CN106383081B - Micro-fluid chip automatic aligning method and system based on binary optical device - Google Patents
Micro-fluid chip automatic aligning method and system based on binary optical device Download PDFInfo
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- CN106383081B CN106383081B CN201610704513.XA CN201610704513A CN106383081B CN 106383081 B CN106383081 B CN 106383081B CN 201610704513 A CN201610704513 A CN 201610704513A CN 106383081 B CN106383081 B CN 106383081B
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- 239000012530 fluid Substances 0.000 title claims abstract description 78
- 230000003287 optical effect Effects 0.000 title claims abstract description 66
- 238000000034 method Methods 0.000 title claims abstract description 31
- 239000004005 microsphere Substances 0.000 claims abstract description 26
- 239000007788 liquid Substances 0.000 claims abstract description 16
- 238000007493 shaping process Methods 0.000 claims abstract description 14
- 238000001514 detection method Methods 0.000 claims abstract description 9
- 238000002347 injection Methods 0.000 claims abstract description 7
- 239000007924 injection Substances 0.000 claims abstract description 7
- 230000005622 photoelectricity Effects 0.000 claims description 8
- 230000008569 process Effects 0.000 claims description 7
- 230000004313 glare Effects 0.000 claims description 6
- 239000000571 coke Substances 0.000 claims description 4
- 238000004140 cleaning Methods 0.000 claims description 3
- 239000012459 cleaning agent Substances 0.000 claims description 3
- 238000012549 training Methods 0.000 claims description 3
- 230000004069 differentiation Effects 0.000 claims 1
- 239000002245 particle Substances 0.000 abstract description 7
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- 238000000684 flow cytometry Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 238000012864 cross contamination Methods 0.000 description 1
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
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Abstract
The invention discloses a kind of micro-fluid chip automatic aligning method and system based on binary optical device, this method comprises: to sample flow and sheath liquid stream of the micro-fluid chip injection comprising standard fluorescence miniflow, sample flow is set to be focused on the center of runner, it controls two-dimentional platform to be moved, until detecting the pulse signal of fluorescent microsphere in photoelectric detector；According to there is the extreme position of fluorescence signal, adjustment is focused the center that sample flow is located at hot spot；Two-dimentional platform movement is controlled according to default signature waveform, adjustment is focused sample flow and is located on focal plane；Two-dimentional platform is finely adjusted until best irradiation position according to the coefficient of variation of fluorescence signal.The present invention has the advantage that using binary optical device as spot shaping device, and the position of the feature decision micro-fluid chip according to the fluorescence signal issued after the irradiation of fluorescent particle stimulated light, and adjust accordingly and be focused sample and flow to optimum position, to realize optimal detection effect and consistency.
Description
Technical field
Invention is related to flow cytometry, physical optics and microfluidic field, and in particular to one kind is based on binary optical device
Micro-fluid chip automatic aligning method and system.
Background technique
Have benefited from the power of the development and flow cytometry of micro-fluidic technologies in terms of cell detection, gushes in recent years
The largely piece flow cytometer based on micro-fluid chip is revealed.These micro-fluid chips can gather the biological particles such as cell
Coke is in the center of runner, and then these biological particles will be irradiated with a laser and launch scattering light and fluorescence.Launch
Optical signal is collected by subsequent photoelectric testing sensor, and incoming analysis system is analyzed.It is generally used for the laser light of irradiation
Spot size is only some tens of pm, this requires the biological particle after being focused just at laser facula irradiation in the range of and
On focal plane.For traditional flow cytometer, this alignment procedures can be manually adjusted, because can will be each after adjusting
Component fixes, subsequent does not need frequently to adjust in addition to correction.But for micro-fluid chip, due to needing to frequently replace miniflow
Body chip to eliminate cross contamination, and manually adjusts time-consuming and laborious and is difficult to ensure consistency.
Summary of the invention
The present invention is directed at least solve one of above-mentioned technical problem.
For this purpose, an object of the present invention is to provide a kind of micro-fluid chips based on binary optical device to be automatically aligned to
Method.
It is another object of the present invention to propose that a kind of micro-fluid chip based on binary optical device is automatically aligned to be
System.
