Abstract:
A method for observing cells, a chip and a device are provided. The method includes: (1) a camera obtaining dynamically the position of cell, the direction and speed of flow in the microchannel within the chip on stage through a microscope and transferring the data to a computer, (2) the computer collecting real-time data from the camera and analyzing it, calculating the relation between the position of cell and pressure needed in the assay, at the same time comparing the result obtained with the real-time pressure fed back by micro pressure devices and received by the computer, and inputting controlling commands to micro pressure devices; (3) based on the commands, micro pressure devices changing the pressure of two ends of microchannel in the chip to adjust the direction and speed of flow in the microchannels of chip so as to regulate the position of cell in the flow. Observing of cells can be completed quickly with accuracy according to the invention. The present invention can be applied in dynamic study for cell in scientific research, medical detection and teaching.

Description:
BACKGROUND OF THE INVENTION 
       [0001]    1. Field of the Invention 
         [0002]    The present invention relates to a method of the observation for cells research, in particularly to a method of proceeding a dynamic research with respect to the observation of cells applied for scientific research and medical studies, to an experimental chip of using same, and to a necessitate device of using same. 
         [0003]    2. Description of the Related Art 
         [0004]    For the biological analysis and the medical researches, the cell development has become a critical technology as those cells are necessary for life. Such technology performing to separate the cells from the living body for executing the extraneous culture usually has obstructions because the existence of the microenvironment around the cells would be facilely out of control. For instance, the injection of liquid reagents into the cells substantially benefits the observation of the cells through a microscope but inevitably renders the cells floated out of the lens of the microscope unless some specific physical or chemical ways are used to constrain them. Thus, the supra interferences may cause detriments to the cell experiment or development, and the efficient development of the cell retention and culture would make an important contribution to the research and application of the life science. 
         [0005]    Thence, a PCT patent application published no. WO/2006/007701, issued by Simon et al., conducts a microfluidic device and its relevant method of using same that mainly applies a microfluidic chip technology to carry out the selection, separation, retention, and the culture of the particle (i.e. single yeast cell) within the fluid channel of the chip, so as to incessantly observe and record biological parameters of the single cell while delivering and shifting the reagents. Base on the control to the microenvironment of the cell and the retention of the cell float on the microfluidic, such technology theoretically solves the aforementioned problems that disturb the cell culture and experiment by applying the microfluidic chip device, in which microchannels and a partial retention structure in curved contour arranged therein are defined attempt to receive the cell within the curved structure for the observation by adjusting the pressure and speed of the fluidic. However, the chip device dedicates neither to perform the visualizing control of the fluid channel nor to show the way how to automatically control the microfluidic of the cell experiment. Since the experiment usually spends for days or weeks and the auto requirement, the conventional application supported by the theory may not be well adapted for the practical experiments, thus requiring improvements. 
       SUMMARY OF THE INVENTION 
       [0006]    The object of the present invention is to provide a method for swiftly and accurately observing cells. 
         [0007]    Another object of the present invention is to provide an experimental chip of observing cells for ensuring the stability of the cells. 
         [0008]    A further object of the present invention is to provide a relevant device for executing the experiment on observing cells, which mainly applies a camera identifying dynamically the position, the direction, and speed of cells and serving as a data transferring to efficiently control the microfluidic in the microchannel within the chip. 
         [0009]    The method for observing cells in accordance with the present invention comprises procedures of: (1) data attaining: a camera is arranged to dynamically capture the position, the flowing orientation and speed of cells in the microchannel within the chip on a stage through a microscope and to transfer the received data to a computer; (2) analyzing and comparing: the computer collects the real-time data from the camera and proceeds to analyze and calculate the relation between the position of cell and pressure complied with the demand of the assay, as well as to compare the result with the real-time pressure fed back from micro pressure devices to the computer, whereby the computer accordingly serves to input controlling commands to micro pressure devices; and (3) controlling and adjusting: according to afore commands, the micro pressure devices changes the pressure supplied to both ends of the microchannel in the chip to adjust the flowing direction and speed of fluid in the microchannel of the chip so as to regulate the position of cell in the fluid. 
         [0010]    The present invention also includes a chip adapted for the method. The chip has a microchannel, an interface system for the chip to communicate with the outside, and a sealing. Wherein, the interface system is divided into an input of reagents or nutrient agar and a controlling interface; the microchannel defines a curved cell control unit for making the cell observation and culture and has both ends thereof discretely connected to the controlling interface, so as to attach a side hole on a side wall of the microchannel face to the opening of the cell control unit with the input of reagents or nutrient agar; the sealing covering the chip obturates the channel of the chip for permitting the controlling interface to connect with micro pressure devices and further to adjust the pressure within the microchannel. Both the microchannel and the cell control unit have respective micropits where microballs can place to conduct the flowing speed and direction of the fluid at specific fluidic port, thereby performing the visualizing control of the fluid channel. 
