1 along the step-length that the x axle moves at every turn, platform is counted n_y=1 along the step-length that the y axle moves at every turn.
2) selection operation point: use indicators such as mouse, directly on the culture plate interface that System Monitor shows, select the set of user's interest operating point.
3) system-computed goes out the coordinate position of selected operating point set.X axle: X_s, y axle: Y_s;
4) system-computed goes out platform and moves to the required mobile step-length of next scanning position from current location:
n_x=|X_s-X|,n_y=|Y_s-Y?|。If X_s-X<0, dir_x=-1 then, otherwise dir_x=1; If Y_s-Y<0, dir_y=-1 then, otherwise dir_y=1;
5) platform along the mobile n_y of a y axle step-length, is gathered the cell image of this position along the mobile n_x of an x axle step-length; Make X=X+n_xgdir_x, Y=Y+n_ygdir_y.
6) repeating step 4) and step 5) finish image acquisition to all selected operating points, system reset, mechanical platform is got back to initial position.
The present invention compared with prior art, have following conspicuous outstanding substantive distinguishing features and remarkable advantage: detector of the present invention removes to control mechanical platform by computing machine output control signal via controller and does two-dimentional moving, realize gathering automatically cell image, to replace loaded down with trivial details manually-operated, time saving and energy saving, and in time detect the growth of cell, avoid the infection between operator and the cell sample.Detection method of the present invention can adopt automatic continuous sweep pattern and flying-spot scanner pattern dual mode to carry out, under automatic continuous sweep pattern, can obtain the image of all cells on the Tissue Culture Plate, under the flying-spot scanner pattern, can obtain the image of selecting by the user on the Tissue Culture Plate that needs observation point.Detector of the present invention and method have advantage fast and automatically, can carry out cell growth detection by automatic batch.
Description of drawings
Fig. 1 is the system architecture diagram of cell-growth automatic detector of the present invention.
Fig. 2 is the structural representation of the mechanical platform among Fig. 1.
Fig. 3 is the vertical view of Fig. 2.
Fig. 4 is the trace routine block diagram under the automatic continuous sweep pattern.
Fig. 5 is the trace routine block diagram under the flying-spot scanner pattern.
Fig. 6 is the automatic continuous sweep single-hole imaging of 96 porocyte culture plates figure.
Fig. 7 is the whole images of the automatic continuous sweep of 96 porocyte culture plates.
Fig. 8 is 96 porocyte culture plate flying-spot scanner single-hole imaging figure.
Fig. 9 is 96 porocyte culture plate flying-spot scanner images.
Figure 10 is the surface chart after the 96 porocyte culture plate flying-spot scanners.
Figure 11 is the comparison diagram that amplify in two holes of 96 porocyte culture plate flying-spot scanners.
Figure 12 showed cell culture plate.
Figure 13 shows that the coordinate that system is set according to Tissue Culture Plate is a discrete coordinates system.
Embodiment
Embodiments of the invention are described in detail as follows in conjunction with the accompanying drawings.
Referring to Fig. 1, this cell-growth automatic detector comprises a mechanical platform and an optical microscope of aiming at mechanical platform of shelving Tissue Culture Plate, its feature connects a video camera at described microscope, the outputting video signal of video camera is through computing machine of image pick-up card input, after described computing machine is handled vision signal, one the tunnel exports display showed cell image to, another road exports printer prints to and goes out cell image, also has one tunnel output through a controller, to the mechanical platform input control signal, the control mechanical platform moves.
Above-mentioned controller can employing section day new Q2HB68MG control module.
Above-mentioned optical microscope adopts the stereomicroscope XTL-500E of Shanghai rectangular optical instrument company.
Above-mentioned video camera adopts JVC colour TV camera TK-C921.
Above-mentioned Tissue Culture Plate comprises 96 porocyte culture plates, a series of Tissue Culture Plates or PCR plate (pcr plate) or elisa plates (enzyme linked immunological adsorption plate) such as 384 porocyte culture plates for for the porous cell culture plate.
