TWI582407B - Three dimensional particle localization and tracking device and method thereof - Google Patents

Description

Particle three-dimensional positioning and tracking device and method thereof

The present invention relates to a particle positioning and tracking device, and more particularly to a particle three-dimensional positioning and tracking device.

In recent years, particle tracking devices have received increasing attention in biological detection. In general, the particle tracking device uses a photoexcited fluorescence microscopy technique to emit an excitation light to the object to be tested, causing electrons in the fluorescent particles or fluorescent molecules of the analyte to transition to an excited state, and then the electrons are excited. The state returns to the ground state, emits a fluorescent beam, and then detects and analyzes the fluorescent beam emitted by the object to be tested. In this way, the two-dimensional position of the particles of the object to be tested can be located and tracked. However, the conventional particle tracking device needs to capture a plurality of two-dimensional images through axial continuous movement, and then perform three-dimensional imaging overlay to obtain the three-dimensional position of the fluorescent particles or fluorescent molecules, which is time consuming and cannot be obtained. Instantly track the three-dimensional movement of particles.

The present disclosure provides a particle three-dimensional positioning and tracking device that can locate and track a three-dimensional movement trajectory of a particle.

In some embodiments of the disclosure, particle three-dimensional positioning and chasing The tracking device comprises a light source module, an imaging lens group and a light detecting module. The light source module is used to excite the object to be tested, so that the object to be tested generates a fluorescent beam. The imaging lens assembly includes a first cylindrical lens, a second cylindrical lens, and an imaging lens, wherein the first cylindrical lens has a first focal length, the first focal length is a positive value, and the second cylindrical lens has a second focal length, The second focal length is a negative value, and the first cylindrical lens and the second cylindrical lens system are used together to adjust the imaging shape of the fluorescent light beam. The imaging lens is used to image the fluorescent light beam to the light detecting module, and the light detecting module is configured to detect the fluorescent light beam passing through the imaging lens group.

In some embodiments of the present disclosure, a method for three-dimensional positioning and tracking of a particle includes the steps of: exciting a sample to be measured to generate a fluorescent beam; and using a focused cylindrical lens to share a divergent cylindrical lens Adjusting the imaging shape of the fluorescent beam; and detecting the passing fluorescent lens of the focused cylindrical lens and the diverging cylindrical lens.

In some embodiments of the present disclosure, the imaging lens assembly has a first cylindrical lens that is focusable, a second cylindrical lens that is divergent, and an imaging lens. That is to say, the focusing ability of the first cylindrical lens that can be focused and the divergent second cylindrical lens are different under different axial directions, by combining such a first cylindrical lens and a second cylindrical lens Controlling the astigmatic effect, so that the imaging of the fluorescent beam passing through the imaging lens group produces appropriate astigmatism, and then imaging the imaging lens to the photodetector, thereby facilitating the establishment of a more accurate calibration curve of the axial position of the particle positioning, increasing the particle The accuracy of 3D positioning and tracking.

10‧‧‧Particle three-dimensional positioning and tracking device

100‧‧‧Light source module

110‧‧‧Test object

112‧‧‧ particles

120‧‧‧ stage

130‧‧‧Light source

142‧‧‧polar mirror

144‧‧ ‧Shutter

151‧‧‧Mirror

152‧‧‧Mirror

153‧‧‧Mirror

154‧‧‧Mirror

160‧‧‧Raster

170‧‧‧First lens group

180‧‧‧Second dichroic mirror

190‧‧‧ objective lens group

200‧‧‧ imaging lens set

200a‧‧‧ imaging lens set

210‧‧‧First cylindrical lens

210a‧‧‧first cylindrical lens

212‧‧‧First rotating support

214‧‧‧Rotary actuator

220‧‧‧second cylindrical lens

220a‧‧‧second cylindrical lens

222‧‧‧Second rotating support

224‧‧‧Rotary actuator

230‧‧‧ imaging lens

300‧‧‧Light detection module

310‧‧‧First Light Detector

320‧‧‧Second light detector

330‧‧‧First dichroic mirror

1122‧‧‧First particle

1124‧‧‧Second particles

3102‧‧‧First fluorescent filter

3202‧‧‧Second Fluorescent Filter

L‧‧‧beam

Light of the Lt‧‧ meridian plane

Light of Ls‧‧, sagittal plane

F‧‧‧Fluorescent beam

F1‧‧‧first focal length

F2‧‧‧second focal length

Fa1‧‧‧first focal length

Fa2‧‧‧second focal length

F1‧‧‧First fluorescent beam

F2‧‧‧second fluorescent beam

Fv‧‧ focus

Fp‧‧ Focus

I1‧‧‧ imaging

I2‧‧‧ imaging

I3‧‧· imaging

S‧‧‧ point light source

T1‧‧‧ central thickness

T2‧‧‧ edge thickness

T3‧‧‧ central thickness

T4‧‧‧Edge thickness

T5‧‧‧ central thickness

T6‧‧‧ edge thickness

T7‧‧‧ central thickness

T8‧‧‧ edge thickness

Op‧‧‧ optical lens

P1‧‧ points

P2‧‧‧ position

P3‧‧‧ position

Z1~Z5‧‧‧Deep

Read the following detailed description and match the corresponding drawings to understand this Reveal the multiple forms. It should be noted that the various features in the drawings do not draw actual proportions in accordance with standard practice in the industry. In fact, the dimensions of the features described can be arbitrarily increased or decreased to facilitate clarity of discussion.

FIG. 1 is a schematic diagram of a three-dimensional particle positioning and tracking device according to some embodiments of the present disclosure.

2A is a schematic diagram of an astigmatic effect in accordance with some embodiments of the present disclosure.

FIG. 2B is a schematic view showing the imaging shape of the point light source according to some embodiments of the present disclosure with respect to different axial positions of the optical lens.

3 is a schematic diagram of an imaging lens group of a particle three-dimensional positioning and tracking device according to some embodiments of the present disclosure.

4A is a schematic diagram of the imaging shape of different fluorescent beams in accordance with some embodiments of the present disclosure.

FIG. 4B is a schematic diagram showing the imaging shape of the fluorescent light beam when the first cylindrical lens and the second cylindrical lens are not rotated relative to each other according to the embodiment of the present disclosure.

FIG. 5 is a calibration graph of the axial position of the particle positioning of the particle three-dimensional positioning and tracking device according to some embodiments of the present disclosure.

Figure 6 is a schematic illustration of another imaging lens assembly of a particle three-dimensional positioning and tracking device in accordance with some embodiments of the present disclosure.