         [0011]    The microchannel and the cell control unit arrange a set of pits to become a pit array. The microballs in the pit would depict the flowing speed and directions of the fluid distributed among the fluidic ports. The pit array would directly show the fluid channel at the specific moment, and the real-time result from the instant fluid observation would assist the controlling system to have an accurate execution. 
         [0012]    The interface system includes an input of reagents or nutrient agar; wherein, the input can be adapted for an injection of common liquid that requires damping buffering or adapted for an injection of specific liquid that is merely applied to the reagents with small and fixed measurement. 
         [0013]    The interface system also includes a controlling interface that connects to two ends of the cell control unit through a damping microchannel. The controlling interface attaches with different positions within the microchannel in order to adapt kinds of cells relative to kinds of damping coefficients. 
         [0014]    The chip is an integrity chip, the interface system includes an input of reagents or nutrient agar and a controlling interface; the microchannel has a cell control unit arranged thereon. 
         [0015]    The microballs are magnetic balls. The balls are subjected to slightly vibrate under the stimuli of an exterior magnetic field, thereby avoiding the adhesions of the balls on inner walls of the pits to affect the indication. 
         [0016]    The cell control unit includes a cell collecting unit attached with the inner wall thereof and serves to gather and save the cells. The cell collecting unit is formed of a gradual wider configuration, of which the narrow portion has one edge joined to the cell control unit; a blocking wall is thence disposed at the joint for interfering the cells flowed at a certain speed. Further, the wider portion thereof has one edge thereof attached to the interface system through the microchannel and also has a blocking wall serving to obstruct the cell flow; the wider portion can also guide the cultured cell within the cell collecting unit out through the interface if necessary. 
         [0017]    The microballs in the pits for the fluid conduction or indication are magnetic balls. The balls are subjected to slightly vibrate under the stimuli of an exterior magnetic field, thereby avoiding the adhesions of the balls on inner walls of the pits to loss the conducting effect. The present invention can control the strength of the magnetic field in the fixed orientation to change the force exerted on the balls and hence to adjust sensitivity thereof. 
         [0018]    The adhesion of the cells inside the microchannel affects the experiment controlling, and the adhesion would be subjected to acoustic wave. Therefore, an acoustic wave generator can be embedded into the chip to eliminate the occurrence of adhesion. 
         [0019]    The present invention also includes a device adapted for the method. The device has a microscope, a camera, a computer, micro pressure devices, and a chip; wherein, the computer connects with the camera through system address buses; the camera is attached to the microscope; the chip is put on a stage; the micro pressure devices have their respective pressure outputs joined to the controlling interface of the chip by lines; the pressure output also connects to the computer by data lines so as to render the feedback of the instant pressure and accept the controlling commands from the computer. 
         [0020]    The micro pressure device can adopt controlling ways regulated by air pressure and hydraulic pressure. 
         [0021]    The micro pressure device is comprised of a pressure source and multiple air pressure controlling units; the pressure source connect with the air pressure controlling units through an air pressure passage; the pressure source mainly consists of a vacuum pump, a vacuum pond, a pressure exchanger, and a gauging apparatus; wherein, the vacuum pump connects with the vacuum pond, the gauging apparatus electrically engages with the vacuum pump and attaches to the vacuum pond through the pressure exchanger; the air pressure controlling unit includes a flow adjusting valve, an electromagnetic valve, a buffering container, a pressure exchanger, and a gauging mean; wherein, there are two sets of the flow adjusting valves in serial connection with the electromagnetic valves, and the two sets thereof are respectively engaged with an input passage and an output passage of the buffering container; the gauging mean is electrically connected with the electromagnetic valve disposed on the input and output passages of the buffering container and is joint to the buffering container through the pressure exchanger. 
         [0022]    The micro pressure devices set by dynamic control could accept an air pressure source in great flow and adjust the air pressure by applying a series of cutoff valves arranged in serial or parallel connections. Alternatively, the devices could adopt a rapid reaction that fixes the serial pressure value to await the shift without exerting the incessant pressure controlling. 
         [0023]    The micro pressure devices proceed to control hydraulic pressure by a displacing detector. The hydraulic pressure regulation has an adjustment more precise than that of the air pressure regulation but essentially has lower modulating ranges. Accordingly, it adopts to use the air pressure control accompanying the hydraulic pressure adjustment to attain the purpose of pressure controlling. Due to the possession of rapid reaction, the air pressure control serves to retrieve the cells to an anticipated location; further, the liquid adjustment takes advantages of executing the precise regulation to obviate the unbalance of the long-term cell culture, for instance the loss balance of the liquid level or of the damping. The air pressure control and the liquid adjustment can be a system embedded into the computer so as to attain an automatic controlling effect. 
         [0024]    The microscope of the present device also includes a dual microscope having an optical path that gathers a coaxial and single light source; wherein, the dual microscope further comprises a reflecting microscope and an inverting microscope. The light of the light source is thrown to the chip for entering into an optical axis of the reflecting microscope through a reflecting mirror; further the reflected light returns to a CCD 1  of the reflecting microscope that serves to observe the dynamic images for satisfying the dynamic control of the chip; the light through the chip is captured by a CCD 2  of the inverting microscope that facilitates the biology observation. 