To achieve the goals above, embodiment of the invention discloses a kind of micro-fluid chips based on binary optical device
Automatic aligning method, including laser, binary optical device, focusing objective len, micro-fluid chip, light collect object lens, optical filter, light
Photodetector, computer, two-dimentional platform and fluorescent microsphere, the laser pass through the binary for emitting laser, the laser
Optical device shaping is simultaneously focused object lens and is focused to required hot spot, and the hot spot irradiation is located at glimmering described in the micro-fluid chip
Light microballoon so that the fluorescent microsphere issues fluorescence, collect object lens collection through the light and filtered out by the optical filter by the fluorescence
Enter the photoelectric detector after veiling glare, fluorescence signal is converted to electric signal by the photoelectric detector, the computer according to
The wave character of the electric signal differentiates the position of micro-fluid chip, and controls the two-dimentional platform and adjust in the micro-fluid chip
The position for being focused sample flow；The described method comprises the following steps: the injection of S1: Xiang Suoshu micro-fluid chip includes standard
The sample flow and sheath liquid stream of fluorescence miniflow make the sample flow be focused on the center of runner, control the two-dimentional platform into
Row movement, until detecting the pulse signal of the fluorescent microsphere in the photoelectric detector；S2: believe according to there is the fluorescence
Number extreme position, control that the two-dimentional platform is mobile until the focusing sample flow is located at the center of the hot spot；S3: root
It is mobile until being located on focal plane by the focusing sample flow according to the default signature waveform control two-dimentional platform；S4: according to described
The coefficient of variation of fluorescence signal is finely adjusted the two-dimentional platform until described be focused sample flow and be located at best irradiation position.
Micro-fluid chip automatic aligning method according to an embodiment of the present invention based on binary optical device, using binary optical
Device is learned as spot shaping device, and according to the feature decision of the fluorescence signal issued after the irradiation of standard fluorescence particle stimulated light
It is focused the position of sample flow in micro-fluid chip, and adjusts micro-fluid chip accordingly and makes to be focused sample flow and be located at optimum bit
It sets, to realize optimal detection effect.
In addition, the micro-fluid chip automatic aligning method according to the above embodiment of the present invention based on binary optical device,
It can also have the following additional technical features:
Further, step S2 further comprises: S201: the control two-dimentional platform is moved to the left, until the photoelectricity is visited
Limit on the left position of the device detection less than fluorescence signal is surveyed, and records the limit on the left position；S202: the control two-dimentional platform is to the right
It is mobile, until limit on the right-right-hand limit position of the photodetector detection less than fluorescence signal, and record the limit on the right-right-hand limit position；
S203: the control two-dimentional platform is mobile, is focused sample flow described in is located at the limit on the left position and the limit on the right-right-hand limit position
Middle position；Wherein, the two-dimentional platform is moved to the left to collect between focus objective lens perpendicular to the focusing objective len and the light
Line be moved to the left, the two dimension platform moves right to collect the company between object lens perpendicular to the focusing objective len and the light
Line moves right.
Further, step S3 further comprises: S301: be focused described in setting sample flow be located on the focal plane and
The wave character for the fluorescent pulse signal that preset fluorescent microsphere when nearby issues；S302: the control two-dimentional platform is forwards, backwards
Direction is mobile, until it is mobile to stop the two-dimentional platform when the computer acquisition wave character is consistent with predetermined waveform feature
Sample is focused described in adjustment to flow on the focal plane of the focusing objective len.
Further, step S4 further comprises: S401: the control two-dimentional platform is adjusted in the lateral direction, and
Acquire the coefficient of variation of the fluorescence signal in adjustment process；S402: the control two-dimentional platform is mobile to be made described to be focused sample flow
Positioned at the minimum corresponding position of the coefficient of variation.
Further, after step s4 further include: S5: being passed through cleaning agent cleaning runner.
To achieve the goals above, embodiment of the invention discloses a kind of micro-fluid chips based on binary optical device
Automatic Alignment System, including laser, binary optical device, focusing objective len, micro-fluid chip, light collect focus objective lens, optical filter,
Photoelectric detector, computer, two-dimentional platform and fluorescent microsphere, the laser pass through described two for emitting laser, the laser
First optical device shaping is simultaneously focused object lens and is focused to required hot spot, and the hot spot irradiation is located at described in the micro-fluid chip
Fluorescent microsphere so that the fluorescent microsphere issues fluorescence, collect object lens collection through the light and filtered by the optical filter by the fluorescence
Except the photoelectric detector is entered after veiling glare, fluorescence signal is converted to electric signal by the photoelectric detector；Pump or other liquid streams
Source makes the sample flow be focused on stream to sample flow and sheath liquid stream of the micro-fluid chip injection comprising standard fluorescence miniflow
Behind the center in road, the computer control two-dimentional platform, which move, detects the fluorescence in the photoelectric detector
The pulse signal of microballoon；The computer is also used to control the two-dimentional platform according to the extreme position for the fluorescence signal occur
It is mobile to be focused the center that sample flow is located at the hot spot up to described；The computer is also used to according to preset fluorescence
The signature waveform control for the fluorescent pulse signal that microballoon the issues two-dimentional platform is mobile until being focused sample flow is located at focal plane
On；The computer is also used to be finely adjusted until being focused sample the two-dimentional platform according to the coefficient of variation of the fluorescence signal
This stream is located at best irradiation position.