The spirit of the disclosure will be clearly described in the following drawings and detailed description, and those skilled in the art will understand the implementation of the disclosure. The invention may be modified and modified by the teachings of the present disclosure without departing from the spirit and scope of the disclosure. For example, the description "the first feature is formed above or above the second feature", in the embodiment, the first feature and the second feature are included in direct contact; and the first feature and the second feature are also included as indirect The contact has additional features formed between the first feature and the second feature. Moreover, the present disclosure will reuse component numbers and/or text in various examples. The purpose of the repetition is to simplify and clarify, and does not in itself determine the relationship between the various embodiments and/or the configurations in question.

1 is a schematic diagram of a particle three-dimensional positioning and tracking device 10 in accordance with some embodiments of the present disclosure. As shown in FIG. 1 , the particle three-dimensional positioning and tracking device 10 includes a light source module 100 , an imaging lens group 200 , and a light detecting module 300 . The light source module 100 is configured to emit a light beam L to excite the object to be tested 110. After the object to be tested 110 absorbs the light beam L passing through the light source module 100, it is excited to generate a fluorescent light beam F. The imaging lens group 200 is for adjusting the imaging shape of the fluorescent light beam F. Further, the imaging lens group 200 includes a first cylindrical lens 210, a second cylindrical lens 220, and an imaging lens 230. The first cylindrical lens 210 has a maximum curvature and refractive power only in a direction perpendicular to the center line of the cylinder, and the second cylindrical lens 220 also has a maximum curvature and refractive power in a direction perpendicular to the center line of the cylinder. The first focal length f1>0 of the first cylindrical lens 210 and the second focal length f2<0 of the second cylindrical lens 220. The first cylindrical lens 210 and the second cylindrical lens 220 are sequentially arranged on the optical path of the fluorescent light beam F, and the two are used together to adjust the imaging shape of the fluorescent light beam F. The imaging lens 230 is used to image the fluorescent light beam to the light detecting module 300. The light detecting module 300 is configured to detect the fluorescent light beam F passing through the imaging lens group 200, thereby positioning and tracking the three-dimensional moving track of the particles.

It is worth noting that the particle three-dimensional positioning and tracking device of the present disclosure is related to the astigmatism effect, so the astigmatic effect is explained below.

When light travels through the imaging system, it can be divided into rays in the tangential direction, that is, rays in the vertical direction, and rays in the sagittal direction, that is, rays in the horizontal direction. The astigmatic effect causes imaging aberrations because the rays in the meridional direction and the rays in the sagittal direction are focused at different positions. Referring to FIG. 2A, FIG. 2A is a schematic diagram of an astigmatic effect according to some embodiments of the present disclosure. For example, as shown in FIG. 2A, the optical system includes an optical lens Op, which has a different focal length in the meridional direction (X direction) and the sagittal direction (Y direction). When the point source S emits a light beam, the light beam may include the light Lt in the meridional plane (XZ plane) and the light Ls in the sagittal plane (YZ plane). Since the refractive power of the optical lens Op on the sagittal plane is different from the refractive power in the meridional plane, the focusing effect of the light Lt on the meridional plane and the light Ls on the sagittal plane are affected. The focusing effect is different, resulting in that the imaging focus of the light ray Lt on the meridional plane is different from the imaging focus of the light ray Ls on the sagittal plane, thus producing a blurred image, which is called astigmatic effect.

As shown in Fig. 2A, in the optical lens Op, when the point source S emits a beam, the beam Lt of the beam on the meridional plane (XZ plane) is focused on the focal plane Fv of the meridional plane, in the sagittal plane (YZ plane) The upper light ray Ls is focused on the focal point Fp of the sagittal plane, and the focal point Fv is different from the focal point Fp (that is, the two are not separated), thus generating astigmatism, that is, the focus Fv and the focus Fp. The imaging between them will be blurred, and the imaging of the point source S will be deformed (for example, from a circle to an ellipse).

Further, when the relative position of the point source S and the optical lens Op changes, the imaging of the point source S produces a different degree of astigmatism on a fixed imaging plane. Refer to both Figures 2A and 2B. FIG. 2B is a schematic view showing the imaging shape of the point light source S according to a part of the embodiment of the present disclosure with respect to the axial direction (Z direction) of the optical lens Op. For example, as shown in FIG. 2A, when the point source S is located at the point P1 of the optical lens Op, the imaging of the point source S can obtain images of equal magnitude in the X and Y directions on the imaging plane of the point Fc. A circular imaging I1 can be presented (as shown in Figure 2B). When the point source S is located at the position P2 (ie, a position farther from the optical lens Op than the point P1), the imaging of the point source S on the imaging plane of the point Fc is changed from a circular shape to an elliptical imaging I2 (eg, Figure 2B shows). When the point source S is further away from the point P1 of the optical lens Op, for example, the point source S is located at the position P3 of the optical lens Op (that is, a position farther from the optical lens Op than the position P2), then the point source S The degree of influence by the astigmatic effect of the optical lens Op changes, and an elliptical imaging I3 (as shown in Fig. 2B) which is flatter than the imaging I2 is presented. In other words, referring to Fig. 2B, the imaging shape of the point light source S at the point P1 is imaged. The imaging shape of the I2 line point source S at the position P2 is imaged. The image shape of the I3 line point source S at the position P3 is imaged. From this, it can be seen that when the point light source S is located at a different position from the axial direction (Z direction) of the optical lens Op, the image shape of the point light source S also changes. Therefore, the relative position of the point light source S and the optical lens Op in the axial direction (Z direction) can be judged by the change in the imaging shape caused by the astigmatic effect.

As described above, some embodiments of the present disclosure analyze the imaging of the fluorescent light beam F emitted by the particles by the difference of the astigmatism, and further obtain the relative position of the particles and the imaging lens group 200, thereby positioning the particles. Axial (Z direction) position. However, when the astigmatic effect is excessive, the imaging of the particles at different positions of the imaging lens group 200 may be too blurred, and the imaging difference of the particles located at different positions cannot be accurately determined, and the relative position of the particles and the imaging lens group 200 cannot be accurately located. Or, when the astigmatic effect is too small, the imaging of the particles at different positions of the imaging lens group 200 may be too similar to accurately determine the imaging difference of the particles at different positions, and the relative position of the particles to the imaging lens group 200 cannot be accurately located. . Accordingly, the embodiments below provide an imaging lens set 200 that can generate appropriate astigmatism effects while effectively utilizing astigmatic effects to locate and track particles.