         [0025]    From the above, the present invention mainly applies the microscope and the camera to initially capture the position and the flowing information of particles, to dynamically identify real-time data of the single- or multi-cell, and to deliver the data to the computer. The computer thence exerts to analyze and make the comparison between the data and the desired pressure value as well as transfers controlling commands to alter the pressure imparted on both ends of the microchannel, which would hence regulate the flowing speed and direction of the fluid and the location of the cells within the fluid. Therefore, a quick and accuracy experiment on cell culture is achieved. 
         [0026]    The relevant device can be efficient developed as an automatic equipment for devoting itself to the dynamic cell studies and experiments, which hence has wide applications on the science research and the medical detection or studies. 
         [0027]    The advantages of the present invention over the known prior arts will become more apparent to those of ordinary skilled in the art by reading the following descriptions with the relating drawings. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0028]      FIG. 1   a  is a schematic view showing the chip of the present invention; 
           [0029]      FIG. 1   b  is a partial schematic view showing the upper portion of the chip of the present invention; 
           [0030]      FIG. 1   c  is a partial schematic view showing the lower portion of the chip of the present invention; 
           [0031]      FIG. 1   d  is a schematic view showing the cell collecting unit within the chip of the present invention; 
           [0032]      FIG. 1   e  is a schematic view showing the microchannel within the chip of the present invention; 
           [0033]      FIG. 1   f  is a schematic view showing the cell control unit within the chip of the present invention; 
           [0034]      FIG. 1   g  is a schematic view showing another cell control unit within the chip of the present invention; 
           [0035]      FIG. 1   h  is a schematic view showing the micro pit within the chip of the present invention; 
           [0036]      FIGS. 2   a - 2   g  are schematic views showing the particle traveling through the microchannel of the chip under the microscope; 
           [0037]      FIG. 3  is a schematic view showing the application of the pit array on indicating the fluid channel; 
           [0038]      FIGS. 4   a - 4   c  are schematic views showing the procedures of controlling the grown cells within the chip; 
           [0039]      FIG. 5  is a schematic view showing the damping fluid channel with manifold structure in the microchannel of the chip; 
           [0040]      FIG. 6  is a schematic view showing the arrangement of the exterior magnetic field round the chip; 
           [0041]      FIG. 7  is a schematic view showing the increment of the acoustic wave generator within the chip; 
           [0042]      FIG. 8  is a flow diagram showing the configuration of the present invention; 
           [0043]      FIG. 9  is a schematic view showing the micro pressure device of a first embodiment; 
           [0044]      FIGS. 10   a - 10   c  are schematic views showing the procedures of attaining the feedback of pictures; 
           [0045]      FIG. 11  is a schematic view showing the micro pressure device of a second preferred embodiment in which cutoff valves are in serial connection to attain the pressure controlling; 
           [0046]      FIG. 12   a  is a schematic view showing the micro pressure device of a second preferred embodiment in which a single fan serves as the air source; 
           [0047]      FIG. 12   b  is a schematic view showing the micro pressure device of a second preferred embodiment in which multiple fans are in serial connection; 
           [0048]      FIG. 13  is a schematic view showing the micro pressure device of a second preferred embodiment applies to fix the serial pressure value to await the shift; 
           [0049]      FIG. 14  is a schematic view showing the micro pressure device of a third preferred embodiment in which cutoff valves are in parallel connection; 
           [0050]      FIG. 15  is a schematic view showing the micro pressure device of a fourth preferred embodiment exerts the conjoint of the air and liquid to attain the regulation; 
           [0051]      FIG. 16  is schematic view showing the dual microscope possessing the feature of gathering a coaxial and single light source; and 
           [0052]      FIG. 17  is a flow diagram showing the method of the present invention. 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
       [0053]    Before describing in greater detail, it should note that the like elements are denoted by the similar reference numerals throughout the disclosure. 
         [0054]    The method of the present invention for observing cells mainly combines the PCT patent application published no. WO/2006/007701 that was issued by the same applicant and disclosed the application of the microfluidic chip technology to carry out the selection, separation, retention, and the culture of the particle (i.e. single yeast cell) within the fluid channel of the chip, so as to incessantly observe and record biological parameter of the single cell while delivering and shifting the reagents. Referring to  FIG. 17  shows the procedures: (1) data attaining: a camera is arranged to dynamically capture the position, the flowing orientation and speed of cells in the microchannel within the chip on a stage through a microscope and transferring the received data to a computer; (2) analyzing and comparing: the computer collects real-time data from the camera and proceeds to analyze and calculate the relation between the position of cell and pressure complied with the demand of the assay, as well as to compare the result with the real-time pressure fed back from micro pressure devices to the computer, whereby the computer accordingly serves to input controlling commands to micro pressure devices; and (3) controlling and adjusting: according to afore commands, the micro pressure devices changes the pressure subjecting to both ends of the microchannel in the chip to adjust the direction and speed of flow in the microchannels of chip so as to regulate the position of cell in the fluid. 