Micro-fluid chip Automatic Alignment System according to an embodiment of the present invention based on binary optical device, using binary optical
Device is learned as spot shaping device, and according to the feature decision of the fluorescence signal issued after the irradiation of standard fluorescence particle stimulated light
It is focused the position of sample flow in micro-fluid chip, and adjusts micro-fluid chip accordingly and makes to be focused sample flow and be located at optimum bit
It sets, to realize optimal detection effect.
In addition, the micro-fluid chip Automatic Alignment System according to the above embodiment of the present invention based on binary optical device,
It can also have the following additional technical features:
Further, the computer is further used for: the control two-dimentional platform is moved to the left, until the photodetection
Device cannot detect the limit on the left position of fluorescence signal, and record the limit on the left position；The two-dimentional platform is controlled to move right,
Until the photodetector cannot detect the limit on the right-right-hand limit position of fluorescence signal, and record the limit on the right-right-hand limit position；Control institute
It is mobile until being focused the middle position that sample flow is located at the limit on the left position and the limit on the right-right-hand limit position to state two-dimentional platform；Its
In, the two dimension platform is moved to the left to collect the line between focus objective lens perpendicular to the focusing objective len and the light to moving to left
Dynamic, the two dimension platform moves right to collect the line between focusing objective len perpendicular to the focusing objective len and light and move right.
Further, the computer is further used for: be focused described in setting sample flow be located on the focal plane and
The predetermined waveform feature for the fluorescent pulse signal that fluorescent microsphere when nearby issues；The two-dimentional platform is controlled to move forward and backward
It is dynamic, until stopping the mobile tune of two-dimentional platform when the computer acquisition wave character is consistent with the predetermined waveform feature
Sample is focused described in whole to flow on the focal plane of the focusing objective len.
Further, the computer is further used for: the control two-dimentional platform is moved in the lateral direction, and is adopted
Collect the coefficient of variation of the fluorescence signal in moving process；The two-dimentional platform is controlled to be moved to until described be focused sample flow and be located at
The minimum corresponding position of the coefficient of variation.
Further, the photoelectric detector is that photoelectricity training increases pipe, photomultiplier tube array, the pale pinkish purple unit of charge or photoelectricity
Diode.
Additional aspect and advantage of the invention will be set forth in part in the description, and will partially become from the following description
Obviously, or practice through the invention is recognized.
Detailed description of the invention
Above-mentioned and/or additional aspect of the invention and advantage will become from the description of the embodiment in conjunction with the following figures
Obviously and it is readily appreciated that, in which:
Fig. 1 is the flow chart of the micro-fluid chip Automatic Alignment System based on binary optical device of the embodiment of the present invention；
Fig. 2 is the structure of the micro-fluid chip Automatic Alignment System based on binary optical device of one embodiment of the invention
Schematic diagram；
Fig. 3 is that the binary optical device of one embodiment of the invention to form the rectangular light of quasi- flat-top to laser shaping and homogenizing
Spot schematic diagram；
Fig. 4 is the inside fluid channel schematic diagram of the micro-fluid chip of one embodiment of the invention；
Fig. 5 is the signal intensity of photoelectricity testing part output in the step S1 of one embodiment of the invention, and after focusing
The change in location schematic diagram of sample flow；
Fig. 6 is the signal intensity of photoelectricity testing part output in the step S2 of one embodiment of the invention, and after focusing
The change in location schematic diagram of sample flow；
Fig. 7 is the signal intensity of photoelectricity testing part output in the step S3 of one embodiment of the invention, and after focusing
The change in location of sample flow, the Energy distribution situation schematic diagram of hot spot when defocus；
Schematic diagram when Fig. 8 is the fine tuning best irradiation position of acquisition in left and right in the step S4 of one embodiment of the invention.
