CN105758834A - Biochip detection method implemented through laser induction and CCD acquisition - Google Patents

Abstract

The invention relates to a biochip detection method implemented through laser induction and CCD acquisition.A biochip two-dimensional scanning device and a CCD camera are provided, a laser light source with fixed power emits laser which conducts two-dimensional scanning according to certain rules in the X direction and the Y direction of a biochip under the control of a two-dimensional scanning mirror, the laser is irradiated on a biochip plane at the angle of inclined 45 degrees under work of the two-dimensional scanning mirror, and CY3 or CY5 fluorochrome is stimulated to generate fluorescence signals which are collected in the modes of imaging and multi-pixel parallel integral of the cooling type CCD camera.By means of the method, the problem of lighting of a complex light path of a laser confocal method and a high-voltage xenon lamp is avoided, and a high detection speed and sensitivity can also be guaranteed.

Description

A kind of induced with laser and the CCD biochip test method gathered

Technical field

The present invention relates to biochip test, particularly a kind of induced with laser and the CCD biochip test method gathered.

Background technology

Patent (application number: CN200410009044.7) proposes a kind of biochip test method with the detection of light hard real time and detection system so that can be detected process when light source intensity change conditions occurs in detection process in biological chips detection system in time and make detection data accurate.Patent (application number: CN03112771.1) proposes a kind of low-density biochip detection system, in conjunction with exciting light system, fluorescence signal collection system and signal detection system, Chief technology is, with fibre-optic bundle, to photomultiplier transit tube-surface and the fluorescence signal collection produced on chip is changed into the signal of telecommunication, draws a kind of method that cost is low, be applicable to detection low-density biochip fluorescence signal.Patent (application number: CN201110398112.3) proposes a kind of bio-chip test device and biochip test method, by judging that identification information is to determine whether biochip can detect, and automatically adjust the setting minimizing mistake of detection module through controller.

Above-mentioned common biochip test method substantially has two kinds, one of which is that the mode adopting laser confocal scanning and photomultiplier tube collection carries out biochip test, but this kind of method is due to the speckle by the laser focusing after expanding to only several micron levels, and rely on two-dimensional scanner that biochip is scanned so can cause that worrying problem of comparison is exactly that the speed of detection is slow.Another kind of method is then adopt the detection mode based on cooling type CCD and high pressure xenon gas lamp, but this kind of detection method has one to be exactly that its scan sensitivity is relatively low than more serious shortcoming a problem is also that the lighting source life-span of high pressure xenon gas lamp that it adopts is comparatively short.

Summary of the invention

It is an object of the invention to provide a kind of directional light adopting laser beam expanding and gather, through vibration mirror scanning and cooling type CCD, the biochip test method that excited fluorescence combines, to overcome the defect existed in prior art.

For achieving the above object, the technical scheme is that a kind of induced with laser and the CCD biochip test method gathered, it is provided that one includes a CCD camera and is arranged at the first galvanometer of described CCD camera both sides and the induced with laser CCD acquisition scans instrument of the second galvanometer for a pair；The LASER Light Source controlling to have constant power by described first galvanometer and described second galvanometer launches laser, in X direction with Y-direction be arranged at immediately below described CCD camera place biochip carry out two-dimensional scan, and laser is irradiated to biochip plane under the control of described first galvanometer and described second galvanometer, and the fluorescent dye in described biochip is excited to produce fluorescence signal；Described fluorescence signal is acquired by described CCD camera by the mode of imaging and many pixel-parallel integration, and by acquired image transmission to the computer being connected with described CCD camera.

In an embodiment of the present invention, described CCD camera includes the light receiving microscopy head and the cooling type CCD camera that set gradually from the bottom to top；Described light receiving microscopy head includes micro-lens and arrowband coated filter.

In an embodiment of the present invention, described first galvanometer and described second galvanometer are in 90 degree of orthogonal settings.

In an embodiment of the present invention, laser Oblique 45 Degree under the control of described first galvanometer and described second galvanometer is irradiated to biochip plane.

In an embodiment of the present invention, described first galvanometer and described second galvanometer are high-speed vibrating mirror, and are all connected with high-speed vibrating mirror drive circuit；Described high-speed vibrating mirror drive circuit is connected with amplification subtraction circuit, control circuit and described computer successively.