         [0055]    The aforementioned chip adapted for the method as shown in  FIG. 1   a  mainly comprises a microchannel  52  (for tens or hundreds micrometers of width), an interface system  51  for the chip to communicate with the outside, and a sealing  502  (see  FIG. 6 ). Wherein, the interface system  51  is divided into an input  511  of reagents or nutrient agar and a controlling interface  515 ; the microchannel  52  communicates with the interface system  51  and distributes cell control units  521  therein for making the observation and cell culture; further, the sealing  502  covers the chip to render the microchannel  52  performed in a closed status, thereby permitting the controlling interface  515  to connect with micro pressure devices and further to regulate the pressure within the microchannel  52 . Both the microchannel  52  and the cell control unit  521  have respective micropits  522  where microballs can place to conduct the flowing speed and direction of the fluid at specific fluidic port, thereby performing the visualizing control of the fluid channel. The micropits  522  can be also arranged merely within the cell control unit  521 . 
         [0056]    The present invention comprises the following principles: 
         [0000]    1. Introducing and indicating flowing speed and direction of the flow according to the location of the particle: 
         [0057]    Referring to  FIG. 2   a - 2   d , if a spherical member, for instance of a ball Q sinking in the fluid, locates at the arc pit  52 A in the microchannel  51 A, it would be lead toward different orientations base on the flowing speed and direction of the fluid within the microchannel  51 A. For example, as shown in  FIG. 2   a , the ball Q (illustrated by a black point) would stay at the lowest part of the pit  52 A detected by a microscope while there is no fluid within the channel  51 A.  FIG. 2   b  depicts that the ball Q would be pushed forward to the left part and retained balance at the left slope when the fluid flows from the right to the left in the microchannel  51 A as arrowed.  FIG. 2   c  describes that the ball Q would be pushed forward to the right part and retained balance at the right slope when the fluid flows from the left to the right in the microchannel  51 A as arrowed.  FIG. 2   d  shows the increment of the flowing speed would result of the departure of the ball Q far from the lowest part and render a prolonged distance between the real location thereof and the lowest part. Such distance would be detected in turn to reckon the exerted flowing speed. 
         [0058]    Referring to  FIG. 2   e - 2   g  shows the microchannel  51 B of the chip  5 B in a 2-dimensioned structure contributed by four microchannels  511 B,  512 B,  513 B,  514 B intersecting at the pit  52 B to accommodate the ball Q that serves to conduct the flowing situation of each channel. For example,  FIG. 2   e  shows that the ball Q stays in the central when no fluid passes through the channels. Under the condition of the left channel  514 B being as an output of flow,  FIG. 2   f  shows that the ball Q shifts to the left part, and the deviating distance from the center would depend on the flowing speed. If the top channel  511 B is as an output of flow,  FIG. 2   g  shows that the ball Q shifts up (but still floats on the water level), and the deviating distance from the center would depend on the flowing speed. 
         [0059]    Referring to  FIG. 3 , the application of the pits  522  on indicating the flowing speed is not restricted and may be arranged to directly present a complex dynamical fluid field. When placing the microballs  57  inside a pit array within a fluid channel, each fluidic port showing of the speed and direction of the fluid would be performed by the balls  57 , so as to calculate the fluid field at the specific moment. 
         [0000]    Regulating the position of the cells by controlling the pressure exerted on two ends of the microchannel: 
         [0060]    Referring to  FIG. 4   a - 4   c  show a cell control unit  6 ; wherein, the fluid enters from a passage  63 . If the cell is in balance as illustrated in  FIG. 4   a , the pressure exertion on both ends  61 ,  62  would be maintained. The cell herein can be as a spherical and heavy ball or in other appropriate performances. Further, if users try to shift it toward the right as shown in  FIG. 4   b , the pressure imparted to the left end  61  would be increased; in like manner,  FIG. 4   c  shows the increased pressure exerted to the right end  62  to render the cell moved to the left. Therefore, the cell would be stationarily set at the desired position beneficial for the observation, thereby ensuring working capability on the nutrition absorption and the metabolism of the cell to smoothly carry out the cell culture under the changeable environment of fluid. 
         [0061]    A liquid damping device, for instance of a fluid channel with manifold structure, serves to buffer the liquid; wherein, the damping device disposed in the controlling flow assists to reduce the sensitivity of liquid to the pressure variation and to enhance the precision of the pressure to the flow speed adjustment. 
         [0062]    Referring to  FIG. 5 , the fluid enters from the point C and travels out of the points A and B, so the way to control the cell is to regulate the pressure to points A and B. With respect to the same pressure variation, the increment of the manifold fluid channel of the damping device would facilitate to decrease the fluctuation of the flowing speed, so as to modulate the sensitivity of the flowing speed to the pressure and precisely control the supplement of the pressure. 