In particular, the embodiment of the present disclosure establishes a more accurate astigmatism effect by the imaging lens group 200, and establishes a calibration curve of the imaging of the more accurate particles along the axial (Z-direction) distance. 3 is a schematic diagram of an imaging lens assembly 200 of a particle three-dimensional positioning and tracking device 10 in accordance with some embodiments of the present disclosure. As shown in FIG. 3 , the imaging lens set 200 is optically coupled between the object to be tested 110 and the light detecting module 300 , so that light from the object to be tested 110 can reach the light detecting module 300 through the imaging lens set 200 . . Further, the imaging lens group 200 includes a first cylindrical lens 210, a second cylindrical lens 220, and an imaging lens 230. The first cylindrical lens 210 and the second cylindrical lens 220 are optically coupled to the imaging lens 230 between the object to be tested 110 and the light detecting module 300. For example, in some embodiments, the first cylindrical lens 210, the second cylindrical lens 220, and the imaging lens 230 are sequentially arranged along the traveling direction of the fluorescent light beam F, so that the fluorescent light from the object to be tested 110 The light beam F can sequentially travel through the first cylindrical lens 210, the second cylindrical lens 220, and the imaging lens 230 to the light detecting module 300. Alternatively, in some embodiments, the imaging lens 230, the first cylindrical lens 210, and the second cylindrical lens 220 are sequentially arranged along the direction of the fluorescent light beam F, such that The fluorescent light beam F from the object to be tested 110 can be sequentially passed through the imaging lens 230, the first cylindrical lens 210 and the second cylindrical lens 220 to the light detecting module 300, but the disclosure is not limited thereto. It can be noted that, as shown in FIG. 3, the focusing ability of the first cylindrical lens 210 in the X direction and the Y direction is different. Similarly, the second cylindrical lens 220 is different in focusing ability in the X direction and the Y direction. The first focal length f1>0 of the first cylindrical lens 210 and the second focal length f2<0 of the second cylindrical lens 220. In other words, the first cylindrical lens 210 has a focusing ability, and the second cylindrical lens 220 has a diverging ability. Therefore, the ability of focusing and diverging the imaging lens group 200 can be adjusted by combining the first cylindrical lens 210 and the second cylindrical lens 220, so that the imaging of the fluorescent light beam F passing through the imaging lens group 200 has a more appropriate image. Dispersion (that is, affected by a more appropriate degree of astigmatism), and facilitates the establishment of a calibration curve of the imaging of the more accurate particles along the axial (Z-direction) distance.

Specifically, in some embodiments, as shown in FIG. 3, the focal lengths of the first cylindrical lens 210 in two perpendicular directions are different. As a result, the focusing effect of the first cylindrical lens 210 on the vertical plane (XZ plane) provided by the fluorescent light beam F is different from that in the horizontal plane (YZ plane). Therefore, when the fluorescent light beam F passes through the first cylindrical lens 210, the imaging focus of the light beam of the fluorescent light beam F on the horizontal plane is different from the imaging focus of the light of the fluorescent light beam F on the vertical plane. Moreover, since the first focal length f1>0 of the first cylindrical lens 210 is a focused lens, the fluorescent light beam F passing through the first cylindrical lens 210 converges. Similarly, in some embodiments, the focal length of the second cylindrical lens 220 in the two perpendicular directions is different. As a result, the diverging effect of the second cylindrical lens 220 on the vertical plane (XZ plane) provided by the fluorescent light beam F is different from the diverging effect in the horizontal plane (YZ plane). Therefore, when the fluorescent beam F passes After passing through the second cylindrical lens 220, the imaging focus of the light beam of the fluorescent light beam F on the horizontal plane is different from the imaging focus of the light of the fluorescent light beam F on the vertical plane. Further, since the second focal length f2 < 0 of the second cylindrical lens 220 is a diverging lens, the fluorescent light beam F passing through the second cylindrical lens 220 is diverged. Therefore, the degree of convergence and divergence of the fluorescent light beam F can be adjusted by the ability of the first cylindrical lens 210 to converge light and the ability of the second cylindrical lens 220 to diverge light, and the fluorescent light beam F can be adjusted. The imaging focus of the imaging lens 230 after imaging on the horizontal plane and the imaging focus of the vertical plane are such that imaging of the fluorescent light beam F through the imaging lens group 200 is affected by a more appropriate degree of astigmatism.

In other words, the imaging lens set 200 is formed by combining the first cylindrical lens 210, the second cylindrical lens 220 and the imaging lens 230, wherein the first focal length f1>0 of the first cylindrical lens 210, and the second cylindrical surface The second focal length f2<0 of the lens 220 is such that the degree of astigmatism of the first cylindrical lens 210 and the degree of astigmatic effect of the second cylindrical lens 220 can cancel or increase each other, thereby adjusting the imaging lens group 200. The extent of the astigmatic effect. In this way, the imaging of the fluorescent light beam F through the imaging lens group 200 can be affected by a more appropriate degree of image scattering effect, and a more accurate particle calibration curve is established, which is advantageous for the accuracy of particle three-dimensional positioning and tracking.

In other words, in some embodiments, as shown in FIG. 3, when the fluorescent light beam F passes through the first cylindrical lens 210, the diopter in the horizontal plane (YZ plane) is different from the diopter in the vertical plane (XZ plane). of. The imaging focus of the fluorescent beam F passing through the first cylindrical lens 210 in the horizontal plane is also different from the imaging focus of the vertical plane. Similarly, when the fluorescent beam F passes through the second cylindrical lens 220, the diopter in the horizontal plane (which may be less than 0) and the diopter in the vertical plane (which may be less than 0) Different. The imaging focus of the fluorescent beam F passing through the second cylindrical lens 220 in the horizontal plane is different from the imaging focus of the vertical plane. By the first cylindrical lens 210 with a positive focal length and the second cylindrical lens 220 with a negative focal length, the imaging focal length of the fluorescent light beam F passing through the horizontal plane of the imaging lens group 200 and the imaging focal length of the vertical surface can be adjusted. For example, in some embodiments, the fluorescent beam F is concentrated by the first cylindrical lens 210, and the fluorescent beam F is diverged by the second cylindrical lens 220, the first cylindrical lens 210 and the second cylindrical surface. The lens 220 can offset the degree of astigmatic effect and reduce the astigmatism of the fluorescent beam F.

For example, in some embodiments, as shown in FIG. 3, in the XZ plane, the central portion of the first cylindrical lens 210 has a central thickness t1, and the first cylindrical lens 210 surrounds the edge of the central portion. The portion has an edge thickness t2. The central thickness t1 of the first cylindrical lens 210 is different from the edge thickness t2 of the first cylindrical lens 210, and the central thickness t1 is greater than the edge thickness t2 such that the first cylindrical lens 210 is a focused lens. Moreover, the thickness of the first cylindrical lens 210 on the YZ plane is substantially equal. Similarly, in the XZ plane, the central portion of the second cylindrical lens 220 has a central thickness t3, and the edge portion of the second cylindrical lens 220 surrounding the central portion has an edge thickness t4. The central thickness t3 of the second cylindrical lens 220 is different from the edge thickness t4 of the second cylindrical lens 220, and the central thickness t3 is smaller than the edge thickness t4 such that the second cylindrical lens 220 is a diverging lens. Moreover, the thickness of the second cylindrical lens 220 on the YZ plane is substantially equal. . The optical axis of the first cylindrical lens 210 coincides with the optical axis of the second cylindrical lens 220. With the first cylindrical lens 210 and the second cylindrical lens 220, the sum of the central thicknesses of the two lenses through which the fluorescent light beam F passes (ie, t1+t3) can be adjusted, and the fluorescent light beam F can be adjusted. The sum of the edge thicknesses of the two lenses (ie t2+t4) to adjust the firefly The imaging focus of the light beam F changes the astigmatism of the imaging of the fluorescent beam F.