In an embodiment of the present invention, directional light laser device laser bundle in described LASER Light Source is collimated to be expanded, and forms rectangular light spot by square diaphragm and under the two-dimensional scan of described first galvanometer and described second galvanometer, described biochip uniform plane is illuminated respectively；Laser beam first deflects into default initial stepping position through described first galvanometer in X direction, and this row is scanned by described second galvanometer again along Y-direction, in this way parallel sweep bio-chip lattice plane line by line.

In an embodiment of the present invention, by trigonometric function mathematical model, the off plumb two-dimensional scan model caused by biochip probe plane and laser beam projects direction out of plumb is modified, and by the angular velocity that Sao Miao stepping spacing and the galvanometer of revised two-dimensional scan model specification the first galvanometer and the second galvanometer deflect, in order to calculate the detection speed of this biochip test method.

In an embodiment of the present invention, described realized in accordance with the following steps by trigonometric function mathematical model correction two-dimensional scan model:

Step S1: note incident intensity is I, projects in probe plane through described first galvanometer and described second galvanometer yaw tilt, and the projected area of laser beam increasesTimes, δ is the deflection angle of described first galvanometer,For the deflection angle of described second galvanometer, and light intensity becomes:

Step S2: described second galvanometer deflection β angle, makes laser beam enter line scans along Y-direction, and:

$L=\frac{h_z}{cos(45+\mathrm{\β})}$

Wherein, h_zFor described second galvanometer center of lens point height；

Step S3: adjust the angle δ of described first galvanometer 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, can be obtained fom the above equation:

$\mathrm{\δ}=\frac{x}{h_z}\·\frac{180}{\mathrm{\π}}\·cos(45+\mathrm{\β});$

Step S4: the deflection angle β that the deflection angle δ of described first galvanometer followed by described second galvanometer is done cosine adjustment, it is horizontal parallel lines to ensure the track that laser beam scans under described second galvanometer effect along Y-direction, and laser beam is definite value at the amount of deflection of X-direction, then the laser beam light path l from the first galvanometer to biochip:

The angular scanning speed of note shoot laser bundle is ω_y, the angular velocity of corresponding described second galvanometer is ω_y/ 2, and scanning physical resolution be A_s=d (um) × d (um), then the linear velocity of laser beam scanning in the Y direction is:

V=ω_y·l

Biochip A_sThe mean irradiation time of area is:

Corresponding scanning resolution A_sThe fluorescent dye of area be stimulated produce photoluminescence number of photons be:

Wherein, the quantum efficiency φ of fluorescence molecule, fluorescent dye cross section σ, q_emFor the fluorescent photon number excited, I is laser beam incident intensity, ω_yFor the angular scanning speed of shoot laser bundle, c is the light velocity, and λ is excitation wavelength, and τ is fluorescence lifetime, fluorescent dye concentration C_s,

Step S5: when the first galvanometer deflection angle that X-direction stepping spacing is corresponding is δ, then when in the Y direction each row being scanned, the angular velocity omega of the second galvanometer_y/ 2 couples of self deflection angle β carry out cosine adjustment, to keep fluorescence response q_emConcordance:

${{\mathrm{\ω}}_y}^{,}=\frac{1}{2}{\mathrm{\ω}}_y{\cos }^2\mathrm{\δ}\·{\cos }^2(45+\mathrm{\β})$

Then photoluminescence number of photons q_emFor:

$q_{em}=\frac{{\mathrm{\φI\σ\λdA}}_s}{{\mathrm{hch}}_z}\·\frac{1}{2{{\mathrm{\ω}}_y}^{,}}\·C_s.$

In an embodiment of the present invention, described CCD camera scans the part in view picture biological chip fluorescent picture every time, and this part fluoroscopic image carries out image acquisition, and by eliminating the gray value of all the other biochip probe planes not scanned, to reduce the accumulation of noise, improve signal to noise ratio；Gather several fluoroscopic images according to this acquisition mode, and after filtering noise, linear superposition forms a complete width biological chip fluorescent picture, completes the collection to view picture biological chip fluorescent picture.

In an embodiment of the present invention, described fluorescent dye is CY3 or CY5.

Compared to prior art, the method have the advantages that a kind of induced with laser proposed by the invention and the CCD biochip test method gathered, both the complex optical path of laser confocal methods and the lighting problem of high pressure xenon gas lamp had been avoided, it also is able to ensure higher detection speed and sensitivity, it is possible to make the sensitivity of detection be better than 1flour/um², the detection time of single channel scanning 22mm*22mm, compared to simple in construction the laser confocal scanning detection mode of mainstream applications, scanning speed was fast less than 55 seconds.This product can be widely applied to the aspects such as medical diagnosis on disease, drug screening, preventive medicine, it is possible to solves the effect that some traditional detection modes are not accomplished.