Specific embodiment
The embodiment of the present invention is described below in detail, examples of the embodiments are shown in the accompanying drawings, wherein from beginning to end
Same or similar label indicates same or similar element or element with the same or similar functions.Below with reference to attached
The embodiment of figure description is exemplary, and for explaining only the invention, and is not considered as limiting the invention.
In the description of the present invention, it is to be understood that, term " center ", " longitudinal direction ", " transverse direction ", "upper", "lower",
The orientation or positional relationship of the instructions such as "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outside" is
It is based on the orientation or positional relationship shown in the drawings, is merely for convenience of description of the present invention and simplification of the description, rather than instruction or dark
Show that signified device or element must have a particular orientation, be constructed and operated in a specific orientation, therefore should not be understood as pair
Limitation of the invention.In addition, term " first ", " second " are used for description purposes only, it is not understood to indicate or imply opposite
Importance.
In the description of the present invention, it should be noted that unless otherwise clearly defined and limited, term " installation ", " phase
Even ", " connection " shall be understood in a broad sense, for example, it may be being fixedly connected, may be a detachable connection, or be integrally connected；It can
To be mechanical connection, it is also possible to be electrically connected；It can be directly connected, can also can be indirectly connected through an intermediary
Connection inside two elements.For the ordinary skill in the art, above-mentioned term can be understood at this with concrete condition
Concrete meaning in invention.
Referring to following description and drawings, it will be clear that these and other aspects of the embodiment of the present invention.In these descriptions
In attached drawing, some particular implementations in the embodiment of the present invention are specifically disclosed, to indicate to implement implementation of the invention
Some modes of the principle of example, but it is to be understood that the scope of embodiments of the invention is not limited.On the contrary, of the invention
Embodiment includes all changes, modification and the equivalent fallen within the scope of the spirit and intension of attached claims.
It is automatically right that the micro-fluid chip according to an embodiment of the present invention based on binary optical device is described below in conjunction with attached drawing
Quasi- method and system.
Fig. 1 is the process of the micro-fluid chip automatic aligning method based on binary optical device of one embodiment of the invention
Figure, Fig. 2 is the structural representation of the micro-fluid chip Automatic Alignment System based on binary optical device of one embodiment of the invention
Figure.
As depicted in figs. 1 and 2, a kind of micro-fluid chip automatic aligning method based on binary optical device, including it is as follows
Hardware: laser 103, binary optical device 104, focusing objective len 105, micro-fluid chip 106, light collect object lens 107, optical filter
113, photoelectric detector 114, computer 116, two-dimentional platform 109 and fluorescent microsphere.Laser 103 is for emitting laser, laser warp
It crosses 104 shaping of binary optical device and is focused object lens 105 and be focused to required hot spot, the shape on the focal plane of focusing objective len 105
At designed hot spot.Hot spot irradiation is located at fluorescent microsphere in micro-fluid chip 106 so that fluorescent microsphere issues fluorescence.Fluorescence warp
Light collects object lens 107 and collects and enter photoelectric detector 114 after filtering out veiling glare by optical filter 113.Photoelectric detector 114 is by fluorescence
Signal is converted to electric signal.Computer 116 differentiates the position of micro-fluid chip 106 according to the wave character of electric signal, and controls
Two-dimentional platform 109 adjusts the position of micro-fluid chip 106.Sample flow and sheath fluid stream after testing enters in waste liquid barrel 110, observation
Screen 111 can be used for observing hot spot.
The micro-fluid chip automatic aligning method based on binary optical device of the embodiment of the present invention the following steps are included:
S1: include the sample flow and sheath liquid stream of standard fluorescence miniflow to micro-fluid chip injection, be focused on sample flow
The center of runner controls two-dimentional platform and is moved, until detecting the pulse signal of fluorescent microsphere in photoelectric detector.
Specifically, micro-fluid chip 106 is mounted on the fixed frame 108 of two-dimentional platform 109.As shown in Figure 3 and Figure 4, lead to
Pump 101 and pump 102 are crossed respectively to 106 injecting sample stream 321 of micro-fluid chip and sheath liquid stream 322, and sheath liquid stream 322 passes through current divider
323 shunt, and so that sample flow 321 is focused on the center 326 of runner after vertical focusing 324 and horizontal focusing 325, most
End form is at being focused sample flow 327.The flow of sample flow 321 and sheath liquid stream 322 is respectively 1 μ l/s and 120 μ l/s, cross section of fluid channel
For 163 μm of 316 μ m, sample flow flow velocity is 4.8m/s.Light 211 is by 104 shaping of binary optical device and is focused lens
105 focus, and focal beam spot 23 is formed on focal plane.When starting, the output of photoelectric detector 114 is noise signal, oscillograph
115 output waveform is as shown in Figure 5.Later, it controls two-dimentional platform 109 or so and is moved forward and backward, until being examined in photoelectric sensor 114
Measure the pulse signal of fluorescent microsphere 431.At this point, being focused sample flow 327 in 212 front and back of focal plane, sample flow 327 after focusing
In the range of exposures of hot spot 23.Generally, according to use experience, it may be determined that an initial adjustable range, so as to as early as possible
Adjust out fluorescent pulse signal.