In some embodiments, the first cylindrical lens 210 and the second cylindrical lens 220 are rotatable for adjusting the astigmatic effect of the imaging lens group 200 to help image generation of the fluorescent light beam F through the imaging lens group 200. Proper astigmatism. Specifically, in some embodiments, as shown in FIG. 3 , the imaging lens set 200 further includes a first rotating support frame 212 and a second rotating support frame 222 . The first cylindrical lens 210 can be located on the first rotating support frame 212, and the first rotating support frame 212 is electrically connected or mechanically coupled to the rotary actuator 214, and can be rotated by the actuation of the rotary actuator 214. Therefore, the first cylindrical lens 210 can be rotated by adjusting the first rotating support frame 212. For example, in some embodiments, as shown in FIG. 3, the first cylindrical lens 210 can be rotated along the XY plane and rotated in the Z direction by adjusting the first rotating support frame 212, thereby changing The fluorescent light beam F passes through the position of the first cylindrical lens 210 to adjust the radius of curvature of the fluorescent light beam F at the passing position of the first cylindrical lens 210. In other words, the first cylindrical lens 210 can be rotated about the direction in which the first cylindrical lens 210 and the second cylindrical lens 220 are arranged. Similarly, the second cylindrical lens 220 can be located on the second rotating support frame 222, and the second rotating support frame 222 is electrically connected or mechanically coupled to the rotary actuator 224, and can be subjected to the rotary actuator 224 The second cylindrical lens 220 can be rotated by adjusting the second rotating support frame 222. For example, in some embodiments, the second cylindrical lens 220 can be rotated along the XY plane and rotated in the Z direction by adjusting the second rotating support 222, thereby changing the fluorescent beam F through the second column. The position of the face lens 220 is to adjust the radius of curvature of the fluorescent beam F at the passing position of the second cylindrical lens 220.

In some embodiments, the first cylindrical lens 210 is rotatable The second cylindrical lens 220 is non-rotatable. In some embodiments, the second cylindrical lens 220 is rotatable and the first cylindrical lens 210 is non-rotatable. In some embodiments, the first cylindrical lens 210 and the second cylindrical lens 220 are both rotatable. By rotating the first cylindrical lens 210, the second cylindrical lens 220, or both, the focusing ability of the imaging lens group 200 provided on the horizontal plane of the fluorescent light beam F and the focusing ability in the vertical plane can be changed, so that The imaging shape of the fluorescent light beam F of the imaging lens group 200 is changed, and the imaging of the fluorescent light beam F through the imaging lens group 200 is affected by an appropriate astigmatic effect.

Referring to FIG. 4A, FIG. 4A is a schematic diagram showing the imaging shape of different fluorescent light beams F according to some embodiments of the present disclosure. Wherein, the vertical axis represents the relative rotation angles of the first cylindrical lens 210 and the second cylindrical lens 220 (for example, the first rotation angle, the second rotation angle, and the third rotation angle), and the horizontal axis represents the emitted fluorescent light beam F. The depth of the particles (i.e., the positions Z1 to Z5 in the Z direction). Referring to FIGS. 3 and 4A simultaneously, when the first cylindrical lens 210 and the second cylindrical lens 220 are relatively rotated (for example, the first cylindrical lens 210, the second cylindrical lens 220, or both), Since the fluorescent light beam F passes through the first cylindrical lens 210 and the second cylindrical lens 220, the surface of the first cylindrical lens 210 through which the fluorescent light beam F passes, the surface of the second cylindrical lens 220, or both The radius of curvature changes with the angle of rotation, so the focusing power of the fluorescent beam F on the horizontal plane and the focusing ability on the vertical plane also change, so the first cylindrical lens 210 and the second cylinder are rotated. The lens 220, or both, can change the imaging pattern of the fluorescent beam F. For example, as shown in FIG. 4A, by rotating the first cylindrical lens 210 and the second cylindrical lens 220, the imaging of the fluorescent light beam F can be converted between imaging I, imaging II, and imaging III. .

It should be noted that, referring to the imaging III of FIG. 4A, when the first cylindrical lens 210 and the second cylindrical lens 220 are at a specific relative rotation angle, the imaging of the fluorescent light beam F may be circular, vertical elliptical or Horizontal ellipse, not oblique ellipse. When the imaging III is not an oblique ellipse, the oblique ellipse can be further converted into a vertical ellipse and a horizontal ellipse, which facilitates the convenience of analyzing the calibration curve and helps to establish a more accurate particle calibration curve. Therefore, in some embodiments, a more accurate particle correction curve can be established by controlling the rotation angle of the first cylindrical lens 210 and the rotation angle of the second cylindrical lens 220. It can be understood that the non-oblique ellipse represents that the long axis of the ellipse is perpendicular or parallel to the length direction when the first cylindrical lens 210 or the second cylindrical lens 220 has not been rotated, that is, the Y direction shown in FIG. In some embodiments, as shown in FIG. 3, the first cylindrical lens 210 and the second cylindrical lens 220 are rotated in the Z direction and the X direction is used as the rotation reference 0 degree direction, when the first column When the rotation angle of the surface lens 210 is 49.0 degrees, and the rotation angle of the second cylindrical lens 220 is 48.5 degrees, the imaging III of the non-oblique elliptical shape can be generated; when the rotation angle of the first cylindrical lens 210 is 46.0 degrees, When the rotation angle of the second cylindrical lens 220 is 45.0 degrees, the imaging III of the non-oblique elliptical shape may also be generated; when the rotation angle of the first cylindrical lens 210 is 46.5 degrees, and the rotation of the second cylindrical lens 220 When the angle is 45.0 degrees, the imaging III of the non-oblique elliptical shape may also be generated; when the rotation angle of the first cylindrical lens 210 is 47.0 degrees, and the rotation angle of the second cylindrical lens 220 is 45.0 degrees, it may also be generated. The imaging III of the non-oblique elliptical shape; when the rotation angle of the first cylindrical lens 210 is 47.5 degrees, and the rotation angle of the second cylindrical lens 220 is 45.0 degrees, the imaging III of the non-oblique elliptical shape may also be generated. It is worth noting that although the above-mentioned rotation angles can produce non-elliptical shapes Image, but the degree of astigmatism of the image is different, so that the image of the non-oblique elliptical shape can be obtained at the same time and the optimal degree of astigmatism can be obtained through adjustment. It is also noted that the above rotation angles are merely illustrative and are not intended to limit the invention.