         [0063]    A preferred embodiment of the present invention as shown in  FIG. 1   a  mainly comprises an integrity chip made of glass, whose dimension is preferably at 63 mm*63 mm. The chip further has an interface system  51  that contains 72 interfaces each with 2 mm diameter distributing around the chip. The interface serves as a pin of CPU for communicating the fluidic chip with the outside. Additionally, the transparent sealing is mainly made of Polydimethylsiloxane (PDMS) covers the entire chip to obturate the microchannel  52  of the chip, so as to adjust the pressure within the microchannel. The interface system  52  is further divided into an input of reagents or nutrient agar and a controlling interface  515  as set forth below. 
         [0064]      FIG. 1   b  depicts the input arranges an entrance  511  adapted for common liquid that requires damping buffering and also provides a distinctive entrance  513  adapted for specific liquid that is merely applied to the reagents with small and fixed measurement. To discharge the redundant liquid before injecting the fluid into the channel of cell culture, the chip has an exit  512  disposed thereon and arranges a set of damping channel interposed between the entrance  511  and the exit  512  for acting as a buffer to eject the redundant liquid. 
         [0065]      FIG. 1   c  shows the controlling interface  515  applied to sampling, growing, controlling, and collecting cells. The controlling interface  515  connects to two ends of the cell control unit through the damping microchannel and engages to different positions within damping the microchannel in order to adapt kinds of cells relative to kinds of damping coefficients. Practically, the controlling interface  515  is not only used as a bridge to connect an exterior micro pressure device but as a medium for entries of the cell and the liquid. Moreover, the microchannel of the chip contains cell control units  521  proceeding to grow and observe the cell, in the middle of which three units  521  have their inner walls attached to a cell collecting unit  53  (shown in  FIG. 1   d ) that serves to collect and save the cells. Wherein, the cell collecting unit is formed of a gradual wider configuration, of which the narrow portion has one edge joined to the cell control unit  521 ; a blocking wall  534  is disposed at the joint for interfering the motion of the cells at a certain flowing speed. Further, the wider portion has one edge thereof attached to the interface system  515  through the microchannel  52  and also has a blocking wall  535  serving to obstruct the cell flow; the wider portion can also guide the cultured cell within the cell collecting unit  53  out through the interface. Referring to  FIG. 1   d  shows the traveling of the fluid from the cell control unit  521  to the cell collecting unit  53 . When the fluid passes through the blocking wall  534  of the narrow portion by a greater flowing velocity, the cell would accompany the fluid to the cell collecting unit  53 . Further, the blocking wall  535  with the wider portion conduces to decrease the flowing speed so as to efficiently separate the cell from the fluid and retain the cell within the cell collecting unit  53 . The wider portion of the cell collecting unit  53  further connects with a cell container  516  (shown in  FIG. 1   c ) for accommodating the cell, from which users can derive it depend on demands. 
         [0066]    The microchannel  52  and the cell control unit  521  arrange a set of pits  522  to become a pit array (shown from  FIG. 1   e  to  1   g ). The microballs in the pits would depict the flowing speed and directions of the fluid distributed among the fluidic ports. The pit array would directly show the fluid channel at the specific moment, and the real-time result would assist the controlling system to have an accurate execution. For example,  FIGS. 1   g  and  1   f  depict that the pit array  522  within the cell controlling unit  521  can perform by various formations, and the arrangement of the pit array at each intersection of the microchannel  52  further proceeds to present the flowing quantity of different fluid channels, thereby efficiently controlling the injection of the reagents in a regular measurement for benefiting the cell culture. 
         [0067]    The microballs placed within the pit  522  are magnetic balls. The balls are subjected to slightly vibrate under the stimuli of an exterior magnetic field, thereby avoiding the adhesions of the balls on inner walls of the pits to affect the conduction and indication. Namely, by regulating the strength of the magnetic field in the fixed orientation, a force applied to the magnetic balls would be changed to adjust the sensitivity thereof. For example, referring to  FIG. 6  shows the configurations of the glass  501  of the chip  5  overlapped with the sealing  502  and a coil  7  disposed below the chip  5 . A current at the intersection of direct current and alternating current is delivered to the coil  7  to create a suitable intensity and magnetic field adapted for controlling the power toward the magnetic ball in the chip, hence retaining the ball within the pit and steadily driving the sensitivity of the ball to the flowing speed and direction of the fluid. 
         [0068]    Essentially, the adhesion of the cells inside the microchannel affects the experiment controlling, and the adhesion would be subjected to acoustic wave. Therefore, an acoustic wave generator  8  as illustrated in  FIG. 7  can be embedded into the chip  5  to eliminate the occurrence of adhesion. 