S2: according to there is the extreme position of fluorescence signal, it is mobile until being focused sample flow 327 to control two-dimentional platform 109
In the left and right center of hot spot.
In one embodiment of the invention, step S2 further comprises:
S201: it controls two-dimentional platform 109 and is moved to the left, until photodetector 114 can't detect the limit on the left of fluorescence signal
Position, and record limit on the left position.
S202: it controls two-dimentional platform and moves right, until photodetector 114 can't detect the limit on the right-right-hand limit position of fluorescence signal
It sets, and records limit on the right-right-hand limit position；
S203: it is mobile until being focused the interposition that sample flow is located at limit on the left position and limit on the right-right-hand limit position to control two-dimentional platform
It sets；
Wherein, two-dimentional platform be moved to the left for collected perpendicular to the focusing objective len 105 and light the line between object lens 107 to
It moves left, two-dimentional platform moves right to collect the line between object lens 107 perpendicular to focusing objective len 105 and light and move right.
Specifically, as shown in fig. 6, gathering since laser uses 104 shaping of binary optical device and is focused objective lens 105
Coke, thus the 212 flat-top rectangular light spot 23 that can obtain a sharpness of border on focal plane, flowing of the long side perpendicular to liquid stream
Direction, short side are parallel with liquid flow path direction.Front and back adjusts two-dimentional platform 109 and adjusts the position of micro-fluid chip 106, until obtaining maximum
Pulse amplitude is focused sample flow 327 at this time and is located near the focal plane 212 of condenser lens 105.Micro-fluid chip is adjusted to the left
106 until not no fluorescence signal limit on the left position, be focused the left-external side that sample flow 327 is located exactly at hot spot at this time；Again to
Right adjusting micro-fluid chip 106, fluorescence signal displays again at this time, continues to adjust to the right until again without fluorescence signal,
It is focused the right outside that sample flow 327 is located exactly at hot spot at this time.According to the limit on the right-right-hand limit position of two not no fluorescence signals, adjust
Micro-fluid chip 106 makes to be focused the middle position that sample flow 327 is located at the two positions, is focused sample flow 327 at this time
In 56 on the left and right middle line of hot spot.It by above-mentioned adjusting, is focused sample flow 327 and is located near focal plane 212, and be located at hot spot
Left and right middle line on.
S3: two-dimentional platform movement is controlled according to default signature waveform and is located on focal plane until being focused sample flow 327.
In one embodiment of the invention, step S3 further comprises:
S301: setting is focused the fluorescent pulse of single fluorescent microsphere 431 when sample flow 327 is located on focal plane and is neighbouring
The predetermined waveform feature of signal；
S302: it controls two-dimentional platform 109 and moves forward and backward, until being preset on computer acquisition wave character and focal plane
The mobile adjustment of the two-dimentional platform of stopping is focused sample flow 327 and is located on the focal plane 212 of focusing objective len 105 when wave character is consistent.
Specifically, as shown in fig. 7, hot spot is only in focal plane 212 since laser uses 104 shaping of binary optical device
It is upper just to have optimal uniformity and accurately size.When defocus, according to the distance and front-rear position apart from focal plane, hot spot can be sent out
The shape that changes 601~605.Thus, when standard fluorescence microballoon 431 by hot spot, the fluorescence signal launched can become
Change 606~610.It can not only differentiate that being focused sample flow 327 is located at before focal plane 212 or after focal plane according to the feature of waveform,
It can also differentiate its distance apart from focal plane.The front-rear position for adjusting micro-fluid chip 106, when the waveform of fluorescence signal meets sample
When flowing wave character when being located on focal plane, it is focused sample flow 327 and has been adjustable accurately on coke flat 212.
S4: two-dimentional platform is finely adjusted according to the coefficient of variation of fluorescence signal until being focused sample flow 327 and is located at most preferably
Irradiation position.
