CN114486892B - A structured illumination microscopy imaging device and method based on acousto-optic deflection scanning - Google Patents

Abstract

The invention discloses a structured light illumination microscopic imaging device and a structured light illumination microscopic imaging method based on acousto-optic deflection scanning. The acousto-optic deflection module in each light path consists of a 4f system formed by two acousto-optic deflectors which are mutually perpendicular and two lenses, and the scanning position of each acousto-optic deflector on the xy plane of the light beam can be changed by controlling the carrier frequency of the two acousto-optic deflectors loaded in the two acousto-optic deflection modules, so that interference fringes in different directions are obtained. The acousto-optic deflector is utilized to scan the light beam, so that a faster scanning speed can be obtained compared with that of galvanometer scanning; in addition, the acousto-optic deflector has higher scanning stability relative to the galvanometer, and can realize more stable illumination stripes.

Description

A structured illumination microscopy imaging device and method based on acousto-optic deflection scanning

Technical field

The present invention relates to the field of super-resolution optical microscopy imaging, and specifically to a structured illumination microscopy imaging method and device based on acousto-optic deflection scanning.

Background technique

Optical microscopy imaging technology plays an important role in biological research and clinical diagnosis applications due to its non-invasive characteristics. However, due to the diffraction limit, the resolution of optical microscopes usually cannot exceed 200 nm. Over the past few decades, many techniques have been invented to overcome this diffraction limit. Among these microscopy techniques, structured illumination microscopy (SIM) is a powerful tool in biomedical imaging because of its The method can provide higher temporal and spatial resolution and can achieve video-rate imaging speeds. Although SIM can only achieve twice the resolution improvement, the optical power it requires is much smaller than stimulated emission depletion microscopy (STED), which often achieves resolutions on the order of several nanometers. Optical power up to gigawatts/cm² is required. Other methods such as photoactivation localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) are much slower than SIM when the field of view is large. They reconstruct a super-resolution image. , requiring thousands of original images, which makes this method difficult to apply in in vivo imaging. Since people usually need to observe the entire life activities of cells, SIM has become a popular imaging technology due to its fast speed, low phototoxicity and photobleaching properties.

The SIM system originally used grating spectroscopy for interference. Since this method mechanically rotates the grating, it is slow and affects the imaging speed. In order to increase the speed, the current mainstream method is to load a grating image on a digital micromirror and image it onto the sample surface. This method of digitally loading images has a very high speed, but DMD itself has diffraction problems, which affects The quality of the illumination spot. In addition, a spatial light modulator is used to generate two beams (2D-SIM) or three beams (3D-SIM) to interfere to produce stripes. With the emergence of ferroelectric liquid crystals, this method can also achieve video-level imaging speeds. , but there is also the problem of low light energy utilization. Another method is to directly split the light source and cause it to interfere to produce fringes. The change in the direction of the fringe changes the position of the beam through galvanometer scanning. This method has high light energy utilization and does not have the problem of diffraction affecting the spot quality. However, due to the vibration of the galvanometer scanning, the stability of the interference fringes is affected.

Contents of the invention

The purpose of the present invention is to provide a structured illumination microscopy device and method based on acousto-optic deflection scanning in view of the shortcomings of the existing technology.

The specific technical solutions of the present invention are as follows:

A structured illumination micro-imaging device based on acousto-optic deflection scanning, including a beam splitting light path for generating interference illumination fringes, an excitation light path for combining the beam light path, and an imaging detection light path,

The excitation light path passes through a laser, a single-mode polarization-maintaining fiber, a collimating lens, a first reflecting mirror, a first half-wave plate, and a polarizing beam splitter in sequence, and the polarizing beam splitter divides the light into two symmetrical optical paths;

One of the two symmetrical optical paths passes through the electro-optical modulator, the second reflector, the third reflector, the second half-wave plate, the first acoustic-optical deflector, the second lens, the third lens, the second acoustic-optical deflector in sequence. The optical deflector, the first scanning lens; the other path passes through the fourth reflector, the fifth reflector, the third half-wave plate, the third acousto-optic deflector, the fourth lens, the fifth lens, the fourth acousto-optic deflector, second scanning lens;

The split light paths are then combined by a beam combiner, and pass through a polarization rotator, a sixth lens, a seventh lens, a dichroic mirror, an objective lens, and a sample in sequence;

The imaging detection light path passes through the sample, objective lens, dichroic mirror, filter, field lens, and camera in sequence;

Also included: computers.

