CN105758834B - A kind of biochip test method of induced with laser and CCD acquisition - Google Patents
A kind of biochip test method of induced with laser and CCD acquisition Download PDFInfo
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- CN105758834B CN105758834B CN201610262872.4A CN201610262872A CN105758834B CN 105758834 B CN105758834 B CN 105758834B CN 201610262872 A CN201610262872 A CN 201610262872A CN 105758834 B CN105758834 B CN 105758834B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N2021/6463—Optics
Abstract
The present invention relates to a kind of biochip test methods of induced with laser and CCD acquisition, biochip two-dimensional scanner and these parts of CCD camera are provided, laser light source with constant power launches laser and carries out two-dimensional scanning according to certain rule in the X-direction and Y-direction of biochip by the control of two-dimensional scanning mirrors, laser Oblique 45 Degree under the work of two-dimensional scanning mirrors is irradiated to biochip plane, and it excites CY3 CY5 fluorescent dye to generate fluorescence signal to be acquired signal by way of the imaging of cooling type CCD camera and more pixel-parallels integral.Method proposed by the invention had both avoided the complex optical path of laser confocal methods and the lighting problem of high pressure xenon gas lamp, can also guarantee higher detection speed and sensitivity.
Description
Technical field
The present invention relates to biochip test, the biochip test method of especially a kind of induced with laser and CCD acquisition.
Background technique
Patent (application number：CN200410009044.7 a kind of biochip test side with light intensity real-time detection) is proposed
Method and detection system, quilt in time when enabling the biological chips detection system to occur light source intensity change conditions in the detection process
Detect processing so that detection data is accurate.Patent (application number：CN03112771.1 a kind of low-density biochip inspection) is proposed
Examining system is to use optical fiber in conjunction with excitation photosystem, fluorescence signal collection system and signal detection system, chief technology
Beam is by the fluorescence signal collection generated on chip to photomultiplier transit pipe surface and is converted to electric signal, obtains a kind of at low cost, suitable
Method for detecting low-density biochip fluorescence signal.Patent (application number：CN201110398112.3 a kind of life) is proposed
Object chip-detecting apparatus and biochip test method, determine whether biochip can be examined by judging identification information
It surveys, and reduces mistake through the setting of controller adjust automatically detection module.
Above-mentioned common biochip test method substantially there are two types of, one of which is using laser confocal scanning and light
The mode of electric multiplier tube acquisition carries out biochip test, but this kind of method is only several due to focusing on the laser after expanding
The spot of micron level, and biochip is scanned by two-dimensional scanner so will lead to a comparison and makes us carrying on a shoulder pole
The problem of heart is exactly that the speed that detects is slow.Another method is then using based on cooling type CCD and high pressure xenon gas lamp
Detection mode, but this kind of detection method have the shortcomings that a comparison it is serious be exactly it scan sensitivity it is relatively low there are one
Problem be exactly the lighting source service life of high pressure xenon gas lamp used by it is shorter.
Summary of the invention
It adopts the purpose of the present invention is to provide a kind of directional light using laser beam expanding and through vibration mirror scanning and cooling type CCD
The biochip test method that collection excited fluorescence combines, to overcome defect existing in the prior art.
To achieve the above object, the technical scheme is that：A kind of biochip test of induced with laser and CCD acquisition
Method, providing one includes a CCD camera and a pair of the first galvanometer for being set to the CCD camera two sides and the second galvanometer
Induced with laser CCD acquisition scans instrument；There is the laser of constant power by first galvanometer and second galvanometer control
Light source launches laser, two-dimentional in the progress for the biochip being set to immediately below the CCD camera with Y-direction in X direction
Scanning, and laser is irradiated to biochip plane under the control of first galvanometer and second galvanometer, and excites
Fluorescent dye in the biochip generates fluorescence signal；The CCD camera is integrated by imaging and more pixel-parallels
Mode is acquired the fluorescence signal, and acquired image is transmitted to the computer being connected with the CCD camera.
In an embodiment of the present invention, the CCD camera includes the light receiving microscopy head set gradually from the bottom to top and cooling
Type CCD camera；The light receiving microscopy head includes micro-lens and narrowband coated filter.