It is worth mentioning that the first cylindrical lens 210 and the second cylindrical lens can be reduced by using the imaging lens group 200 having the first cylindrical lens 210 and the second cylindrical lens 220 compared to adjusting a single lens. When the 220 is rotated, the fluorescent light beam F passes through the offset of the imaging focal plane after the imaging lens group 200. That is, the imaging focal plane of the fluorescent light beam F does not excessively shift, for example, when the first cylindrical lens 210 and the second cylindrical lens 220 rotate, the imaging focal plane of the fluorescent light beam F The offset is less than 0.1 mm, and such a small offset does not affect the detection function of the light detecting module 300. In this way, the position of the light detecting module 300 can be further adjusted, and the convenience of using the particle three-dimensional positioning and tracking device can be increased.

Moreover, in some embodiments, the absolute value of the first focal length f1 is substantially equal to the absolute value of the second focal length f2. When the first cylindrical lens 210 and the second cylindrical lens 220 are not relatively rotated (that is, neither the first cylindrical lens 210 nor the second cylindrical lens 220 is rotated, or when the first cylindrical lens 210 and the second When the cylindrical lens 220 rotates at the same angle), the astigmatic effect of the first cylindrical lens 210 and the astigmatic effect of the second cylindrical lens 220 can cancel each other out. Referring to FIG. 4B, FIG. 4B is a schematic diagram showing the imaging shape of the fluorescent light beam F when the first cylindrical lens 210 and the second cylindrical lens 220 are not rotated relative to each other according to the embodiment of the present disclosure. As shown in FIG. 4B, when the first cylindrical lens 210 and the second cylindrical lens 220 are not relatively rotated, and the absolute values of the first focal length f1 and the absolute value of the second focal length f2 are substantially equal, different depths (Z1~Z5) particles The imaging of the incident fluorescent light beam F through the imaging lens group 200 is not affected by the astigmatic effect, but maintains a circular imaging shape. It can be seen that the astigmatic effect can be eliminated by combining the first cylindrical lens 210 with the positive focal length of the absolute focal length and the second cylindrical lens 220 with the negative focal length, thereby preventing the imaging of the fluorescent light beam F from being astigmatized. The effect of the effect. Alternatively, the astigmatic effect of the imaging lens group 200 can be adjusted by rotating the first cylindrical lens 210, the second cylindrical lens 220, or both, so that the imaging of the fluorescent light beam F is subjected to an appropriate degree of astigmatism. The effect is to create a more accurate particle calibration curve. The term "about" or "substantially" as used herein is used to modify any error that may vary slightly, but such minor changes do not alter its nature.

In some embodiments, as shown in FIG. 3 , the light source module 100 further includes a stage 120 . The object to be tested 110 is located on the stage 120, and the object to be tested 110 contains particles 112. The particles 112 are located above the stage 120. The stage 120 is movable in the Z direction while being close to or away from the imaging lens group 200. The imaging lens set 200 is optically coupled between the particles 112 and the light detecting module 300. Further, the imaging lens set 200 is located between the stage 120 and the light detecting module 300. The first cylindrical lens 210 and the second cylindrical lens 220 of the imaging lens group 200 are rotatable such that the imaging lens group 200 can produce a more appropriate astigmatic effect. Subsequently, the imaging lens group 200 is fixed, and the stage 120 is moved such that the particles 112 are relatively displaced with the imaging lens group 200, thereby changing the relative position of the particles 112 and the imaging lens group 200. For example, in some embodiments, the moving frequency of the stage 120 may be 200 Hz, but the disclosure is not limited thereto. After the stage 120 is moved, the light detecting module 300 can detect the fluorescent light beam F passing through the particles 112 of the imaging lens group 200, capture the imaging of the fluorescent light beam F, and establish the particles 112 through subsequent image processing. A calibration plot of the imaging of the emitted fluorescent beam F as a function of depth Z.

Referring to FIG. 5, FIG. 5 is a calibration graph of the axial position of the particle positioning of the particle three-dimensional positioning and tracking device 10 according to some embodiments of the present disclosure. As shown in Fig. 5, the X curve represents the curve of the width of the horizontal component of the fluorescent beam F emitted by the particle as a function of the depth Z of the particle, and the Y curve represents the vertical component of the fluorescent beam F emitted by the particle. The curve of the width as a function of the depth Z of the particle. By moving the stage 120, the particles 112 are located at different depths Z, and the imaging shapes of the fluorescent beams F emitted by the particles 112 at different depths Z are different. For example, in some embodiments, as shown in FIG. 5, based on the astigmatic effect of the imaging lens group 200, when the particle 112 is located at the depth Z1, the imaging system of the fluorescent beam F of the particle 112 is a vertical ellipse. . Similarly, when the particle 112 is at depth Z2, the imaging of the fluorescent beam F of the particle 112 is circular. Similarly, when the particle 112 is at depth Z3, the imaging of the fluorescent beam F of the particle 112 is a horizontal ellipse. Therefore, the imaging of the fluorescent light beam F of the particles 112 under different depths Z can be captured by the light detecting module 300, thereby obtaining the imaging width variation value of the particle 112 as a function of the depth Z, and the particles 112 are respectively recorded. The width of the horizontal component imaging below the different depths Z is the width of the vertical component imaging of the particles 112 below the different depths Z, thereby obtaining a particle calibration curve of the particle three-dimensional positioning and tracking device 10.

Therefore, in some embodiments of the present disclosure, based on the appropriate astigmatism effect of the imaging lens group 200, an accurate particle calibration curve of the three-dimensional particle localization and tracking device 10 can be established. In this way, when actually applied to particle positioning and tracking, the actual object to be tested can be placed on the particle three-dimensional positioning and tracking device 10, and the fluorescent beam emitted by the specific particles of the actual object to be tested can be compared. The imaging is compared to the particle calibration curve described above to obtain the axial (Z-direction) position of the particular particle. Specifically, in some embodiments, the imaging width of the horizontal component of the fluorescent beam emitted by the specific particle of the actual analyte and the X curve of the particle calibration curve may be compared and compared with the specific particle of the actual analyte. The imaging width of the vertical component of the emitted fluorescent beam is plotted against the Y curve of the particle calibration curve to obtain the axial (Z-direction) position at which the particular particle is located.

