CN105758834B - A kind of biochip test method of induced with laser and CCD acquisition - Google Patents

Abstract

The invention relates to a biochip detection method based on laser induction and CCD acquisition, which provides a two-dimensional scanning device for biochips and a CCD camera. The X-direction and Y-direction of the chip perform two-dimensional scanning according to certain rules. The laser is irradiated obliquely at 45 degrees to the plane of the biochip under the work of the two-dimensional scanning galvanometer, and excites CY3 or CY5 fluorescent dyes to generate fluorescent signals through the cooling CCD. The signal is collected by means of camera imaging and multi-pixel parallel integration. The method proposed by the invention not only avoids the complex optical path of the laser confocal method and the lighting problems of the high-pressure xenon lamp, but also can ensure higher detection speed and sensitivity.

Description

A Biochip Detection Method Based on Laser Induction and CCD Acquisition

technical field

The invention relates to biochip detection, in particular to a biochip detection method of laser induction and CCD collection.

Background technique

The patent (application number: CN200410009044.7) proposes a biochip detection method and detection system with real-time light intensity detection, so that the biochip detection system can be detected in time when the light intensity of the light source changes during the detection process. The data is accurate. The patent (application number: CN03112771.1) proposes a low-density biochip detection system, which combines the excitation light system, fluorescence signal collection system and signal detection system. The main key technology is to use optical fiber bundles to collect the fluorescence signals generated on the chip The surface of the photomultiplier tube is converted into an electrical signal, and a low-cost method suitable for detecting the fluorescent signal of a low-density biochip is obtained. The patent (application number: CN201110398112.3) proposes a biochip detection device and a biochip detection method, which determines whether the biochip can be detected by judging the identification information, and automatically adjusts the settings of the detection module through the controller to reduce errors .

There are roughly two common biochip detection methods mentioned above, one of which is to use laser confocal scanning and photomultiplier tube acquisition for biochip detection, but this method focuses the expanded laser to only a few microns spots, and relying on a two-dimensional scanning device to scan the biochip will cause a more worrying problem, that is, the detection speed is relatively slow. Another method is to use the detection method based on cooling CCD and high-pressure xenon lamp, but this detection method has a serious disadvantage that its scanning sensitivity is relatively low. Another problem is the high-pressure xenon lamp it uses. The life of the lighting source is relatively short.

Contents of the invention

The purpose of the present invention is to provide a biochip detection method which adopts the parallel light of laser beam expansion, scans through the vibrating mirror and collects the stimulated fluorescence with the cooled CCD, so as to overcome the defects in the prior art.

In order to achieve the above object, the technical solution of the present invention is: a biochip detection method of laser induction and CCD collection, providing a first vibrating mirror and a second vibrating mirror including a CCD camera and a pair of being arranged on both sides of the CCD camera. The laser-induced CCD acquisition scanner of the vibrating mirror; the laser light source with a fixed power is controlled by the first vibrating mirror and the second vibrating mirror to emit laser light, and is arranged directly below the CCD camera along the X direction and the Y direction Two-dimensional scanning of the biochip at the location, and the laser is irradiated onto the plane of the biochip under the control of the first vibrating mirror and the second vibrating mirror, and excites the fluorescent dye in the biochip to generate a fluorescent signal; The CCD camera collects the fluorescence signal through imaging and multi-pixel parallel integration, and transmits the collected image to a computer connected to the CCD camera.

In an embodiment of the present invention, the CCD camera includes a light-receiving lens and a cooled CCD camera arranged in sequence from bottom to top; the light-receiving lens includes a macro lens and a narrow-band coated filter.

In an embodiment of the present invention, the first oscillating mirror and the second oscillating mirror are arranged perpendicular to each other at 90 degrees.

In an embodiment of the present invention, under the control of the first vibrating mirror and the second vibrating mirror, the laser light is irradiated to the plane of the biochip obliquely at 45 degrees.