In an embodiment of the present invention, first galvanometer and second galvanometer are in 90 degree of orthogonal settings.
In an embodiment of the present invention, laser Oblique 45 Degree under the control of first galvanometer and second galvanometer
It is irradiated to biochip plane.
In an embodiment of the present invention, first galvanometer and second galvanometer are high-speed vibrating mirror, and with height
Fast galvanometer driving circuit is connected；The high-speed vibrating mirror driving circuit successively with amplification subtraction circuit, control circuit and the meter
Calculation machine is connected.
In an embodiment of the present invention, directional light laser device laser beam in the laser light source is collimated to be expanded, and is passed through
Square diaphragm forms rectangular light spot respectively under the two-dimensional scanning of first galvanometer and second galvanometer to the life
Object chip plane Uniform Illumination；Laser beam first deflects into preset initial stepping position through first galvanometer in X direction, described
Second galvanometer is again scanned the row along Y-direction, in this way parallel sweep bio-chip lattice plane line by line.
In an embodiment of the present invention, it is thrown by trigonometric function mathematical model by biochip probe plane and laser beam
The two-dimensional scanning model of out of plumb caused by the out of plumb of direction is penetrated to be modified, and passes through revised two-dimensional scanning model
The angular speed for setting Sao Miao stepping spacing and the galvanometer deflection of the first galvanometer and the second galvanometer, to calculate the biochip
The detection speed of detection method.
In an embodiment of the present invention, described that two-dimensional scanning model is corrected according to following step by trigonometric function mathematical model
It is rapid to realize：
Step S1：Note incident intensity is I, projects probe through first galvanometer and the second galvanometer yaw tilt
In plane, the projected area of laser beam increasesTimes, δ is the deflection angle of first galvanometer,It is described second
The deflection angle of galvanometer, and light intensity becomes：
Step S2：Second galvanometer deflects β angle, makes laser beam along Y-direction into line scans, and：
Wherein, hzFor the second galvanometer center of lens point height；
Step S3：The angle δ of first galvanometer is adjusted, so that laser beam carries out stepping deflection in X direction, and is deflected
Amount：
X=(L+e) tan δ
Due to L+e ≈ L, tan δ ≈ δ, as available from the above equation：
Step S4：The deflection angle δ of first galvanometer deflection angle β for being followed by second galvanometer is made into cosine tune
Whole, to guarantee that laser beam is horizontal parallel lines along the track of Y-direction scanning under second galvanometer effect, and laser beam exists
The amount of deflection of X-direction is definite value, then light path l of the laser beam from the first galvanometer to biochip：
The angular scanning speed for remembering shoot laser beam is ωy, the angular speed of corresponding second galvanometer is ωy/ 2, and scan object
Reason resolution ratio is As=d (um) × d (um), then the linear velocity of the scanning of laser beam in the Y direction be：
V=ωy·l
Biochip AsThe mean irradiation time of area is：
Corresponding scanning resolution AsThe be stimulated photoluminescence number of photons of generation of the fluorescent dye of area is：
Wherein, the quantum efficiency φ of fluorescent molecule, fluorescent dye section σ, qemFor the fluorescent photon number of excitation, I is laser
Beam incident intensity, ωyFor the angular scanning speed of shoot laser beam, c is the light velocity, and λ is excitation wavelength, and τ is fluorescence lifetime, fluorescence
Dye strength Cs,
Step S5：When the corresponding first galvanometer deflection angle of X-direction stepping spacing is δ, then every row is swept in the Y direction
When retouching, the angular velocity omega of the second galvanometery/ 2 carry out cosine adjustment to itself deflection angle β, to keep fluorescence response qemConsistency：
Then photoluminescence number of photons qemFor：
In an embodiment of the present invention, the CCD camera scans a part in whole picture biological chip fluorescent picture every time,
Image Acquisition, and the gray scale by eliminating remaining biochip probe plane not scanned are carried out to the part fluorescent image
Value improves signal-to-noise ratio to reduce the accumulation of noise；Several fluorescent images are acquired according to the acquisition mode, and filter out line after noise
Property be superimposed to form complete width biological chip fluorescent picture, complete acquisition to whole picture biological chip fluorescent picture.