Referring to Fig. 2 and Fig. 3, the structure of above-mentioned mechanical platform is: fix a y and mate to the sliding shoe slip to guide rail and a y on a substrate, y is fixedlyed connected with a middle layer platform to sliding shoe, the middle layer platform is fixedlyed connected with a nut, this nut and a y revolve to the silk letter and join, and y connects a motor output shaft that is fixed in substrate to the leading screw end and connects; There is a top platform platform top, middle layer, fix an x on the platform of middle layer to guide rail, mate to the sliding shoe slip with an x, x is fixedlyed connected with top platform to sliding shoe, top platform is fixedlyed connected with a nut, this nut and an x revolve to the silk letter and join, and by the supporting of the bearing in the supporting base that is fixed in the middle layer platform, x connects a motor output shaft that is fixed in the middle layer platform to the leading screw end and connects x to leading screw; All there are the printing opacity breach in described substrate and middle layer platform central authorities, and there is the breach of configuration Tissue Culture Plate in top platform central authorities.
Referring to Fig. 4, this cell growth automatic testing method adopts the cell-growth automatic detector of embodiment 1 to detect, and detects and adopts automatic continuous sweep pattern, and step is as follows:
1) initialization: the moving step length that mechanical platform is set is the distance (as Figure 12) between the adjacent culture hole of Tissue Culture Plate; The coordinate that system is set according to Tissue Culture Plate is a discrete coordinates system (as Figure 13); The platform initial position is set: x axial coordinate X=0, y axial coordinate Y=0; Platform moving direction parameter d ir_x=1 is set, and (platform moves along x axle positive dirction dir_y=1 during dir_x=1, and platform moves in the other direction along the x axle during dir_x=-1; Platform moves along y axle positive dirction during dir_y=1, and platform moves in the other direction along the y axle during dir_y=-1); Platform is set counts n_x=1 along the step-length that the x axle moves at every turn, platform is counted n_y=1 along the step-length that the y axle moves at every turn.
2) image of seizure current location makes X=X+n_xgdir_x.
3) if x axial coordinate X>11 of platform and dir_x=1, perhaps, X≤0 and dir_x=1 then judge Y: if Y 〉=7 then skip to step 5); If Y<7, then platform makes Y=Y+n_ygdir_y along moving ny the step-length of y-axis shift.
4) if x axial coordinate 0≤X≤11 and the Y of platform are less than or equal to 7, then Y is carried out odd even and judge, if Y is an odd number, dir_x=-1 then, platform is along the mobile n_x of an x axle step-length; If Y is even number, dir_x=1 then, platform is along the mobile n_x of an x axle step-length; Be back to step 2);
5) mechanical platform resets: n_y=7 is set, and dir_y=-1, platform is along moving n_y the step-length of y-axis shift.This example is carried out automatic continuous sweep to 96 porocyte culture plates, and enlargement factor adopts about 1.7 times.
Scanning result is kept under hard disk F:cellgrowth 1 catalogue automatically, and the image in every hole can be opened in scanning software.
Fig. 6 illustrates the automatic continuous sweep single-hole imaging of these 96 porocyte culture plates figure.Wherein have a large amount of cells in the pattern coil inner region, also being scattered in other zone in the hole has a large amount of cells,
Fig. 7 illustrates the whole images of this automatic continuous sweep of 96 porocyte culture plates.