Preferably, the first acousto-optic deflector, the second lens, the third lens and the second acousto-optic deflector constitute a first 4f system.

Preferably, the first acousto-optic deflector and the second acousto-optic deflector are placed perpendicularly to each other.

Preferably, the third acousto-optic deflector, the fourth lens, the fifth lens and the fourth acousto-optic deflector constitute the second 4f system.

Preferably, the third acousto-optic deflector and the fourth acousto-optic deflector are placed perpendicularly to each other.

Preferably, the center of the second acousto-optic deflector is located at the front focal plane of the first scanning lens.

Preferably, the center of the fourth acousto-optic deflector is located at the front focal plane of the second scanning lens.

Preferably, the common focal plane of the first scanning lens and the second scanning lens, the sixth lens, the seventh lens, and the entrance pupil plane of the objective lens form a third 4f system.

A structured illumination microscopy imaging method based on acousto-optic deflection scanning includes the following steps:

(1) The laser emits laser light that passes through the polarization-maintaining single-mode fiber and then is collimated by a collimating lens. The collimated light enters the first half-wave plate after being reflected by the first reflector. By rotating the first half-wave plate The optical axis is used to change the polarization direction of the excitation light so that the horizontal and vertical polarizations of the excitation light each account for half of the laser energy, and then are divided into two symmetrical light paths through a polarization beam splitter. The horizontally polarized light is transmitted through the polarization beam splitter, and the vertical polarization light is transmitted through the polarization beam splitter. Polarized light is reflected through a polarizing beam splitter;

(2) The vertically polarized light passes through the electro-optical modulator, and then passes through the second reflective mirror and the third reflective mirror, so that it is incident on the first acousto-optical deflector at the Bragg angle on the incident plane that is perpendicular to the xy plane and passes through the y axis. on, the light is scanned along the y direction. After the scanning light passes through the second lens and the third lens, the scanning fixed point is imaged onto the second acousto-optic deflector, and is imaged on the vertical xy plane and passes through the incident surface of the x axis. is incident on the second acousto-optic deflector at a Bragg angle, causing the light to scan along the x-direction. By setting the carrier frequency loaded on the first acousto-optic deflector and the second acousto-optic deflector, the first acousto-optic deflector The second half-wave plate placed in front of the device is used to change the polarization direction of the incident light so that its polarization direction is consistent with the deflection direction of the first acousto-optic deflector. The scanning light emitted by the second acousto-optic deflector passes through the first Convergence is performed after scanning the lens;

(3) The horizontally polarized light is loaded with carrier frequencies of different frequencies on the third acousto-optical deflector and the fourth acousto-optical deflector to achieve scanning of light spots at different positions in the xy plane, and is converged through the second scanning lens ;

(4) The two paths of light pass through the beam combiner to combine, then pass through the deflection rotator to change the polarization direction of the light, then pass through the sixth lens and the seventh lens to relay, and pass through the dichroic mirror to scan the first The two light spots converged by the lens and the second scanning lens are imaged at a symmetrical position at the center of the entrance pupil surface of the objective lens, and are then collimated by the objective lens into two beams of parallel light, each with an angle close to the numerical aperture of the objective lens. The parallel light is reflected on the sample Interference occurs on the surface to form interference fringes in a certain direction to illuminate the sample;

(5) The fluorescence excited by the sample is also received by the objective lens, is reflected by the dichroic mirror, filters out the excitation light and ambient stray light through a filter, and then passes through the field lens and is imaged on the camera.