In an embodiment of the present invention, both the first vibrating mirror and the second vibrating mirror are high-speed vibrating mirrors, and both are connected to a high-speed vibrating mirror driving circuit; The control circuit is connected with the computer.

In an embodiment of the present invention, the laser beam of the parallel laser in the laser light source is collimated and expanded, and passes through a square diaphragm to form a rectangular spot on the two-dimensional surfaces of the first vibrating mirror and the second vibrating mirror respectively. Under scanning, the plane of the biochip is uniformly illuminated; the laser beam is first deflected to a preset initial step position along the X direction through the first vibrating mirror, and then the second vibrating mirror scans the row along the Y direction, In this way, the biochip dot matrix plane is scanned in parallel row by row.

In one embodiment of the present invention, the non-perpendicular two-dimensional scanning model caused by the non-perpendicularity between the plane of the biochip probe and the projection direction of the laser beam is corrected by the trigonometric function mathematical model, and the corrected two-dimensional scanning model The scanning step pitch of the first vibrating mirror and the second vibrating mirror and the angular velocity of the vibrating mirror deflection are set to calculate the detection speed of the biochip detection method.

In an embodiment of the present invention, the correction of the two-dimensional scanning model through the trigonometric function mathematical model is implemented according to the following steps:

Step S1: record the incident intensity as I, deflect and obliquely project onto the probe plane through the first galvanometer and the second galvanometer, and the projected area of the laser beam increases times, δ is the deflection angle of the first vibrating mirror, is the deflection angle of the second vibrating mirror, and the light intensity becomes:

Step S2: The second galvanometer is deflected by an angle β, so that the laser beam is scanned along the Y direction, and:

Wherein, _hz is the height of the center point of the second vibrating mirror lens;

Step S3: Adjust the angle δ of the first vibrating mirror so that the laser beam is deflected step by step along the X direction, and the deflection amount is:

x=(L+e)tanδ

Since L+e≈L, tanδ≈δ, it can be obtained from the above formula:

Step S4: Make a cosine adjustment of the deflection angle δ of the first galvanometer following the deflection angle β of the second galvanometer to ensure that the trajectory of the laser beam scanned in the Y direction under the action of the second galvanometer is Horizontal parallel straight lines, and the deflection of the laser beam in the X direction is a constant value, then the optical path l of the laser beam from the first vibrating mirror to the biochip:

Note that the scanning angular velocity of the outgoing laser beam is ω _y , the angular velocity corresponding to the second vibrating mirror is ω _y /2, and the scanning physical resolution is A _s =d(um)×d(um), then the laser beam is in Y The linear velocity of scanning in the direction is:

v=ω _y ·l

The average irradiation time of the biochip A _s area is:

The number of photofluorescent photons generated by the excitation of the fluorescent dye corresponding to the scanning resolution A _s area is:

Among them, the quantum efficiency φ of the fluorescent molecule, the cross-section σ of the fluorescent dye, q _em is the number of excited fluorescent photons, I is the incident intensity of the laser beam, ω _y is the scanning angular velocity of the outgoing laser beam, c is the speed of light, and λ is the wavelength of the excitation light, τ is the fluorescence lifetime, the fluorescent dye concentration C _s ,

Step S5: When the deflection angle of the first vibrating mirror corresponding to the step pitch in the X direction is δ, when scanning each row in the Y direction, the angular velocity ω _y /2 of the second vibrating mirror is cosine-adjusted to its own deflection angle β , to keep the fluorescence response q _em consistent:

Then the number of photons q _em of photoluminescence is:

In one embodiment of the present invention, the CCD camera scans a part of the entire biochip fluorescence image each time, and performs image acquisition on this part of the fluorescence image, and eliminates the gray levels of the remaining biochip probe planes that have not been scanned. In order to reduce the accumulation of noise and improve the signal-to-noise ratio; according to this acquisition method, multiple fluorescence images are collected, and after filtering out the noise, they are linearly superimposed to form a complete fluorescence image of the biochip, and the collection of the entire fluorescence image of the biochip is completed. .