In one embodiment of the invention, step S4 further comprises:
S401: it controls two-dimentional platform and is finely adjusted in the lateral direction, and acquire the variation of the fluorescence signal in moving process
Coefficient；
S402: it controls two-dimentional platform and is moved to the minimum corresponding position of the coefficient of variation.
Specifically, although being adjustable accurately on focal plane 212 as shown in figure 8, being focused sample flow 327,
Since laser uses binary optical device shaping, therefore hot spot is not absolute flat-top (not being absolute uniform).This will affect irradiation
Quality and test result.Therefore, it is possible to which left and right fine adjustments are focused the position 741 of sample flow 327.Take the area of fluorescent pulse
711 be the intensity of fluorescence signal, collects the fluorescence signal 72 for calculating 10000 fluorescent microspheres 431.Draw these signal strengths
Histogram 73 and the signal for deviateing mean value with a removal of door 731, calculate the coefficient of variation of signal in door.Left and right fine adjustments are micro-
Fluid chip 106 had both been focused the position 741 of sample flow 327, the as optimal irradiation position when coefficient of variation minimum.This
When, it is focused sample flow 327 and is not only adjustable accurately on focal plane, also in optimal irradiation position, so far complete to adjust
Section.
In one embodiment of the invention, after step s4 further include:
S5: it is passed through cleaning agent cleaning runner.So that the sample comprising biological particle can be passed through again to runner after the washing
This stream and sheath fluid stream are detected.
The embodiment of the present invention also discloses a kind of micro-fluid chip Automatic Alignment System based on binary optical device, packet
Include following hardware: laser 103, binary optical device 104, focusing objective len 105, micro-fluid chip 106, light collect object lens 107,
Optical filter 113, photoelectric detector 114, computer 116, two-dimentional platform 109 and fluorescent microsphere 431.Laser 103 is sharp for emitting
Light, laser, which passes through 104 shaping of binary optical device and is focused object lens 105, is focused to required hot spot 23.Hot spot irradiation is located at micro-
Fluorescent microsphere in fluid chip 106 is so that fluorescent microsphere issues fluorescence.Fluorescence is collected object lens 107 through light and is collected and by optical filter
113 filter out and enter photoelectric detector 114 after veiling glare.Fluorescence signal is converted to electric signal by photoelectric detector 114.Pump 101 and 102
Or other liquid flowing sources are including the sample flow and sheath liquid stream of standard fluorescence miniflow to 106, microfluid core injections, make sample flow quilt
After focusing on the center of runner, computer 116, which controls two-dimentional platform and be moved in photoelectric detector, detects that fluorescence is micro-
The pulse signal of ball；Computer 116 is also used to according to there is the extreme position of fluorescence signal, and controlling two-dimentional platform movement makes to be focused
Sample flow 327 is located at the center of hot spot；Computer 116, which is also used to control two-dimentional platform movement according to preset signature waveform, to be made
Sample flow 327 is focused to be located on focal plane；Computer 116 is also used to carry out two-dimentional platform according to the coefficient of variation of fluorescence signal
Fine tuning is located at best irradiation position until being focused sample flow 327.
In one embodiment of the invention, computer 116 is further used for: it controls two-dimentional platform 109 and is moved to the left, until
Photoelectric detector 114 can't detect the limit on the left position of fluorescence signal, and record limit on the left position；Control two-dimentional platform 109 to the right
It is mobile, until photoelectric detector 114 can't detect the limit on the right-right-hand limit position of fluorescence signal, and record limit on the right-right-hand limit position；Control two dimension
The movement of platform 109 makes to be focused the middle position that sample flow 327 is located at limit on the left position and limit on the right-right-hand limit position；Wherein, two-dimentional platform 109
It is moved to the left to collect the line between object lens 107 perpendicular to the focusing objective len 105 and the light and being moved to the left, two-dimentional platform
109 move right to collect the line between object lens perpendicular to the focusing objective len and the light and moving right.
In one embodiment of the invention, computer 116 is further used for: setting is focused fluorescence in sample flow 327
Microballoon 431 is located at the predetermined waveform feature on focal plane and when neighbouring；Two-dimentional platform 109 is controlled to move forward and backward, until
Computer 116 acquires the two-dimentional platform 109 of stopping mobile adjustment when wave character is consistent with predetermined waveform feature on focal plane and is focused
Sample flow 327 is located on the focal plane 212 of the first focusing objective len 105.