In some embodiments of the present disclosure, referring to FIG. 1 , the light source module 100 can provide a light beam L, and the intensity, polarization angle, wavelength, frequency, and dispersion compensation of the light beam L have been adjusted by components of the light source module 100. The light beam L adjusted by the light source module 100 can provide sufficient energy to excite the object to be tested 110. That is, the specific particles 112 of the analyte 110 can absorb the energy of the light beam L such that the electrons in the particle 112 transition to the excited state, and then the electrons return from the excited state to the ground state, releasing the fluorescent light beam F. Then, based on the astigmatic effect of the imaging lens group 200, the imaging shape of the fluorescent light beam F transmitted to the light detecting module 300 is changed, and the light detecting module 300 is corrected by the imaging shape of the matching particles 112 and the above-mentioned particle. The curve is obtained to obtain the axial (Z direction) position of the fluorescent light beam F. Further, the plane position (XY) of the particles 112 can be obtained by fitting the light intensity of the fluorescent beam F to a two-dimensional elliptical Gaussian function. It is worth noting that the elliptical Gaussian distribution of light here refers to the intensity distribution of light in space, and the center point of the distribution of the elliptical Gaussian function is the plane position (XY) of the particle 112. In this way, the particle three-dimensional positioning and tracking device of the present disclosure can determine the particle 112 to be tested based on the ellipsoidal distribution of the imaged astigmatism of the fluorescent beam F emitted by the particle 112 and the light intensity of the fluorescent beam F. The three-dimensional position (XYZ) in the object 110, in turn, locates and tracks the three-dimensional movement trajectory of the particles 112.

For example, in some embodiments of the present disclosure, as shown in FIG. 1 , the light beam L emitted by the light source module 100 is focused to a focus range of the object 110 to be a non-dot region, so that a fluorescent beam is generated. The range covers a two-dimensional area on all layers of the object to be tested 110, wherein the slice layer is perpendicular to the arrangement direction of one of the first cylindrical lens 210 and the second cylindrical lens 220 (ie, the Z direction of FIG. 3) ). In other words, since the multiphoton fluorescence excitation mechanism requires a strong excitation energy, it is possible to achieve a two-dimensional region in which the excitation fluorescence region is an axial (Z-direction) slice by exciting the fluorescence only in the focal plane. That is, the light beam L is focused to a wide area of the object to be tested 110, not a point. As a result, the range in which the object to be tested 110 is excited by the light beam L to generate the fluorescent light beam F is a two-dimensional region of the slice in the axial direction (Z direction) of the object to be tested 110. For example, the two-dimensional plane of the object to be tested 110 may have at least two specific particles 112, so that the light beam L can excite two particles 112 of the same or different absorption spectrum, and emit fluorescent beams F of the same or different wavelengths. . In other words, the particles 112 can be classified into a first particle 1122 and a second particle 1124. For example, in some embodiments, the object to be tested 110 includes a plurality of first particles 1122, and each of the first particles 1122 is located at different positions of the object to be tested 110 (ie, different XYZ coordinates), and each of the first particles 1122 can absorb the light of the first spectrum of the light beam L to generate the fluorescent light beam F. Or, in some embodiments, the object to be tested 110 includes first particles 1122 and second particles 1124, the first particles 1122 can absorb light of the first spectrum of the light beam L, and the second particles 1124 can absorb the second light beam L The light of the spectrum produces a fluorescent beam F of a different spectrum. Since the light beam L of the light source module 100 is focused to the non-point region of the object to be tested 110, the particle three-dimensional positioning and tracking device can simultaneously detect at least two particles 112 of the object to be tested 110 (for example, a plurality of first particles 1122) Or a plurality of first particles and second particles) The relative position of at least two particles 112 is tracked.

For example, in some embodiments of the present disclosure, the object 110 of the light source module 100 is excited to generate a fluorescent beam F. The fluorescent light beam F can be classified into a first fluorescent light beam F1 and a second fluorescent light beam F2. That is, the light source module 100 emits the light beam L to the object to be tested 110, so that the first particles 1122 and the second particles 1124 of the object to be tested 110 are excited, and the first fluorescent beam F1 and the second fluorescent beam F2 are emitted. . Since the light beam L can excite the first particles 1122 and the second particles 1124 of two different absorption spectra, the fluorescent light beams F of different wavelengths can be emitted. For example, the first particle 1122 can absorb the light of the first spectrum of the light beam L and emit the first fluorescent light beam F1. The second particle 1124 can absorb the light of the second spectrum of the light beam L and emit the second fluorescent light beam F2. In some embodiments of the present disclosure, since the absorption spectra of the first particles 1122 and the second particles 1124 are different, the wavelengths of the first fluorescent beam F1 and the wavelength of the second fluorescent beam F2 respectively emitted by the two are respectively different.

In some embodiments, as shown in FIG. 1 , the light detecting module 300 further includes a first photodetector 310 , a second photodetector 320 , and a first dichroic mirror 330 . The first dichroic mirror 330 optically couples the first photodetector 310 and the second photodetector 320. Further, the first dichroic mirror 330 is disposed in front of the first photodetector 310 and the second photodetector 320. The first dichroic mirror 330 is caused by the difference between the wavelength of the first fluorescent beam F1 and the wavelength of the second fluorescent beam F2, so that the optical path of the first fluorescent beam F1 passing through the first dichroic mirror 330 passes through the first The optical path of the second fluorescent beam F2 of the dichroic mirror 330 is separated. The first photodetector 310 is disposed on the optical path of the first fluorescent beam F1 passing through the first dichroic mirror 330. The first photodetector 310 is configured to detect and analyze the first fluorescent beam F1. Similarly, the first The two photodetectors 320 are disposed on the optical path of the second fluorescent beam F2 passing through the first dichroic mirror 330. The second photodetector 320 is configured to detect and analyze the second fluorescent light beam F2. In some embodiments of the present disclosure, the optical path of the first fluorescent beam F1 and the optical path of the second fluorescent beam F2 may be different by 90 degrees, but the disclosure should not be limited thereto. In some embodiments, the photodetection module 300 further includes a first fluorescent filter 3102 and a second fluorescent filter 3202. The first fluorescent filter 3102 is optically coupled between the first photodetector 310 and the dichroic mirror 330. The first fluorescent filter 3102 is for limiting the light beam reaching the first light detecting 310, and only passes the first fluorescent light beam F1. Similarly, the second fluorescent filter 3202 is optically coupled between the second photodetector 320 and the dichroic mirror 330. The second fluorescent filter 3102 is for limiting the light beam reaching the second light detecting 320, and only passes the second fluorescent light beam F2.