In an embodiment of the present invention, the fluorescent dye is CY3 or CY5.
Compared to the prior art, the invention has the advantages that：Proposed by the invention a kind of induced with laser and CCD
The biochip test method of acquisition had both avoided the complex optical path of laser confocal methods and the lighting problem of high pressure xenon gas lamp,
Also it can guarantee higher detection speed and sensitivity, the sensitivity of detection can be made better than 1flour/um2, single channel sweeps
The detection time for retouching 22mm*22mm is no more than 55 seconds, compared to tying for the laser confocal scanning detection mode of mainstream applications
Structure is simple, and scanning speed is fast.The product can be widely applied to medical diagnosis on disease, drug screening, preventive medicine etc., can solve
The effect that certainly some traditional detection modes are not accomplished.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of induced with laser and the bio-chip test device of CCD acquisition in the present invention.
Fig. 2 is the drive control circuit schematic diagram of two-dimensional scanning mirrors in the present invention.
Fig. 3 is X-direction step-scan diagrammatic cross-section in the present invention.
Fig. 4 is Y-direction continuous scanning diagrammatic cross-section in the present invention.
Fig. 5 is the work flow diagram of induced with laser and the scanner of the biochip test method of CCD acquisition in the present invention.
Specific embodiment
With reference to the accompanying drawing, technical solution of the present invention is specifically described.
The present invention provides the bio-chip test device of a kind of induced with laser and CCD acquisition, including excitation light source, biological core
Piece two-dimensional scanner, light receiving microscopy head, these parts of cooling type CCD camera, system block diagram are as shown in Figure 1.By counting
Pre-set two-dimensional scanning model is configured in calculation machine so that the laser light source with constant power launches laser and passes through two dimension
The control of scanning galvanometer carries out two-dimensional scanning according to certain rule in the X-direction and Y-direction of biochip, and laser is swept in two dimension
It retouches Oblique 45 Degree under the work of galvanometer and is irradiated to biochip plane, and CY3 CY5 fluorescent dye is excited to generate fluorescence letter
Number cooling type CCD camera imaging and more pixel-parallels integral by way of signal is acquired.Laser is both avoided in this way
The lighting problem of the complex optical path of focus method and high pressure xenon gas lamp altogether, can also guarantee higher detection speed and sensitivity.
Further, in the present embodiment, by using CY3 and CY5 fluorescent dye by this after excitation
The principle of lentor displacement carries out exciting light irradiation, Ke Yitong to the biochip of labeled CY3 CY5 fluorescent dye
It crosses exciting light and excites generated fluorescence point number of words to carry out theoretical reckoning fluorescent dye, it can in conjunction with cooling type CCD camera
The smallest fluorescent intensity coefficient detected, can calculate using the directional light of laser beam expanding and through vibration mirror scanning and cooling type
The theoretical values of the detection sensitivity for the biochip test method that CCD acquisition excited fluorescence combines.
Further, in the present embodiment, fluorescent dye be stimulated caused by fluorescence intensity and excitating light strength I, wave
Long λexcAnd quantum efficiency φ, extinction coefficient epsilon and the dye strength C of fluorescent moleculesClose relation.It is dense in fluorescent dye
When spending lower, ignore quenching effect, when being stimulated, the excitation rate of each fluorescent molecule in ground state can be expressed as：
C is the light velocity, λ in formulaexcExcitation wavelength, the unit of light intensity I are W/cm2, hv is the energy for absorbing photon, fluorescence
The unit of dyestuff section σ is cm2, the relational expression of σ and extinction coefficient epsilon：
σ=3.8 × 10-21ε(cm2) (3)
If τ is fluorescence lifetime, 1/ τ is the relaxation rate of fluorescent molecule, and the fluorescent molecule that N is irradiated by exciting light is total,
N1For the fluorescent molecule number in excitation state.When fluorescent dye excitation process reaches stable state, it is excited rate and disappears to swash speed
Rate is equal：
There is normalized fluorescence excitation ratio：
Therefore being excited generation fluorescence rate (normalization) is：
φ is the quantum efficiency of fluorescent dye in formula.