Accompanying drawing explanation

Fig. 1 is the structural representation of the bio-chip test device that induced with laser and CCD gather in the present invention.

Fig. 2 is the driving control circuit schematic diagram of two-dimensional scanning mirrors in the present invention.

Fig. 3 is X-direction step-scan generalized section in the present invention.

Fig. 4 is that in the present invention, Y-direction scans generalized section continuously.

Fig. 5 is the workflow diagram of the scanner of the biochip test method that induced with laser and CCD gather in the present invention.

Detailed description of the invention

Below in conjunction with accompanying drawing, technical scheme is specifically described.

The present invention provides a kind of induced with laser and the CCD bio-chip test device gathered, and including excitation source, biochip two-dimensional scanner, light receiving microscopy head, these parts of cooling type CCD camera, its system block diagram is as shown in Figure 1.By configuring the two-dimensional scan model pre-set in a computer, make the LASER Light Source with constant power launch laser and carry out two-dimensional scan by X-direction and the Y-direction at biochip that control of two-dimensional scanning mirrors according to certain rule, laser Oblique 45 Degree under the work of two-dimensional scanning mirrors is irradiated to biochip plane, and excites the mode that CY3 or CY5 fluorescent dye produces the fluorescence signal imaging by cooling type CCD camera and many pixel-parallel integration that signal is acquired.So both avoid the complex optical path of laser confocal methods and the lighting problem of high pressure xenon gas lamp, it is also possible to ensure higher detection speed and sensitivity.

Further, in the present embodiment, by utilizing the principle of CY3 and CY5 fluorescent dye Stokes shift after being subject to excitation, the biochip of labelling CY3 or CY5 fluorescent dye is carried out excitation light irradiation, produced fluorescence point number of words can be excited to carry out theoretical reckoning fluorescent dye by exciting light, in conjunction with the minimum fluorescent intensity coefficient that cooling type CCD camera can detect, the theoretical values of the directional light that can calculate employing laser beam expanding the detection sensitivity gathering the biochip test method that excited fluorescence combines through vibration mirror scanning and cooling type CCD.

Further, in the present embodiment, fluorescent dye is stimulated produced fluorescence intensity and excitating light strength I, wavelength X_exc, and the quantum efficiency φ of fluorescence molecule, extinction coefficient epsilon and dye strength C_sClose relation.When fluorescent dye concentration is relatively low, ignoring quenching effect, when it is stimulated, the excitation rate of each fluorescence molecule being in ground state can be expressed as:

$\mathrm{\α}=\frac{I\mathrm{\σ}}{h\mathrm{\ν}}---\left(1\right)$

$\mathrm{\ν}=\frac{c}{{\mathrm{\λ}}_{exc}}---\left(2\right)$

In formula, c is the light velocity, λ_excExcitation wavelength, the unit of light intensity I is W/cm², hv is the energy absorbing photon, and the unit of fluorescent dye cross 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 fluorescence molecule, and N is the fluorescence molecule sum that exciting light irradiates, N₁For being in the fluorescence molecule number of excited state.When fluorescent dye excitation process reaches steady statue, it is equal with the sharp speed that disappears to be excited speed:

$\frac{{\mathrm{dN}}_1}{dt}=(N-N_1)\mathrm{\α}-N_1\frac{1}{\mathrm{\τ}}---\left(4\right)$

$\frac{d(N-N_1)}{dt}=N_1\frac{1}{\mathrm{\τ}}-(N-N_1)\mathrm{\α}---\left(5\right)$

Namely there is normalized fluorescence excitation ratio:

$N_f=\frac{N_1}{N}=\frac{\mathrm{\α}\mathrm{\τ}}{1+\mathrm{\α}\mathrm{\τ}}---\left(6\right)$

Therefore be excited produce fluorescence speed (normalization) be:

$p_f=\frac{\mathrm{\φ}\·N_f}{\mathrm{\τ}}---\left(7\right)$

In formula, φ is the quantum efficiency of fluorescent dye.

If the surface area (physical resolution) of CCD biochip that pixel imaging is corresponding is A_s(um²), fluorescent dye concentration C_s(flour/um²), sweep time t_s(s), then the fluorescent photon number q that can excite_emFor:

q_em=p_f·A_s·C_s·t_s(8)

Fluorescent photon arrives CCD pixel by optical lens imaging optical path and is formed response electron number q_sFor:

$q_s=D_{ccd}\·K_{em}\·\frac{1-cos\mathrm{\θ}}{2}\·q_{em}---\left(9\right)$

θ=sin^-1(NA)(10)

D in formula_ccdFor the quantum efficiency of CCD, K_emFor the light transmission efficiency of optical glass, NA is the numerical aperture of object lens.