         [0069]    From the above, the present invention has following advantages:
   1. By means of the arrangements of pits within the microchannel and the cell control unit for receiving microballs, the flowing speed and direction of the fluidic port would be conducted by the pits or pit array at the specific moment to generate a result. Thus, the real-time result would assist the controlling system to have an accurate execution, so as to perform the visualizing control of the fluid channel   2. The integrity chip accompanying with the configuration of the interface system that includes an input of reagents or nutrient agar and a controlling interface as well as the cell controlling unit of the microchannel assists the chip to be widely applied for technique fields.   3. The fluid damping device disposed in the controlling flow assists to reduce the sensitivity of liquid to the pressure variation and to enhance the precision of the pressure to the flow speed adjustment.   4. The chip also has the damping microchannel attached to the input of reagents or nutrient agar for achieving the effect of buffering liquid.   5. The cell controlling unit not only renders the cell to be stationarily retained therein beneficial for the observation but efficiently exerts the cell culture under the dynamic fluidic environment.   6. The microchannel within the chip comprises a plurality of doors communicated with the outside, so as to eliminate the air inside the channel and fill the channel with liquid.   7. The microchannel within the chip comprises a plurality of doors for the alternate utilization, thereby maintaining the operation of the experiment.   8. The microballs are magnetic balls. The balls are subjected to slightly vibrate under the stimuli of an exterior magnetic field, thereby avoiding the adhesions of the balls on inner walls of the pits to affect the conduction.   9. The embedment of the acoustic wave generator into the chip serves to eliminate the occurrence of adhesion.   
 
         [0079]    Referring to  FIG. 8 , the device of the present invention for observing cells mainly comprises a microscope  1 , a camera  2 , a computer  3 , micro pressure devices  4 , and a chip  5 ; wherein, the chip  5  is as aforementioned formed of an integrity chip; the computer  3  connects with a USB of the camera  2  through system address buses; the camera  2  is attached to the microscope  1 ; the chip  5  is put on a stage between an eyepiece  31  and an object glass, so that the eyepiece  31  could aim at the cell control unit  521  of the chip  5 ; the micro pressure devices  4  are joined to the controlling interface  515  of the chip  5  by pneumatic lines for regulating the pressure within the microchannel  52 . 
         [0080]    The present invention can further increase some auxiliary equipments, such as a temperature controlling device, an oxygen pressure division controlling device, an automatic injecting device, a grown cell gathering device, an automatic reagents shifter, an auto reagents mixing apparatus, fluorescent signal measuring device, an auto chip cleaning apparatus, and etc. 
         [0081]    The device of the present invention includes following principles: 
         [0082]    The microscope  1  is adopted by an inverting microscope, of which the object glass would be arranged under the chip  5 . The chip  5  would thence connect with the micro pressure devices  4 . Further, the camera  2  belongs to a digital inspecting apparatus for receiving both dynamic and static signals and accordingly delivers the received data to the computer  3  for the analysis. The camera  2  also possesses the characteristic of recording the process of the experiment and culture. The micro pressure devices  4  connect to the computer  3  by data lines, and the computers includes a software adapted for analyzing the position and shifting speed of the target cell or particle and further compares the analyzed result with the real-time pressure fed back from the micro pressure devices  4  to the computer  3 , whereby the computer  3  accordingly serves to input proper controlling commands to the micro pressure devices  4 . Thence, the micro pressure devices  4  would change the pressure subjecting to both ends of the microchannel in the chip according to afore commands sent from the computer software to adjust the flowing direction and speed of fluid in the microchannels of chip  5  so as to regulate the position of cell in the fluid. 
         [0083]    The micro pressure device herein can adopt controlling ways regulated by air pressure and hydraulic pressure. 
         [0084]    Referring to  FIG. 9 , the micro pressure devices  4  of a first preferred embodiment comprises a pressure source  41  and multiple air pressure controlling units  42 , and herein merely three units  42 A,  42 B,  42 C are illustrated; the pressure source  41  connects with the air pressure controlling units  42  by an air pressure passage  43 . 
         [0085]    Still further, the pressure source  41  mainly consists of a vacuum pump  411 , a vacuum pond  412 , a pressure exchanger  413 , and a gauging apparatus  414 ; wherein, the vacuum pump  411  connects with the vacuum pond  412 , the gauging apparatus  414  electrically engages with the vacuum pump  411  and attaches to the vacuum pond  412  through the pressure exchanger  413 . 
         [0086]    The air pressure controlling unit  42  includes a flow adjusting valve  421 , an electromagnetic valve  422 , a buffering container  423 , a pressure exchanger  424 , and a gauging mean  425 ; wherein, there are two sets of the flow adjusting valves  421  in serial connection with the electromagnetic valves  422 , and the two sets thereof are respectively engaged with an input passage and an output passage of the buffering container  423 . The sequential correlation of the interrelated elements engaged to the input passage is: the electromagnetic valve  422 , the flow adjusting valve  421 , and the buffering container  423 ; in addition, the sequential correlation of the interrelated elements engaged to the output passage is: the flow adjusting valve  421 , the electromagnetic valve  422 , and the buffering container  423 . The gauging mean  425  electrically communicates with the electromagnetic valve  422  disposed on the input and output passages of the buffering container  423  and are joint to the buffering container  423  through the pressure exchanger  424 . 