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

Compared with the prior art, the present invention has the following beneficial effects: a biochip detection method of laser induction and CCD acquisition proposed by the present invention not only avoids the complex optical path of the laser confocal method and the lighting problems of the high-pressure xenon lamp, but also It can guarantee high detection speed and sensitivity, and can make the detection sensitivity better than 1 flour/um ² , and the detection time of single-channel scanning 22mm*22mm does not exceed 55 seconds, compared with the mainstream laser confocal scanning detection method. The structure is simple and the scanning speed is fast. This product can be widely used in disease diagnosis, drug screening, preventive medicine, etc., and can solve some effects that traditional detection methods cannot do.

Description of drawings

Fig. 1 is a schematic structural diagram of a biochip detection device for laser induction and CCD acquisition in the present invention.

FIG. 2 is a schematic diagram of a driving control circuit of a two-dimensional scanning vibrating mirror in the present invention.

Fig. 3 is a schematic cross-sectional view of step scanning in the X direction in the present invention.

Fig. 4 is a schematic cross-sectional view of continuous scanning in the Y direction in the present invention.

Fig. 5 is a working flow chart of the scanner of the biochip detection method of laser induction and CCD acquisition in the present invention.

Detailed ways

The technical solution of the present invention will be specifically described below in conjunction with the accompanying drawings.

The present invention provides a biochip detection device for laser induction and CCD acquisition, which includes an excitation light source, a two-dimensional biochip scanning device, a light collection lens, and a cooling CCD camera. The system block diagram is shown in FIG. 1 . By configuring the pre-set two-dimensional scanning model in the computer, the laser light source with fixed power emits laser light and performs two-dimensional scanning in the X direction and Y direction of the biochip according to certain rules through the control of the two-dimensional scanning galvanometer. Under the work of the two-dimensional scanning galvanometer, it is irradiated obliquely at 45 degrees to the plane of the biochip, and the CY3 or CY5 fluorescent dye is excited to generate a fluorescent signal. The signal is collected by imaging with a cooled CCD camera and multi-pixel parallel integration. This not only avoids the complex optical path of the laser confocal method and the lighting problems of the high-pressure xenon lamp, but also ensures a high detection speed and sensitivity.

Further, in this embodiment, by using the principle of the Stokes shift of CY3 and CY5 fluorescent dyes after being excited by the excitation light, the biochips that have been labeled with CY3 or CY5 fluorescent dyes are irradiated with excitation light, which can be achieved by exciting Theoretical calculation of the number of fluorescent numbers generated by the excitation of fluorescent dyes by light, combined with the minimum fluorescent light intensity coefficient that can be detected by the cooled CCD camera, can calculate the parallel light that uses laser beam expansion and scans through the galvanometer and cools. The theoretical value of the detection sensitivity of the biochip detection method combined with CCD acquisition and stimulated fluorescence.

Further, in this embodiment, the fluorescence intensity generated by the excitation of the fluorescent dye is closely related to the excitation light intensity I, the wavelength λ _exc , the quantum efficiency φ, the extinction coefficient ε, and the dye concentration C _s of the fluorescent molecule. When the concentration of the fluorescent dye is low, ignoring the quenching effect, when it is excited, the excitation rate of each fluorescent molecule in the ground state can be expressed as:

In the formula, c is the speed of light, λ _exc is the wavelength of excitation light, the unit of light intensity I is W/cm ² , hv is the energy of absorbed photons, the unit of cross section σ of fluorescent dye is cm ² , and the relationship between σ and extinction coefficient ε:

σ＝3.8×10 ^-21 ε(cm ² ) (3)

Let τ be the fluorescence lifetime, ₁ /τ be the relaxation rate of the fluorescent molecules, N be the total number of fluorescent molecules irradiated by the excitation light, and N1 be the number of fluorescent molecules in the excited state. When the fluorochrome excitation process reaches a steady state, the excitation and de-excitation rates are equal:

That is, there is a normalized fluorescence excitation ratio:

Therefore, the excited fluorescence rate (normalized) is:

where φ is the quantum efficiency of the fluorescent dye.