Referring to Fig. 5, this cell growth automatic testing method adopts the cell-growth automatic detector of embodiment 1 to detect, and detects and adopts the flying-spot scanner pattern, and step is as follows:
1) initialization: the moving step length that mechanical platform is set is the distance (as Figure 12) between the adjacent culture hole of Tissue Culture Plate; The coordinate that system is set according to Tissue Culture Plate is a discrete coordinates system (as Figure 13); The platform current location is set: x axial coordinate X=0, y axial coordinate Y=0; Platform moving direction parameter d ir_x=1 is set, and (platform moves along x axle positive dirction dir_y=1 during dir_x=1, and platform moves in the other direction along the x axle during dir_x=-1; Platform moves along y axle positive dirction during dir_y=1, and platform moves in the other direction along the y axle during dir_y=-1); Platform is set counts n_x=1 along the step-length that the x axle moves at every turn, platform is counted n_y=1 along the step-length that the y axle moves at every turn.
2) selection operation point: use indicators such as mouse, directly on the culture plate interface that System Monitor shows, select the set of user's interest operating point.
3) system-computed goes out the coordinate position of selected operating point set.X axle: X_s, y axle: Y_s;
4) system-computed goes out platform and moves to the required mobile step-length of next scanning position from current location:
n_x=|X_s-X?|,n_y=|Y_s-Y?|。If X_s-X<0, dir_x=-1 then, otherwise dir_x=1; If Y_s-Y<0, dir_y=-1 then, otherwise dir_y=1;
5) platform along the mobile n_y of a y axle step-length, is gathered the cell image of this position along the mobile n_x of an x axle step-length; Make X=X+n_xgdir_x, Y=Y+n_ygdir_y.
6) repeating step 4) and step 5) finish image acquisition to all selected operating points, system reset, mechanical platform is got back to initial position.
This example is that 96 porocyte culture plates are carried out flying-spot scanner, and enlargement factor also adopts about 1.7 times.
Scanning result is kept under the hard disk F automatically: under cellgrowth 2 catalogues, the image in every hole can be opened in scanning software.
Fig. 8 illustrates this 96 porocyte culture plate flying-spot scanner single-hole imaging figure.There are a large amount of cells in the picture in picture shape coil inner region.
Fig. 9 illustrates these 96 porocyte culture plates, and all select the flying-spot scanner image.
Figure 10 illustrates the surface chart of these 96 porocyte culture plate flying-spot scanner imagings.The use mouse is double-clicked the arbitrarily small figure in the 96 hole synoptic diagram, can be observed the image that amplify in this hole.
Figure 11 illustrates the figure that clicks in two holes, can carry out amplification ratio.
Claims (5)
1. cell-growth automatic detector, comprise a mechanical platform (1) and an optical microscope (3) of aiming at mechanical platform (1) of shelving Tissue Culture Plate (2), its feature connects a video camera (4) at described microscope, the outputting video signal of video camera (4) is through image pick-up card (a 5) input computing machine (6), after described computing machine (6) is handled vision signal, one the tunnel exports display (7) showed cell image to, another road exports printer (8) to and prints cell image, also has one tunnel output through a controller (9), to the mechanical platform input control signal, control mechanical platform (1) moves.
2. cell-growth automatic detector according to claim 1 is characterized in that described controller is by segmenting stepping motor control card and program module is formed.
3. cell-growth automatic detector according to claim 1, the mechanism that it is characterized in that described mechanical platform (1) is: fix a y and mate to sliding shoe (12) slip with a y to guide rail (13) on a substrate (17), y is fixedlyed connected with a middle layer platform (16) to sliding shoe (12), middle layer platform (16) is fixedlyed connected with a nut (20), this nut (20) revolves to leading screw (19) with a y joins, y to leading screw (19) by the bearing in the supporting base that is fixed in substrate (17) (18,22) supporting, y connects motor (a 21) output shaft that is fixed in substrate (17) to leading screw (19) end and connects; There is a top platform (10) middle layer platform (16) top, fix an x on the middle layer platform (16) to guide rail, mate to the sliding shoe slip with an x, x is fixedlyed connected with top platform (10) to sliding shoe, top platform (10) is fixedlyed connected with a nut (15), this nut (15) revolves to leading screw (14) with an x joins, by the supporting of the bearing in the supporting base that is fixed in middle layer platform (16), x connects motor (a 11) output shaft that is fixed in middle layer platform (16) to the leading screw end and connects x to leading screw (14); All there are the printing opacity breach in described base version (17) and middle layer platform (16) central authorities, and there is the breach of configuration Tissue Culture Plate (2) in top platform (10) central authorities.