If the surface area (physical resolution) that corresponding biochip is imaged in a pixel of CCD is As(um2), fluorescence
Dye strength Cs(flour/um2), sweep time ts(s), then the fluorescent photon number q that can be excitedemFor：
qem=pf·As·Cs·ts (8)
Fluorescent photon reaches CCD pixel by optical lens imaging optical path and forms response electron number qsFor：
θ=sin-1(NA) (10)
D in formulaccdFor the quantum efficiency of CCD, KemFor the light transmission efficiency of optical mirror slip, NA is the numerical aperture of object lens.
Wherein, the response electron number q in formula 8 and formula 9 about CCDsWith the fluorescence intensity C of bio-chip latticesRelationship be
The basis of scanner design.
Further, in the present embodiment, the light beam of parallel light laser is collimated expands, and is formed by square diaphragm
Rectangular light spot realizes the Uniform Illumination of biochip plane under the two-dimensional scanning of galvanometer, and scanner operating room, laser beam is first along X
Direction deflects into determining initial stepping position through the galvanometer a in two-dimensional scanning mirrors, and the galvanometer b in two-dimensional scanning mirrors is right again
Y-direction carries out the scanning of a line, so can parallel sweep bio-chip lattice plane line by line.Due to biochip probe plane
With laser beam projects direction out of plumb, so each position of biochip probe plane projection distance and crevice projection angle not
Together, cause area and the intensity of laser beam projects inconsistent, can using the mathematical model of trigonometric function relational expression come to this not
Vertical two-dimensional scanning model is modified.By revised two-dimensional scanning model can set its scan stepping spacing and
The angular speed of galvanometer deflection is excited glimmering to extrapolate using the directional light of laser beam expanding and through vibration mirror scanning and cooling type CCD acquisition
The detection speed for the biochip test method that light combines.
As shown in Fig. 2, being the drive control circuit schematic diagram of galvanometer a and galvanometer b.Since laser is typical point light
Source, the light beam of laser is collimated to be expanded to form directional light, is by sectionInscribed square diaphragm after form square
Shape hot spot realizes the Uniform Illumination of plane under the two-dimensional scanning of galvanometer a and galvanometer b.The 90 degree of orthogonal installations of double galvanometers, galvanometer
A is responsible for X-direction scanning, and galvanometer b is responsible for Y-direction scanning.The center of lens spacing e of galvanometer a and galvanometer b, wherein X-direction stepping are swept
Section is retouched as shown in figure 3, Y-direction continuous scanning section is as shown in Figure 4：When scanner works, laser beam edge is first deflected by galvanometer a
For X-direction to determining initial stepping position, galvanometer b carries out a line continuous scanning to Y-direction again.Galvanometer a can step by step modulating arrival
New position, galvanometer b successively parallel sweep biochip probe plane line by line.
Further, in the present embodiment, 22 × 22mm of the effective probe plane of biochip is set2, in galvanometer b eyeglass
Heart point height hz=55mm, the center spacing e=8mm of galvanometer eyeglass.Laser beam edgeDirection projection, in the Y direction,
Light path L is 70.0 to 85.9mm, the crevice projection angle of galvanometer bFor 38.66 to 50.20 degree；In X-direction, the deflection angle δ of galvanometer a
It is -8.03 to 8.03 degree.
In the present embodiment, it due to biochip probe plane and laser beam projects direction out of plumb, is visited in biochip
The projection distance and crevice projection angle of each position of needle plane are different, cause the area of laser beam projects and intensity inconsistent.
If laser beam incident intensity is I, projected in probe plane through galvanometer a and b yaw tilt, the perspective plane of laser beam
Product increasesTimes, light intensity becomes：
As shown in figure 4, galvanometer b deflects β angle, laser beam may be implemented and scanned by y1 to y2 a line along Y-direction, wherein
Have：
As shown in figure 3, laser beam is by the position x1, by adjusting the angle δ of galvanometer a, can by the stepping spacing of setting, by
X1 to the direction x2 carry out stepping deflection, amount of deflection：
X=(L+e) tan δ (15)
Such as Fig. 3 and convolution 13, smaller relative to L, e and δ, then L+e ≈ L, tan δ ≈ δ can be obtained by formula 15：
To guarantee that laser beam is horizontal parallel lines along the track y1 to y2 of Y-direction scanning under galvanometer b effect, i.e. laser
Beam is definite value in the offset x of X-direction, then the deflection angle β that the deflection angle δ of galvanometer a should be followed by galvanometer b makees cosine adjustment.