Assuming that the area (physical resolution) of the biochip corresponding to one pixel imaging of the CCD is A _s (um ² ), the concentration of fluorescent dye C _s (flour/um ² ), and the scanning time t _s (s), the excitable The number of fluorescent photons q _em is:

q _em ＝p _f ·A _s ·C _s ·t _s (8)

The number of response electrons q _s formed by the fluorescent photons reaching the CCD pixel through the imaging optical path of the optical lens is:

θ=sin ^-1 (NA) (10)

In the formula, D _ccd is the quantum efficiency of CCD, K _em is the light transmission efficiency of the optical lens, and NA is the numerical aperture of the objective lens.

Among them, the relationship between the number of CCD response electrons q _s and the fluorescence concentration C _s of the biochip lattice in Formula 8 and Formula 9 is the basis of scanner design.

Further, in this embodiment, the beam of the parallel light laser is collimated and expanded, and a rectangular spot is formed through a square diaphragm to realize uniform illumination of the plane of the biochip under the two-dimensional scanning of the vibrating mirror. In the scanner studio, the laser beam First deflect to the determined initial step position by the galvanometer a in the two-dimensional scanning galvanometer along the X direction, and then scan the Y direction in a row by the galvanometer b in the two-dimensional scanning galvanometer, so that the biological can be scanned line by line Chip lattice plane. Since the plane of the biochip probe is not perpendicular to the projection direction of the laser beam, the projection distance and projection angle of each position on the plane of the biochip probe are different, resulting in inconsistent projected area and intensity of the laser beam. The trigonometric function relationship can be used A mathematical model is used to correct the non-perpendicular two-dimensional scanning model. Through the revised two-dimensional scanning model, the scanning step distance and the angular velocity of the galvanometer deflection can be set to calculate the biochip that uses the parallel light of laser beam expansion, scans through the galvanometer and collects stimulated fluorescence with a cooled CCD The detection speed of the detection method.

As shown in FIG. 2 , it is a schematic diagram of the driving control circuit of the vibrating mirror a and the vibrating mirror b. Since the laser is a typical point light source, the beam of the laser is collimated and expanded to form parallel light, and the cross section is A rectangular light spot is formed after the inscribed square diaphragm of , and the uniform illumination of the plane is realized under the two-dimensional scanning of the vibrating mirror a and the vibrating mirror b. The double vibrating mirrors are installed orthogonally at 90 degrees, the vibrating mirror a is responsible for scanning in the X direction, and the vibrating mirror b is responsible for scanning in the Y direction. The lens center distance e between the vibrating mirror a and the vibrating mirror b, where the X-direction step scanning section is shown in Figure 3, and the Y-direction continuous scanning section is shown in Figure 4: when the scanner is working, the laser beam is first deflected by the vibrating mirror a Along the X direction to the determined initial stepping position, the vibrating mirror b performs a row of continuous scanning in the Y direction. The vibrating mirror a can be adjusted step by step to reach a new position, and the vibrating mirror b sequentially scans the probe plane of the biochip line by line.

Further, in this embodiment, it is set that the effective probe plane of the biochip is 22×22mm ² , the center point height h _z of the vibrating mirror b lens is 55mm, and the center distance e of the vibrating mirror lens is 8mm. laser beam edge Directional projection, in the Y direction, the optical path L is 70.0 to 85.9mm, the projection angle of the vibrating mirror b is 38.66 to 50.20 degrees; in the X direction, the deflection angle δ of the vibrating mirror a is -8.03 to 8.03 degrees.

In this embodiment, since the plane of the biochip probe is not perpendicular to the projection direction of the laser beam, the projection distance and projection angle of each position on the plane of the biochip probe are different, resulting in inconsistent projected area and intensity of the laser beam.