In some embodiments, the first photodetector 310 and the second photodetector 320 are substantially simultaneously detected. In other words, the first photodetector 310 detects and captures the imaging of the first fluorescent beam F1 through the imaging lens group 200, and at the same time, the second photodetector 320 detects and captures the second fluorescent beam. F2 is imaged by the imaging lens group 200. Therefore, there is no time difference between the imaging of the first photodetector 310 capturing the first fluorescent beam F1 and the imaging of the second photodetector 320 capturing the second fluorescent beam F2. In this way, by substantially synchronous detection of the first photodetector 310 and the second photodetector 320, the particle three-dimensional positioning and tracking device 10 can simultaneously track the interaction between the two particles.

In some embodiments of the present disclosure, as shown in FIG. 1 , the light source module 100 includes a light source 130 , a polarizing mirror group 142 , a shutter 144 , a plurality of mirrors 151 , 152 , 153 , and 154 , a grating 160 , and a first lens The group 170, the second dichroic mirror 180 and the objective lens group 190. Light source 130 is used to emit light beam L. Polar mirror The 142 and the shutter 144 are optically coupled to the light source 130 to receive the light beam L from the light source 130 and to adjust the intensity of the light beam L. The mirror 151 is optically coupled to the shutter 144 to receive the light beam L from the shutter 144 and adjust the optical path of the light beam L. The mirror 152 is optically coupled to the mirror 151 and is capable of receiving the light beam L from the mirror 151 and adjusting the optical path of the light beam L. The grating 160 is optically coupled to the mirror 152 to receive the light beam L from the mirror 152 and to split the light so that the optical paths of the different wavelengths of the light beam L are separated. Mirror 153 is optically coupled to grating 160 and is capable of receiving beam L from grating 160 and adjusting the optical path of beam L. The first lens group 170 is optically coupled to the mirror 153 and can receive the light beam L from the mirror 153 and adjust the focal length of the light beam L. The mirror 154 is optically coupled to the first lens group 170 and is capable of receiving the light beam L from the first lens group 170 and adjusting the optical path of the light beam L. The second dichroic mirror 180 is optically coupled to the mirror 154 and the objective lens group 190, and can receive the light beam L from the mirror 154 and the subsequent fluorescent light beam F from the object to be tested 110, and separate the light path and the fluorescent light of the light beam L. The optical path of the beam F. The objective lens group 190 is optically coupled to the second dichroic mirror 180, and can receive the light beam L from the second dichroic mirror 180 and adjust the light beam L such that the light beam L passing through the objective lens group 190 is focused and irradiated to the object to be tested 110. By the above description, the light source module 100 can provide the light beam L with the appropriate intensity, angle, focal length, illumination area and spectrum to the object to be tested 110, thereby exciting the object to be tested 110.

In some embodiments of the present disclosure, the light source 130 may be a laser or an ultra-fast pulsed laser, but the disclosure should not be limited thereto. For example, in some embodiments of the disclosure, the light source module 100 can be a multiphoton fluorescent excitation microscopy device. That is to say, the light source module 100 can emit a light beam L with an ultra-fast pulse (a pulse width of about 80 to 150 fs and a frequency of about 10 to 100 MHz), but the disclosure is disclosed. Not limited to this. By the super-segment of the light beam L and providing the characteristics of high photon density and flux, the particles 112 of the object to be tested 110 can absorb at least two photons emitted by the light beam L, so that the particles 112 of the object to be tested 110 obtain sufficient energy. And the transition. In addition, the ultra-fast pulse laser as the light source 130 can more effectively focus the beam L to the range of the object to be tested 110 into a non-dot region. It is worth noting that since the particles 112 absorb the energy of at least two photons of the light beam L, the energy of one photon of the fluorescent light beam F released by the particles 112 is greater than the energy of one photon of the light beam L. In other words, the photon energy of the beam L is different from the photon energy of the fluorescent beam F, so the spectrum of the beam L is different from that of the fluorescent beam F1, so that the beam can be effectively filtered by the spectroscopic system. The L and the fluorescent beam F reduce the noise of the imaging of the fluorescent beam F, which facilitates more accurate detection of the three-dimensional moving trajectory of the particles. In addition, since the wavelength of the light beam L emitted by the light source module 100 is long, the longer wavelength light beam L can reach the deeper depth of the object to be tested 110, which facilitates the detection of the particles 112 of the object to be tested 110.

In some embodiments of the present disclosure, the particle three-dimensional positioning and tracking device 10 provides a method for three-dimensional positioning and tracking of particles, comprising the following steps. Exciting the object to be tested 110 such that the object to be tested 110 generates a fluorescent beam F, using a focused cylindrical lens (ie, the first cylindrical lens 210) and a diverging cylindrical lens (ie, the second cylindrical surface) The lens 220) collectively adjusts the imaging of the fluorescent beam F, and detects through the focused cylindrical lens (ie, the first cylindrical lens 210), a diverging second cylindrical lens (ie, the second cylindrical lens) 220) A fluorescent beam F with the imaging lens 230. Specifically, referring to FIG. 1 , the light source module 100 emits and adjusts the light beam L such that the light beam L excites the object to be tested 110 , and the object to be tested 110 generates the fluorescent light beam F. Imaging lens set 200 (ie, focused first cylindrical lens 210, diverging The second cylindrical lens 220 and the imaging lens 230) adjust the imaging of the fluorescent light beam F, and the light detecting module 300 detects the fluorescent light beam F passing through the imaging lens group 200. Then, based on the astigmatic effect of the imaging lens group 200, the imaging shape of the fluorescent light beam F transmitted to the light detecting module 300 is changed, and the light detecting module 300 is corrected by the imaging shape of the matching particles 112 and the above-mentioned particle. The curve is obtained to obtain the axial (Z direction) position of the fluorescent light beam F. Further, the plane position (XY) of the particles 112 can be obtained by fitting the light intensity of the fluorescent light beam F to a one-two elliptic dimensional Gaussian function. In this way, the particle three-dimensional positioning and tracking device 10 can locate the three-dimensional position of the particle 112 in the object to be tested 110 based on the elliptical Gaussian distribution of the imaged astigmatism of the fluorescent light beam F and the light intensity of the fluorescent light beam F. The three-dimensional movement trajectory of the particle 112 is tracked. It should be noted that the imaging lens assembly 200 includes a first cylindrical lens 210, a second cylindrical lens 220 and an imaging lens 230, and the first cylindrical lens 210 is a cylindrical lens that is focused (ie, f1>0). And the second cylindrical lens 220 is a cylindrical lens that is diverging (that is, f2 < 0).