As shown in figure 3, light path l of the laser beam from galvanometer a to biochip：
The angular scanning speed of laser beam is set out as ωy(angular speed of corresponding galvanometer b is ωy/ 2), the physics of scanning is differentiated
Rate is As=d (um) × d (um), then the linear velocity of the scanning of laser beam in the Y direction be：
V=ωy·l (18)
Biochip AsThe mean irradiation time of area is：
Scanning resolution A can must be corresponded to by formula 8 and formula 12sThe fluorescent dye of area is stimulated the photoluminescence photon of generation
Number：
To keep fluorescence response qemConsistency, when the corresponding galvanometer a deflection angle of X-direction stepping spacing is determined as δ, Y
The angular velocity omega of galvanometer b when the every scanning a line in directiony/ 2 should carry out the cosine adjustment of itself deflection angle β, i.e., had according to formula 20：
So the relational expression of photoluminescence number of photons is rewritten as：
I.e. in the angular scanning speed ω of galvanometer by' fluorescence response qemWith probe dye concentration CsFor determining linear pass
System.
Further, in the present embodiment, there are also cooling by micro-lens, narrowband coated filter for the acquisition system of image
Type CCD camera is constituted, and by a part in scanning whole picture biological chip fluorescent picture and carries out Image Acquisition to it in time,
The accumulation that gray value by eliminating the biochip probe plane that remaining is not scanned can greatly reduce noise improves letter
Make an uproar ratio, by this acquisition mode can collect several fluorescent images and to after filtering out noise linear superposition form complete one
Width biological chip fluorescent picture.
Further, in the present embodiment, the work of induced with laser and the scanner of the biochip test method of CCD acquisition
It is as shown in Figure 5 to make flow chart.After placing biochip to be detected, by being configured to computer, using through above-mentioned side
The revised two-dimensional scanning model of method controls two-dimensional scanning high-speed vibrating mirror, carries out two-dimensional scanning to biochip probe plane,
Stop scanning a period of time after scanning a part of entire probe plane and carry out Image Acquisition, will be counted immediately after collecting image
It is stored according to being transmitted in computer, progress two-dimensional scanning is further continued for after storage and is so recycled until will entire biological core
Piece probe flat scanning finishes, and the denoising of image is carried out into computer system and by the image integration of piecemeal at a width
Complete biological chip fluorescent picture.
The above are preferred embodiments of the present invention, all any changes made according to the technical solution of the present invention, and generated function is made
When with range without departing from technical solution of the present invention, all belong to the scope of protection of the present invention.
Claims (6)
1. a kind of biochip test method of induced with laser and CCD acquisition, it is characterised in that, providing one includes a CCD camera
And a pair is set to the first galvanometer of the CCD camera two sides and the induced with laser CCD acquisition scans instrument of the second galvanometer；It is logical
Crossing first galvanometer and second galvanometer control, there is the laser light source of constant power to launch laser, in X direction and Y
Direction is in the carry out two-dimensional scanning for the biochip being set to immediately below the CCD camera, and laser is in first galvanometer
And it is irradiated to biochip plane under the control of second galvanometer, and the fluorescent dye in the biochip is excited to produce
Raw fluorescence signal；The CCD camera is acquired the fluorescence signal by way of imaging and more pixel-parallels integral,
And acquired image is transmitted to the computer being connected with the CCD camera；
First galvanometer and second galvanometer are in 90 degree of orthogonal settings；
Laser Oblique 45 Degree under the control of first galvanometer and second galvanometer is irradiated to biochip plane；
By trigonometric function mathematical model to by caused by biochip probe plane and laser beam projects direction out of plumb not
Vertical two-dimensional scanning model is modified, and passes through revised the first galvanometer of two-dimensional scanning model specification and the second galvanometer
Sao Miao stepping spacing and galvanometer deflection angular speed, to calculate the detection speed of the biochip test method；