         [0087]    With respect to the principle of the micro pressure devices  4 , multiple air pressure controls  42  of the micro pressure devices  4  would be applied to fit with demands of the channel of the chip  5 . Each channel thereof is controlled relative to the each air pressure control  42  of the micro pressure devices  4  (shown of one in  FIG. 9 ). The air pressure of the vacuum pond  412  transfers the information of air pressure from the pressure exchanger  413  to the gauging apparatus  414 . The gauging apparatus  414  accordingly institutes the data base on the information and outputs controlling currents to turn on or off the vacuum pump  411  (or a compressing apparatus), so as to control and balance the air pressure within the vacuum pond  412 . The pressure of the vacuum pond  412  is merely deemed as the pressure source but is not directly transferred to the chip  5 . The buffering container  423  is the critical element attached to the chip  5  for determining the air pressure imparted to the chip  5 . The buffering container  423  has one end thereof connected with the vacuum pond  412  to attain the real pressure similar to that of the vacuum pond  412  and the other end thereof communicated with the outside to attain the pressure similar to the atmospheric pressure. As a result, the pressure within the buffering container  423  would be strictly restricted amidst the atmospheric pressure and the pressure of the vacuum pond  412 , and the speed of the pressure variation would also be accurately regulated by the flow adjusting valve  421 , so as to well control the pressure intensity and the velocity exerted on the chip  5 . Additionally, the impulsion attendant with the pressure regulation would be relatively declined gradient to the desired value under the buffer incurred by the buffering container  423 . That is, the gauging mean  425  proceeds to compare the pressure information from the pressure exchanger  424  with a predetermined threshold and transfers the results to control the electromagnetic valve  422 , hence to regulate the pressure within the buffering container  423 . Wherein, each gauging mean  425  connects to RS  485  control buses and provides the threshold through the system address buses of the computer  3 . Concurrently, the pressure information detected by the gauging mean  425  is transferred to the computer  3  through the address buses as well. In like manner, hundreds of air pressure controlling units can be processed on RS  485  buses. 
         [0088]    In practical, each micro pressure device of the present invention may tend to apply a static pressure controlling, that is, the air keeps quiescent within the container without floating when no pressure regulation persists. Such static state would be liable to temperature, pressure, and efflux of the gas. Oppositely, a dynamic control mainly renders an incessant flowing of the air, in which the air would gradually be changed under the motivation of the pressure incurred by resistances or frictions while the air is flowed from the higher voltage to the lower voltage. 
         [0089]    the micro pressure devices of a second preferred embodiment applies the aforementioned dynamic control to gradually change the air under the motivation of the pressure incurred by resistances or frictions while flowing it from the higher voltage to the lower voltage. If the pressure device serially connects with the cutoff valves, the pressure of each section would be efficiently controlled, namely the sectional pressure passes through the valves would be stable as long as the pressure differences on the beginning and the end portions remains balance. For example,  FIG. 11  depicts that the gas floats or transits from P 0  to P 1  by the serial of cutoff valves V 1 , V 2 , V 3 , V 4  proceeding to control the pressure intensity P 2 , P 3 , P 4 , so that the air pressure from P 0  to P 1  would be distributed to P 2 , P 3 , P 4  for satisfying the requirements. If the flowing quantity of V 3  decreases, the pressure of P 4  would be decreased toward the orientation of P 1  and those of P 2 , P 3  would also be relevantly raised toward the orientation of P 0 . Therefore, the regulation of the flow adjusting valve stably facilitates the reduction of the pressure. 
         [0090]    To meet the demands of supplying large amounts of the air pressure source, the vacuum pump serving to generate the air pressure can be substituted by proper devices with greater quantities, such as a fan. Each fan can be in serial connection to raise the pressure difference and the adjustment on the rotation speed thereof can promote to adjust the air pressure. Further, the pressure difference would also depend on the power capability, rotation speed, and numbers of the fan. For example,  FIG. 12   a ,  12   b  depicts that the more fans are serially connected, the greater pressure difference is attained, namely the pressure difference between P 2  ( FIG. 12   b ) and the air is greater than the pressure difference between P 1  ( FIG. 12   a ) and the air. 
         [0091]    Besides the application on pressure regulation to fit with the characteristic of such serial connection, the present invention can also apply a rapid reaction that fixes the serial pressure value to await the shift. As shown in  FIG. 13 , the adjustment of the pressure substantially depends on the position of the cell. For example, when the cell is at the position A, the P 10  would thence be adopted; similarly, when the cell is respectively located at the position B, C, D, and E, the P 20 , P 30 , P 40 , and P 50  would thence be respectively adopted as well. Herein the P 10 , P 20 , and P 30  are relative to the pressure output from P 2 , P 3 , and P 4  (shown in  FIG. 11 ). 