In one embodiment of the invention, computer 116 is further used for: controlling two-dimentional platform 109 in the lateral direction
It is moved to adjust and be focused the position of sample flow 327 in left and right directions, and acquires the change of the fluorescence signal in moving process
Different coefficient；It controls two-dimentional platform 109 and is moved to the minimum corresponding position of the coefficient of variation.
In one embodiment of the invention, photoelectric detector 114 is that photoelectricity training increases pipe, photomultiplier tube array, charge
Pale pinkish purple unit or photodiode.
It should be noted that the micro-fluid chip Automatic Alignment System based on binary optical device of the embodiment of the present invention
The specific reality of the micro-fluid chip automatic aligning method based on binary optical device of specific embodiment and the embodiment of the present invention
It is identical to apply mode, repeats no more.
In addition, the embodiment of the present invention the micro-fluid chip automatic aligning method based on binary optical device and system its
It constitute and effect be all for a person skilled in the art it is known, in order to reduce redundancy, do not repeat them here.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
Centainly refer to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be any
One or more embodiment or examples in can be combined in any suitable manner.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that: not
A variety of change, modification, replacement and modification can be carried out to these embodiments in the case where being detached from the principle of the present invention and objective, this
The range of invention is by claim and its equivalent limits.
Claims (10)
1. a kind of micro-fluid chip automatic aligning method based on binary optical device, which is characterized in that including laser, binary
Optical device, focusing objective len, micro-fluid chip, light collect object lens, optical filter, photoelectric detector, computer, two-dimentional platform and fluorescence
Microballoon, for the laser for emitting laser, the laser is by the binary optical device shaping and is focused object lens focusing
For required hot spot, the hot spot irradiation is located at fluorescent microsphere described in the micro-fluid chip so that fluorescent microsphere sending is glimmering
Light, the fluorescence are collected after object lens are collected and filter out veiling glare by the optical filter through the light into the photoelectric detector, institute
It states photoelectric detector and fluorescence signal is converted into electric signal, the wave character differentiation of the computer according to the electric signal is gathered
The position of burnt sample flow, and control the two-dimentional platform and adjust the position for being focused sample flow in the micro-fluid chip；
It the described method comprises the following steps:
The injection of S1: Xiang Suoshu micro-fluid chip includes the sample flow and sheath liquid stream of standard fluorescence miniflow, gathers the sample flow
Coke controls the two-dimentional platform and is moved, until detecting the fluorescence in the photoelectric detector in the center of runner
The pulse signal of microballoon；
S2: according to the extreme position for the fluorescence signal occur, the two-dimentional platform movement is controlled, is focused sample flow described in adjustment
To the left and right center of the hot spot；
S3: sample flow is focused according to the control of default signature waveform and is moved on the focal plane of focusing objective len；
S4: the two-dimentional platform is finely adjusted according to the coefficient of variation of the fluorescence signal, until described be focused sample flow position
In best irradiation position.
2. the micro-fluid chip automatic aligning method according to claim 1 based on binary optical device, which is characterized in that
Step S2 further comprises:
S201: the control two-dimentional platform is moved to the left, until limit on the left position of the photodetector detection less than fluorescence signal
It sets, and records the limit on the left position；
S202: the control two-dimentional platform moves right, until limit on the right-right-hand limit position of the photodetector detection less than fluorescence signal
It sets, and records the limit on the right-right-hand limit position；
S203: the control two-dimentional platform, which is moved to, described to be focused sample flow and is located at the limit on the left position and the limit on the right-right-hand limit position
The middle position set；
Wherein, the two-dimentional platform is moved to the left to collect the line between object lens perpendicular to the focusing objective len and the light to the left
Mobile, the two dimension platform moves right to collect the line between object lens perpendicular to the focusing objective len and the light and move right
It is dynamic.
3. the micro-fluid chip automatic aligning method according to claim 2 based on binary optical device, which is characterized in that
Step S3 further comprises:
S301: it is focused the fluorescent microsphere that sample flow is located on the focal plane of the focusing objective len and when neighbouring described in setting and sends out
The predetermined waveform feature of fluorescent pulse signal out；
S302: the control two-dimentional platform moves forward and backward, until presetting in the computer acquisition wave character with described
Stop being focused the focal plane that sample flow to the focusing objective len described in the mobile adjustment of two-dimentional platform when wave character is consistent
On.
4. the micro-fluid chip automatic aligning method according to claim 3 based on binary optical device, which is characterized in that
Step S4 further comprises:
S401: the control two-dimentional platform is adjusted in the lateral direction, and acquires the variation of the fluorescence signal in adjustment process
Coefficient；
S402: the control two-dimentional platform is mobile to make the sample flow that is focused be located at the minimum corresponding position of the coefficient of variation.