It is described to be realized in accordance with the following steps by trigonometric function mathematical model amendment two-dimensional scanning model：
Step S1：Note incident intensity is I, projects probe plane through first galvanometer and the second galvanometer yaw tilt
On, the projected area of laser beam increasesTimes, δ is the deflection angle of first galvanometer,For second galvanometer
Deflection angle, and light intensity becomes：
Step S2：Second galvanometer deflects β angle, makes laser beam along Y-direction into line scans, and：
Wherein, hzFor the second galvanometer center of lens point height；
Step S3：The angle δ of first galvanometer is adjusted, so that laser beam carries out stepping deflection, and amount of deflection in X direction：
X=(L+e) tan δ
Due to L+e ≈ L, tan δ ≈ δ, as available from the above equation：
Step S4：The deflection angle δ of first galvanometer deflection angle β for being followed by second galvanometer is made into cosine adjustment, with
Guarantee that laser beam is horizontal parallel lines along the track of Y-direction scanning under second galvanometer effect, and laser beam is in X-direction
Amount of deflection be definite value, then light path l of the laser beam from the first galvanometer to biochip：
The angular scanning speed for remembering shoot laser beam is ωy, the angular speed of corresponding second galvanometer is ωy/ 2, and scan physics point
Resolution is As=d (um) × d (um), then the linear velocity of the scanning of laser beam in the Y direction be：
V=ωy·l
Biochip AsThe mean irradiation time of area is：
Corresponding scanning resolution AsThe be stimulated photoluminescence number of photons of generation of the fluorescent dye of area is：
Wherein, the quantum efficiency φ of fluorescent molecule, fluorescent dye section σ, qemFor the fluorescent photon number of excitation, I enters for laser beam
Penetrate intensity, ωyFor the angular scanning speed of shoot laser beam, c is the light velocity, and λ is excitation wavelength, and τ is fluorescence lifetime, fluorescent dye
Concentration Cs,
Step S5：When the corresponding first galvanometer deflection angle of X-direction stepping spacing is δ, then every row is scanned in the Y direction
When, the angular velocity omega of the second galvanometery/ 2 carry out cosine adjustment to itself deflection angle β, to keep fluorescence response qemConsistency：
Then photoluminescence number of photons qemFor：
2. the biochip test method of a kind of induced with laser according to claim 1 and CCD acquisition, which is characterized in that
The CCD camera includes the light receiving microscopy head set gradually from the bottom to top and cooling type CCD camera；The light receiving microscopy head includes micro-
Away from camera lens and narrowband coated filter.
3. the biochip test method of a kind of induced with laser according to claim 1 and CCD acquisition, which is characterized in that
First galvanometer and second galvanometer are high-speed vibrating mirror, and are connected with high-speed vibrating mirror driving circuit；The high speed
Galvanometer driving circuit is successively connected with amplification subtraction circuit, control circuit and the computer.
4. the biochip test method of a kind of induced with laser according to claim 1 and CCD acquisition, which is characterized in that
Directional light laser device laser beam in the laser light source is collimated to be expanded, and is formed rectangular light spot by square diaphragm and is existed respectively
The biochip uniform plane is illuminated under the two-dimensional scanning of first galvanometer and second galvanometer；Laser beam elder generation edge
X-direction deflects into preset initial stepping position through first galvanometer, and second galvanometer again sweeps the row along Y-direction
It retouches, in this way parallel sweep bio-chip lattice plane line by line.
5. the biochip test method of a kind of induced with laser according to claim 1 and CCD acquisition, which is characterized in that
The CCD camera scans a part in whole picture biological chip fluorescent picture every time, carries out image to the part fluorescent image and adopts
Collection, and the gray value by eliminating remaining biochip probe plane not scanned improve letter to reduce the accumulation of noise
It makes an uproar ratio；Several fluorescent images are acquired according to the acquisition mode, and linear superposition forms complete width biology core after filtering out noise
Piece fluorescent image completes the acquisition to whole picture biological chip fluorescent picture.
6. the biochip test method of a kind of induced with laser according to claim 1 and CCD acquisition, which is characterized in that
The fluorescent dye is CY3 or CY5.
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