Assuming that the incident intensity of the laser beam is I, it is deflected and projected onto the probe plane by the galvanometer a and b, and the projected area of the laser beam increases times, its light intensity becomes:

As shown in Figure 4, the galvanometer b is deflected by an angle of β, and the laser beam can be scanned along the Y direction from y1 to y2 for one line, where:

As shown in Figure 3, the laser beam can be deflected step by step from x1 to x2 according to the set step distance by adjusting the angle δ of the galvanometer a from the position of x1, and the deflection amount is:

x=(L+e)tanδ (15)

As shown in Figure 3 and combined with formula 13, relative to L, e and δ are both small, then L+e≈L, tanδ≈δ, from formula 15:

In order to ensure that the trajectory y1 to y2 scanned by the laser beam along the Y direction under the action of the galvanometer b is a horizontal parallel straight line, that is, the offset x of the laser beam in the X direction is a constant value, the deflection angle δ of the galvanometer a should follow The deflection angle β of the vibrating mirror b is adjusted by cosine.

As shown in Figure 3, the optical path l of the laser beam from the galvanometer a to the biochip:

Assuming that the scanning angular velocity of the laser beam is ω _y (corresponding to the angular velocity of the vibrating mirror b is ω _y /2), the physical resolution of scanning is A _s =d(um)×d(um), then the laser beam is in the Y direction The linear speed of the scan is:

v = ω _y l (18)

The average irradiation time of the biochip A _s area is:

From Equation 8 and Equation 12, the number of photofluorescence photons generated by the excitation of the fluorescent dye corresponding to the scanning resolution A _s area can be obtained:

In order to maintain the consistency of the fluorescent response q _em , when the deflection angle of the galvanometer a corresponding to the step spacing in the X direction is determined to be δ, the angular velocity ω _y /2 of the galvanometer b for each line in the Y direction should be adjusted by its own deflection angle β Cosine adjustment, that is, according to formula 20:

Therefore, the relational expression of the photon photon number is rewritten as:

That is, the fluorescence response q _em at the scanning angular velocity ω _y ' of the galvanometer b has a definite linear relationship with the probe dye concentration C _s .

Further, in this embodiment, the image acquisition system is composed of a macro lens, a narrow-band coated filter and a cooled CCD camera. By scanning a part of the entire biochip fluorescence image and performing image acquisition on it in time, The accumulation of noise can be greatly reduced and the signal-to-noise ratio can be greatly reduced by eliminating the gray value of the rest of the biochip probe plane that has not been scanned. Through this acquisition method, multiple fluorescent images can be collected and linearly superimposed after filtering out the noise to form a complete image. A fluorescent image of a biochip.

Further, in this embodiment, the working flow chart of the scanner for the biochip detection method of laser induction and CCD acquisition is shown in FIG. 5 . After placing the biochip to be detected, configure the computer, use the two-dimensional scanning model corrected by the above method to control the two-dimensional scanning high-speed galvanometer, and perform two-dimensional scanning on the probe plane of the biochip. Stop scanning a part of the needle plane for a period of time and carry out image acquisition. Immediately after the image is collected, the data is transferred to the computer for storage. After the storage is completed, the two-dimensional scanning is continued until the entire biochip probe plane is scanned. The computer system performs image denoising processing and integrates the block images into a complete biochip fluorescence image.

The above are the preferred embodiments of the present invention, and all changes made according to the technical solution of the present invention, when the functional effect produced does not exceed the scope of the technical solution of the present invention, all belong to the protection scope 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, h_zFor 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 ω_y, the angular speed of corresponding second galvanometer is ω_y/ 2, and scan physics point Resolution is A_s=d (um) × d (um), then the linear velocity of the scanning of laser beam in the Y direction be：

V=ω_y·l

Biochip A_sThe mean irradiation time of area is：

Corresponding scanning resolution A_sThe 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, q_emFor the fluorescent photon number of excitation, I enters for laser beam Penetrate intensity, ω_yFor 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 C_s,

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 galvanometer_y/ 2 carry out cosine adjustment to itself deflection angle β, to keep fluorescence response q_emConsistency：

Then photoluminescence number of photons q_emFor：

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.