4. a cell-growth automatic detector method adopts cell-growth automatic detector according to claim 1 to detect, and it is characterized in that detecting adopting automatic continuous sweep pattern, and step is as follows:
1) initialization: the moving step length that mechanical platform is set is the distance (as Figure 12) between the adjacent culture hole of Tissue Culture Plate; The coordinate that system is set according to Tissue Culture Plate is a discrete coordinates system (as Figure 13); The platform initial position is set: x axial coordinate X=0, y axial coordinate Y=0; Platform moving direction parameter d ir_x=1 is set, and (platform moves along x axle positive dirction dir_y=1 during dir_x=1, platform during dir_x=-1
Move in the other direction along the x axle; Platform moves along y axle positive dirction during dir_y=1, and platform moves in the other direction along the y axle during dir_y=-1); Platform is set counts n_x=1 along the step-length that the x axle moves at every turn, platform is counted n_y=1 along the step-length that the y axle moves at every turn.
2) image of seizure current location makes X=X+n_xgdir_x.
3) if x axial coordinate X>11 and the dir_x=-1 of platform, perhaps, X≤0 and dir_x=1 then judge Y: if Y 〉=7, then skip to step 5); If Y<7, then platform makes Y=Y+n_ygdir_y along moving n_y the step-length of y-axis shift.
4) if x axial coordinate 0≤X≤11 and the Y of platform are less than or equal to 7, then Y is carried out odd even and judge, if Y is an odd number, dir_x=-1 then, platform is along the mobile n_x of an x axle step-length; If Y is even number, dir_x=1 then, platform is along the mobile n_x of an x axle step-length; Be back to step 2);
5) mechanical platform resets: n_y=7 is set, and dir_y=-1, platform is along moving n_y the step-length of y-axis shift.
5. a cell-growth automatic detector method adopts cell-growth automatic detector according to claim 1 to detect, and it is characterized in that detecting adopting the flying-spot scanner pattern, and step is as follows:
1) initialization: the moving step length that mechanical platform is set is the distance (as Figure 12) between the adjacent culture hole of Tissue Culture Plate; The coordinate that system is set according to Tissue Culture Plate is a discrete coordinates system (as Figure 13); The platform current location is set: x axial coordinate X=0, y axial coordinate Y=0; Platform moving direction parameter d ir_x=1 is set, and (platform moves along x axle positive dirction dir_y=1 during dir_x=1, and platform moves in the other direction along the x axle during dir_x=-1; Platform moves along y axle positive dirction during dir_y=1, and platform moves in the other direction along the y axle during dir_y=-1); Platform is set counts n_x=1 along the step-length that the x axle moves at every turn, platform is counted n_y=1 along the step-length that the y axle moves at every turn.
2) selection operation point: use indicators such as mouse, directly on the culture plate interface that System Monitor shows, select the set of user's interest operating point.
3) system-computed goes out the coordinate position of selected operating point set.X axle: X_s, y axle: Y_s;
4) system-computed goes out platform and moves to the required mobile step-length of next scanning position from current location:
n_x=|X_s-X|,n_y=|Y_s-Y|。If X_s-X<0, dir_x=-1 then, otherwise dir_x=1; If Y_s-Y<0, dir_y=-1 then, otherwise dir_y=1;
5) platform along the mobile n_y of a y axle step-length, is gathered the cell image of this position along the mobile n_x of an x axle step-length; Make X=X+n_xgdir_x, Y=Y+n_ygdir_y.
6) repeating step 4) and step 5) finish image acquisition to all selected operating points, system reset, mechanical platform is got back to initial position.
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