         [0092]    However, the difficulty attendant on such serial connection is that the sectional pressure on the entire channel would be changed while regulating any one of the cutoff valves. The micro pressure device of a third preferred embodiment can thence arrange the cutoff valves into parallel connections for discretely controlling the sectional pressure. For example,  FIG. 14  shows that the valves V 1  and V 2  serve to regulate the P 2 , the valves V 3  and V 4  serve to regulate the P 3 , and the V 5  and V 6  valves serve to regulate the P 4 . The pressure source of this preferred embodiment can also be in serial connection. 
         [0093]    The micro pressure device of a fourth preferred embodiment mainly proceeds to control hydraulic pressure by a displacing detector. The hydraulic pressure regulation has an adjustment more precise than that of the air pressure regulation but essentially has lower modulating ranges. Accordingly, it adopts to use the air pressure control accompanying the hydraulic pressure adjustment to attain the purpose of pressure controlling. Due to the possession of rapid reaction, the air pressure control serves to control the cells back to an anticipated location; further, the liquid adjustment takes advantages of executing the precise regulation to obviate the unbalance of the long-term cell culture, for instance the loss balance of the liquid level or of the damping. The air pressure control and the liquid height adjustment can be a system embedded into the computer so as to attain an automatic controlling effect. The liquid height is an incessant adjustment on the hydraulic pressure so as to continuously control the position of the cell within a certain the range adjacent to the center of the working place. If a further range far from the center is required, an air pressure controlling is thence needed. Referring to  FIG. 15 , the height of the liquid can be controlled by the computer and the online data information can be achieved by the displacing detector, whereby, the computer would reckon the hydraulic pressure A, and simultaneously the air pressure B is output by the air pressure device. These two pressures would be incorporated into a synthetic pressure C (C=A+B) and be transferred to the chip. 
         [0094]    The present device can not only use the single microscope to observe the optical path but applies a dual microscope for gathers a coaxial and single light source. Referring to  FIG. 16 , different charge coupled devices (CCDs) essentially make observations in different magnifications under the optical microscope. The high magnification usually serves to capture pictures of cells or particles for the micro-observation, and the low magnification with large visual field capturing the instant pictures thereof often serves to control cells or particles. In this figure, when a light emitted from the light source  30  (white or colorful light) enters a reflecting microscope  32  through a reflecting mirror  31 . Further the reflected light returns to a CCD 1  of the reflecting microscope that serves to observe the dynamic images for satisfying the dynamic control of the chip  5 ; the light through the chip  5  is captured by a CCD 2  of the inverting microscope  33  that serves to attain the biology observing analysis. It can also apply a fluorescent reverting microscope for analyzing fluorescent pictures. 
         [0095]    Since the microball  57  within the pit array of the microchannel  52  of the chip  5  is visualized by the microscope  3 , the method of the present invention as shown from  FIG. 10   a  to  FIG. 10   c  mainly applies the software of the computer to determine the flowing speed and direction of the fluid according to the position of the microball  57  detected by the camera and subsequently exerts to transfer controlling commands to alter the pressure imparted on both ends of the microchannel, which would hence regulate the speed and direction of the flow and the location of the cells within the microchannel at the desired value. 
         [0096]      FIG. 4   a  to  4   c  depicts the pressure controlling procedures of the cell within the microchannel and shows a configuration of a cell control unit  6 ; wherein, the fluid enters from a passage  63 , and the cell retained on the arc pit would conduct the fluid field shape and intensity around the cell. Thence, the dynamic analysis facilitates to control the cell culture and experiment by utilizing the proper and instant regulating and feedback. 
         [0097]    If the cell is in balance as illustrated in  FIG. 4   a , the pressure exertion on both ends  61 ,  62  would be maintained. The cell herein can be as a heavy ball or other appropriate shapes. Further, if users try to shift it toward the right as shown in  FIG. 4   b , the pressure imparted to the left end  61  would be increased; in like manner,  FIG. 4   c  shows the increased pressure exerted to the right end  62  to render the cell shifting to the left. 
         [0098]    To sum up, the method for observing cells in accordance with the present invention comprises procedures of: (1) data attaining: a camera  2  is arranged to dynamically capture the position, the flowing orientation and speed of cells in the microchannel within the chip  5  on a stage through a microscope  1  and transferring the received data to a computer  3 ; (2) analyzing and comparing: the computer  3  collects real-time data from the camera  2  and proceeds to analyze and calculate the relation between the position of cell and pressure complied with the demand of the assay, as well as to compare the result with the real-time pressure fed back from micro pressure devices to the computer, whereby the computer accordingly serves to input controlling commands to micro pressure devices; and (3) controlling and adjusting: according to afore commands, the micro pressure devices  4  change the pressure subjecting to both ends of the microchannel in the chip  5  to adjust the flowing direction and speed of fluid in the microchannels of chip  5  so as to regulate the position of cell in the fluid. 
         [0099]    While we have shown and described the embodiment in accordance with the present invention, it should be clear to those skilled in the art that further embodiments may be made without departing from the scope of the present invention.