Wherein, about the response electron number q of CCD in formula 8 and formula 9_sFluorescence intensity C with bio-chip lattice_sRelation be the basis of scanner design.

Further, in the present embodiment, the light beam of directional light laser instrument is collimated to be expanded, form rectangular light spot by square diaphragm under the two-dimensional scan of galvanometer, realize the Uniform Illumination of biochip plane, scanner operating room, laser beam first in X direction the galvanometer a in two-dimensional scanning mirrors deflect into the initial stepping position determined, Y-direction is carried out the scanning of a line by galvanometer b in two-dimensional scanning mirrors again, so can parallel sweep bio-chip lattice plane line by line.Due to biochip probe plane and laser beam projects direction out of plumb, so the projection distance of each position and crevice projection angle in biochip probe plane are all different, cause that area and the intensity of laser beam projects are inconsistent, it is possible to adopt the mathematical model of trigonometric function relational expression that this off plumb two-dimensional scan model is modified.Can set that the angular velocity of its scanning stepping spacing and galvanometer deflection is extrapolated the directional light adopting laser beam expanding and gathers the detection speed of the biochip test method that excited fluorescence combines through vibration mirror scanning and cooling type CCD by revised two-dimensional scan model.

As in figure 2 it is shown, be the driving control circuit schematic diagram of galvanometer a and galvanometer b.Owing to laser is typical point source, the light beam of laser instrument is collimated expands formation directional light, by cross section isInscribed square diaphragm after formed rectangular light spot, under the two-dimensional scan of galvanometer a and galvanometer b, realize the Uniform Illumination of plane.The 90 degree of orthogonal installations of double; two 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 step-scan section is as shown in Figure 3, Y-direction scans section as shown in Figure 4 continuously: during scanner work, is first deflected laser beam in X direction to the initial stepping position determined by galvanometer a, and Y-direction is carried out a line and scans continuously by galvanometer b again.Galvanometer a can arrive new position by step by step modulating, galvanometer b parallel sweep biochip probe plane line by line successively.

Further, in the present embodiment, the effective probe plane 22 × 22mm of biochip is set², the central point height h of galvanometer b eyeglass_z=55mm, the center distance e=8mm of galvanometer eyeglass.Laser beam edgeDirection projects, and in the Y direction, light path L is 70.0 to 85.9mm, the crevice projection angle of galvanometer bIt is 38.66 to 50.20 degree；In X-direction, the deflection angle δ of galvanometer a is-8.03 to 8.03 degree.

In the present embodiment, due to biochip probe plane and laser beam projects direction out of plumb, all different at the projection distance of each position and the crevice projection angle of biochip probe plane, cause that the area of laser beam projects and intensity are inconsistent.

If laser beam incident intensity is I, projecting in probe plane through galvanometer a and b yaw tilt, the projected area of laser beam increasesTimes, its light intensity becomes:

As shown in Figure 4, galvanometer b deflects β angle, it is possible to achieve laser beam is scanned y2 a line along Y-direction by y1, Qi Zhongyou:

$L=\frac{h_z}{cos(45+\mathrm{\β})}---\left(13\right)$

As it is shown on figure 3, laser beam is by x1 position, by adjusting the angle δ of galvanometer a, it is possible to by the stepping spacing set, x1 to x2 direction carry out stepping deflection, its amount of deflection:

X=(L+e) tan δ (15)

Such as Fig. 3 convolution 13, all less relative to L, e and δ, then L+e ≈ L, tan δ ≈ δ, formula 15 can obtain:

$\mathrm{\δ}=\frac{x}{h_z}\·\frac{180}{\mathrm{\π}}\·cos(45+\mathrm{\β})---\left(16\right)$

Being horizontal parallel lines for ensureing the track y1 to y2 that laser beam scans under galvanometer b effect along Y-direction, namely laser beam is definite value at the side-play amount x of X-direction, then the deflection angle δ of galvanometer a should followed by the deflection angle β of galvanometer b and does cosine adjustment.