5. the micro-fluid chip automatic aligning method according to claim 1-4 based on binary optical device,
It is characterized in that, after step s4 further include:
S5: it is passed through cleaning agent cleaning runner.
6. a kind of micro-fluid chip Automatic Alignment System based on binary optical device, which is characterized in that including laser, binary
Optical device, focusing objective len, micro-fluid chip, light collect object lens, optical filter, photoelectric detector, computer, two-dimentional platform and fluorescence
Microballoon, for the laser for emitting laser, the laser is by the binary optical device shaping and is focused object lens focusing
For required hot spot, the hot spot irradiation is located at fluorescent microsphere described in the micro-fluid chip so that fluorescent microsphere sending is glimmering
Light, the fluorescence are collected after object lens are collected and filter out veiling glare by the optical filter through the light into the photoelectric detector, institute
It states photoelectric detector and fluorescence signal is converted into electric signal；Pump or other liquid flowing sources are including mark to micro-fluid chip injection
The sample flow and sheath liquid stream of quasi- fluorescence miniflow, after so that the sample flow is focused on the center of runner, the computer control
It makes the two-dimentional platform to be moved, the pulse signal of the fluorescent microsphere is detected in the photoelectric detector；The computer
It is also used to control the two-dimentional platform movement, until in the micro-fluid chip according to the extreme position for the fluorescence signal occur
It is focused the center that sample flow is located at the hot spot；The computer is also used to according to the control of preset signature waveform
Two-dimentional platform is mobile, is focused sample flow described in and is located on the focal plane of the focusing objective len；The computer is also used to root
The focusing sample flow is finely adjusted until best irradiation position according to the coefficient of variation of the fluorescence signal.
7. the micro-fluid chip Automatic Alignment System according to claim 6 based on binary optical device, which is characterized in that
The computer is further used for:
The two-dimentional platform is controlled to be moved to the left, until limit on the left position of the photodetector detection less than fluorescence signal, and
Record the limit on the left position；It controls the two-dimentional platform to move right, until the photodetector is detected less than fluorescence signal
Limit on the right-right-hand limit position, and record the limit on the right-right-hand limit position；Control the two-dimentional platform it is mobile, until described be focused sample flow and be located at
The middle position of the limit on the left position and the limit on the right-right-hand limit position；Wherein, the two-dimentional platform is moved to the left as perpendicular to described
The line that focusing objective len and the light are collected between object lens is moved to the left, and the two dimension platform moves right as perpendicular to the focusing
The line that object lens and the light are collected between object lens moves right.
8. the micro-fluid chip Automatic Alignment System according to claim 7 based on binary optical device, which is characterized in that
The computer is further used for:
It sets the sample flow and is located at the fluorescent pulse that on the focal plane of the focusing objective len and fluorescent microsphere issues when neighbouring
The predetermined waveform feature of signal；It controls the two-dimentional platform to move forward and backward, until in the computer acquisition wave character
Sample flow position is focused up to described with the two-dimentional platform mobile adjustment is stopped when wave character is consistent on the default focal plane
In on the focal plane of the focusing objective len.
9. the micro-fluid chip Automatic Alignment System according to claim 8 based on binary optical device, which is characterized in that
The computer is further used for:
It controls the two-dimentional platform to be moved in the lateral direction, and acquires the coefficient of variation of the fluorescence signal in moving process；
It is mobile up to the sample flow that is focused is located at the minimum corresponding position of the coefficient of variation to control the two-dimentional platform.
10. according to the described in any item micro-fluid chip Automatic Alignment Systems based on binary optical device of claim 6-9,
It is characterized in that, the photoelectric detector is that photoelectricity training increases pipe, photomultiplier tube array, the pale pinkish purple unit of charge or photodiode.
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| CN108519332B (en) * | 2018-04-28 | 2019-12-03 | 南通市肺科医院（南通市第六人民医院） | Full-automatic microwell plate system and the computer-readable medium for the system |
| CN110487725B (en) * | 2019-09-04 | 2024-05-28 | 新羿制造科技（北京）有限公司 | Automatic focusing detection device and corresponding automatic focusing method |
| CN113008851B (en) * | 2021-02-20 | 2024-04-12 | 大连海事大学 | Device for improving weak signal detection signal-to-noise ratio of confocal structure based on oblique-in excitation |
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