Fig. 6 is a schematic view of an imaging lens group 200a according to another embodiment of the present disclosure. As shown in Fig. 6, the main difference between the present embodiment and the foregoing embodiment is that the first focal length fa1 < 0 of the first cylindrical lens 210a and the second focal length fa2 > 0 of the second cylindrical lens 220a. Specifically, in some embodiments, the first cylindrical lens 210a has a first focal length fa1 and the first focal length fa1 <0. The refracting power of the first cylindrical lens 210a in the horizontal plane (YZ plane) (which may be less than 0) is different from the diopter in the vertical plane (XZ plane) (may be less than 0), and therefore, the focus and vertical of the fluorescent light beam F in the horizontal plane The focus of the face is different. Similarly, the second cylindrical lens 220a has a second focal length fa2 and the second focal length fa2 > 0. The diopter of the second cylindrical lens 220 in the horizontal plane and the curvature in the vertical plane The luminosity is different, and therefore, the focal point of the fluorescent beam F in the horizontal plane is different from the focal point of the vertical plane. The focal length of the focal plane of the fluorescent light beam F passing through the horizontal plane of the imaging lens group 200a and the focal length of the vertical plane can be adjusted by the first cylindrical lens 210a having the first focal length fa1<0 and the second cylindrical lens 220a having the second focal length fa2>0. . For example, in some embodiments, the fluorescent beam F is diverged by the first cylindrical lens 210a, and the fluorescent beam F is focused by the second cylindrical lens 220a, thereby canceling the astigmatism of the imaging lens group 200a. Effect, while reducing the degree of astigmatism of the image.

For example, in some embodiments, as shown in FIG. 6, in the XZ plane, the central thickness t5 of the first cylindrical lens 210a is different from the edge thickness t6 of the first cylindrical lens 210a, and the central thickness is T5 is smaller than the edge thickness t6 such that the first cylindrical lens 210a is a diverging lens. Further, the thickness of the first cylindrical lens 210a on the YZ plane is substantially equal. Similarly, in the XZ plane, the central thickness t7 of the second cylindrical lens 220a is different from the edge thickness t8 of the second cylindrical lens 220, and the central thickness t7 is greater than the edge thickness t8 such that the second cylindrical lens 220a is in focus. Lens. Further, the thickness of the second cylindrical lens 220a on the YZ plane is substantially equal. The optical axis of the first cylindrical lens 210a coincides with the optical axis of the second cylindrical lens 220a. The diverging first cylindrical lens 210a and the focused second cylindrical lens 220a are sequentially arranged between the stage 120 and the light detecting module 300. With the first cylindrical lens 210a and the second cylindrical lens 220a, the central thickness of the two lenses passing through the fluorescent light beam F can be adjusted (that is, t5+t7), and the fluorescent light beam F can be adjusted. The sum of the edge thicknesses of the two lenses (i.e., t6 + t8), thereby adjusting the imaging focus of the fluorescent beam F, changes the imaged astigmatism of the fluorescent beam F.

Although the disclosure has been disclosed above in the embodiments, it is not In order to limit the disclosure, any person skilled in the art can make various changes and refinements without departing from the spirit and scope of the disclosure. Therefore, the scope of protection of the disclosure is subject to the definition of the patent application scope. .

10‧‧‧Particle three-dimensional positioning and tracking device

100‧‧‧Light source module

110‧‧‧Test object

112‧‧‧ particles

120‧‧‧ stage

130‧‧‧Light source

142‧‧‧polar mirror group

144‧‧ ‧Shutter

151‧‧‧Mirror

152‧‧‧Mirror

153‧‧‧Mirror

154‧‧‧Mirror

160‧‧‧Raster

170‧‧‧First lens group

180‧‧‧Second dichroic mirror

190‧‧‧ objective lens group

200‧‧‧ imaging lens set

210‧‧‧First cylindrical lens

220‧‧‧second cylindrical lens

230‧‧‧ imaging lens

300‧‧‧Light detection module

310‧‧‧First Light Detector

320‧‧‧Second light detector

330‧‧‧First dichroic mirror

1122‧‧‧First particle

1124‧‧‧Second particles

3102‧‧‧First fluorescent filter

3202‧‧‧Second Fluorescent Filter

L‧‧‧beam

F‧‧‧Fluorescent beam

F1‧‧‧First fluorescent beam

F2‧‧‧second fluorescent beam

Claims (10)

A particle three-dimensional positioning and tracking device comprises: a light source module for exciting a sample to be tested to generate a fluorescent beam; and an imaging lens group comprising a first cylindrical lens and a second a cylindrical lens and an imaging lens, wherein the first cylindrical lens has a first focal length, the first focal length is a positive value, and the second cylindrical lens has a second focal length, the second focal length is a negative value, The first cylindrical lens cooperates with the second cylindrical lens system to adjust an imaging shape of the fluorescent light beam; and a light detecting module, wherein the imaging lens is configured to image the fluorescent light beam The light detecting module is configured to detect the fluorescent light beam passing through the imaging lens group. The particle three-dimensional positioning and tracking device of claim 1, wherein the absolute value of the first focal length is substantially equal to the absolute value of the second focal length. The particle three-dimensional positioning and tracking device of claim 1, wherein the first cylindrical lens and the second cylindrical lens are relatively rotatable to adjust the image shape of the fluorescent beam. The particle three-dimensional positioning and tracking device of claim 3, further comprising: a rotating support frame, the first cylindrical lens is located on the rotating support frame; A rotary actuator for actuating the rotation of the first rotary support. The particle three-dimensional positioning and tracking device of claim 3, further comprising: a rotating support frame, the second cylindrical lens is located on the rotating support frame; and a rotary actuator for actuating the Rotate the support frame to rotate. The particle three-dimensional positioning and tracking device of claim 3, wherein a rotation angle of the first cylindrical lens and a rotation angle of the second cylindrical lens are selected such that the imaging shape of the fluorescent beam In the case of a non-oblique ellipse, the long axis of the non-oblique ellipse is perpendicular or parallel to the length direction of the first cylindrical lens or the second cylindrical lens when it has not been rotated. The particle three-dimensional positioning and tracking device of claim 1, wherein the focus of the light beam by the light source module is focused on a non-dot region of the object to be tested, so that the range of the fluorescent beam is generated. A two-dimensional region is covered on all layers of the object to be tested, wherein the slice layer is perpendicular to an arrangement direction of one of the first cylindrical lens and the second cylindrical lens. The particle three-dimensional positioning and tracking device of claim 1, wherein the light detecting module comprises a first photodetector, a second photodetector, and a dichroic mirror, the dichroic mirror The method is configured to distribute two fluorescent beams of different wavelengths to the first photodetector and the second photodetector. The particle three-dimensional positioning and tracking device of claim 8, wherein the first photodetector and the second photodetector are substantially synchronously detected. A method for three-dimensional positioning and tracking of a particle comprises: exciting a sample to be tested to generate a fluorescent beam; and using a focused cylindrical lens to adjust an image of the fluorescent beam together with a diverging cylindrical lens Shape; and detecting the fluorescent beam passing through the focused cylindrical lens and the diverging focusing lens.