As it is shown on figure 3, the light path l that laser beam is from galvanometer a to biochip:

Set out the angular scanning speed of laser beam as ω_y(angular velocity of corresponding galvanometer b is ω_y/ 2), the physical resolution of scanning is A_s=d (um) × d (um), then the linear velocity of laser beam scanning in the Y direction is:

V=ω_y·l(18)

Biochip A_sThe mean irradiation time of area is:

Corresponding scanning resolution A can be obtained by formula 8 and formula 12_sThe fluorescent dye of area be stimulated produce photoluminescence number of photons:

For keeping fluorescence response q_emConcordance, when the galvanometer a deflection angle that X-direction stepping spacing is corresponding is defined as δ, Y-direction often scans the angular velocity omega of galvanometer b during a line_y/ 2 cosine adjustment that should carry out self deflection angle β, namely have according to formula 20:

${{\mathrm{\ω}}_y}^{,}=\frac{1}{2}{\mathrm{\ω}}_y{\cos }^2\mathrm{\δ}\·{\cos }^2(45+\mathrm{\β})---\left(21\right)$

So the relational expression of photoluminescence number of photons is rewritten as:

$q_{em}=\frac{{\mathrm{\φI\σ\λdA}}_s}{{\mathrm{hch}}_z}\·\frac{1}{2{{\mathrm{\ω}}_y}^{,}}\·C_s---\left(22\right)$

Namely at the angular scanning speed ω of galvanometer b_y' fluorescence response q_emWith probe dye concentration C_sFor the linear relationship determined.

Further, in the present embodiment, the acquisition system of image is also had cooling type CCD camera to constitute by micro-lens, arrowband coated filter, by the part scanned in view picture biological chip fluorescent picture and in time it is carried out image acquisition, the accumulation that can greatly reduce noise by eliminating the gray value of all the other biochip probe planes not scanned improves signal to noise ratio, can collect several fluoroscopic images by this acquisition mode and linear superposition after filtering noise is formed a complete width biological chip fluorescent picture.

Further, in the present embodiment, the workflow diagram of the scanner of the biochip test method that induced with laser and CCD gather is as shown in Figure 5.After placing biochip to be detected, by computer is configured, utilize through the revised two-dimensional scan model of said method, control two-dimensional scan high-speed vibrating mirror, biochip probe plane is carried out two-dimensional scan, after the part scanning whole probe plane, stop scanning a period of time and carry out image acquisition, transfer data to immediately after collecting image computer stores, it is further continued for carrying out two-dimensional scan after storage and so circulates until by complete for whole biochip probe flat scanning, the denoising carrying out image in computer system and the biological chip fluorescent picture becoming a width complete the image integration of piecemeal.

It is above presently preferred embodiments of the present invention, all changes made according to technical solution of the present invention, when produced function is without departing from the scope of technical solution of the present invention, belong to protection scope of the present invention.

Claims (10)

1. an induced with laser and the CCD biochip test method gathered, it is characterised in that, provide one include a CCD camera and be arranged at the first galvanometer of described CCD camera both sides and the induced with laser CCD acquisition scans instrument of the second galvanometer for a pair；The LASER Light Source controlling to have constant power by described first galvanometer and described second galvanometer launches laser, in X direction with Y-direction be arranged at immediately below described CCD camera place biochip carry out two-dimensional scan, and laser is irradiated to biochip plane under the control of described first galvanometer and described second galvanometer, and the fluorescent dye in described biochip is excited to produce fluorescence signal；Described fluorescence signal is acquired by described CCD camera by the mode of imaging and many pixel-parallel integration, and by acquired image transmission to the computer being connected with described CCD camera.

2. a kind of induced with laser according to claim 1 and the CCD biochip test method gathered, it is characterised in that described CCD camera includes the light receiving microscopy head and the cooling type CCD camera that set gradually from the bottom to top；Described light receiving microscopy head includes micro-lens and arrowband coated filter.

3. a kind of induced with laser according to claim 1 and the CCD biochip test method gathered, it is characterised in that described first galvanometer and described second galvanometer are in 90 degree of orthogonal settings.

4. a kind of induced with laser according to claim 3 and the CCD biochip test method gathered, it is characterised in that laser Oblique 45 Degree under the control of described first galvanometer and described second galvanometer is irradiated to biochip plane.

5. a kind of induced with laser according to claim 1 and the CCD biochip test method gathered, it is characterised in that described first galvanometer and described second galvanometer are high-speed vibrating mirror, and are all connected with high-speed vibrating mirror drive circuit；Described high-speed vibrating mirror drive circuit is connected with amplification subtraction circuit, control circuit and described computer successively.

6. a kind of induced with laser according to claim 1 and the CCD biochip test method gathered, it is characterized in that, directional light laser device laser bundle in described LASER Light Source is collimated to be expanded, and forms rectangular light spot by square diaphragm and under the two-dimensional scan of described first galvanometer and described second galvanometer, described biochip uniform plane is illuminated respectively；Laser beam first deflects into default initial stepping position through described first galvanometer in X direction, and this row is scanned by described second galvanometer again along Y-direction, in this way parallel sweep bio-chip lattice plane line by line.

7. a kind of induced with laser according to claim 4 and the CCD biochip test method gathered, it is characterized in that, by trigonometric function mathematical model, the off plumb two-dimensional scan model caused by biochip probe plane and laser beam projects direction out of plumb is modified, and by the angular velocity that Sao Miao stepping spacing and the galvanometer of revised two-dimensional scan model specification the first galvanometer and the second galvanometer deflect, in order to calculate the detection speed of this biochip test method.

8. the biochip test method that a kind of induced with laser according to claim 5 and CCD gather, it is characterised in that described realized in accordance with the following steps by trigonometric function mathematical model correction two-dimensional scan model:

Step S1: note incident intensity is I, projects in probe plane through described first galvanometer and described second galvanometer yaw tilt, and the projected area of laser beam increasesTimes, δ is the deflection angle of described first galvanometer,For the deflection angle of described second galvanometer, and light intensity becomes:

Step S2: described second galvanometer deflection β angle, makes laser beam enter line scans along Y-direction, and:

$L=\frac{h_z}{cos(45+\mathrm{\β})}$

Wherein, h_zFor described second galvanometer center of lens point height；

Step S3: adjust the angle δ of described first galvanometer 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, can be obtained fom the above equation:

$\mathrm{\δ}=\frac{x}{h_z}\·\frac{180}{\mathrm{\π}}\·cos(45+\mathrm{\β});$

Step S4: the deflection angle β that the deflection angle δ of described first galvanometer followed by described second galvanometer is done cosine adjustment, it is horizontal parallel lines to ensure the track that laser beam scans under described second galvanometer effect along Y-direction, and laser beam is definite value at the amount of deflection of X-direction, then the laser beam light path l from the first galvanometer to biochip:

The angular scanning speed of note shoot laser bundle is ω_y, the angular velocity of corresponding described second galvanometer is ω_y/ 2, and scanning physical resolution be A_s=d (um) × d (um), then the linear velocity of laser beam scanning in the Y direction is:

V=ω_y·l

Biochip A_sThe mean irradiation time of area is:

Corresponding scanning resolution A_sThe fluorescent dye of area be stimulated produce photoluminescence number of photons be:

Wherein, the quantum efficiency φ of fluorescence molecule, fluorescent dye cross section σ, q_emFor the fluorescent photon number excited, I is laser beam incident intensity, ω_yFor the angular scanning speed of shoot laser bundle, c is the light velocity, and λ is excitation wavelength, and τ is fluorescence lifetime, fluorescent dye concentration C_s,

Step S5: when the first galvanometer deflection angle that X-direction stepping spacing is corresponding is δ, then when in the Y direction each row being scanned, the angular velocity omega of the second galvanometer_y/ 2 couples of self deflection angle β carry out cosine adjustment, to keep fluorescence response q_emConcordance:

${{\mathrm{\ω}}_y}^{,}=\frac{1}{2}{\mathrm{\ω}}_y{\cos }^2\mathrm{\δ}\·{\cos }^2(45+\mathrm{\β})$

Then photoluminescence number of photons q_emFor:

$q_{em}=\frac{{\mathrm{\φI\σ\λdA}}_s}{{\mathrm{hch}}_z}\·\frac{1}{2{{\mathrm{\ω}}_y}^{,}}\·C_s.$

9. a kind of induced with laser according to claim 1 and the CCD biochip test method gathered, it is characterized in that, described CCD camera scans the part in view picture biological chip fluorescent picture every time, this part fluoroscopic image is carried out image acquisition, and by eliminating the gray value of all the other biochip probe planes not scanned, to reduce the accumulation of noise, improve signal to noise ratio；Gather several fluoroscopic images according to this acquisition mode, and after filtering noise, linear superposition forms a complete width biological chip fluorescent picture, completes the collection to view picture biological chip fluorescent picture.

10. a kind of induced with laser according to claim 1 and the CCD biochip test method gathered, it is characterised in that described fluorescent dye is CY3 or CY5.