Preferably, by changing the voltage loaded on the electro-optic modulator, the stripe phase of the illumination sample is changed to achieve three-step phase shift, and the fluorescence image under corresponding stripe excitation is recorded each time; changing the voltage loaded on the first acousto-optic deflector, the second The carrier frequency on the acousto-optic polarizer, the third acousto-optic deflector and the fourth acousto-optic deflector can change the scanning direction of the incident light, thereby changing the direction of the illumination stripes, thereby obtaining illumination stripes in three directions, each direction The corresponding fluorescence images were recorded, and 9 images were finally obtained.

Compared with the existing technology, the present invention has the following beneficial technical effects:

(1) By using an acousto-optic deflector instead of a galvanometer for beam scanning, it has the advantage of fast scanning speed, thereby increasing the imaging speed;

(2) By using an acousto-optic deflector instead of a galvanometer for beam scanning, it has the advantage of stable beam scanning, which can ensure the stability of the illumination stripes. At the same time, an electro-optical modulator is used to adjust the beam phase without using piezoelectric ceramics. The optical path has no jitter caused by vibration, which can also further improve the stability of the light spot.

It has good applications in beam scanning, especially in two-photon microscopy imaging. Applying it to the scanning of two interference beams can ensure that the fringe switching speed is increased while maintaining the stability of the fringes, and has good application prospects in structured illumination microscopy imaging.

Description of the drawings

Figure 1 is a schematic diagram of a device for microscopic imaging based on acousto-optical deflection scanning structured light imaging according to the present invention;

Figures 2a and 2b are respectively schematic diagrams of the corresponding optical path details in the first acousto-optical deflection module and the second acousto-optical deflection module from different perspectives;

Figure 3 The distribution of scanning light spots at the entrance pupil surface of the objective lens.

Detailed ways

The present invention will be described in detail below with reference to the embodiments and drawings, but the present invention is not limited thereto.

As shown in Figure 1, the structured illumination microscopy imaging device based on acousto-optic deflection scanning of the present invention includes:

Beam splitting light path, beam combining light path, excitation light path and imaging detection light path used to generate interference illumination fringes;

The excitation light path sequentially passes through the laser 1, the single-mode polarization-maintaining fiber 2, the collimating lens 3, the first mirror 4, the first half-wave plate 5, and the polarizing beam splitter 6. The polarizing beam splitter 6 divides the light into symmetrical optical paths. The two-way split beam path;

The devices arranged in the two symmetrical optical paths are basically the same, one of which passes through the electro-optical modulator 7, the second reflecting mirror 8, the third reflecting mirror 9, the second half-wave plate 10, and the first acousto-optical deflection module 11 (including The first acousto-optical deflector 29, the second lens 30, the third lens 31, the second acousto-optical deflector 32), the first scanning lens 12; the other components are the same as the symmetrical optical path described above except that no electro-optical modulator is provided. , that is, through the fourth reflector 13, the fifth reflector 14, the third half-wave plate 15, the second acousto-optic deflection module 16 (including the third acousto-optic deflector 33, the fourth lens 34, the fifth lens 35, the Four acoustic light deflectors 36), second scanning lens 17;

The split light path is then combined into a combined light path through the beam combiner 18, and passes through the polarization rotator 19, the sixth lens 20, the seventh lens 21, the dichroic mirror 22, the objective lens 23, and the sample 24 in sequence;

The imaging detection light path sequentially passes through the sample 24, objective lens 23, dichroic mirror 22, filter 25, field lens 26, and camera 27;

It also includes: a computer 28, which is electrically connected to the electro-optical modulator 7, the first acousto-optical deflection module 11, the second acousto-optical deflection module 16 and the camera 27 respectively.

The structured light microscopy imaging method based on acousto-optic deflection scanning of the present invention includes the following steps:

(1) The laser 1 emits laser light as the excitation light of the sample 24. After passing through the polarization-maintaining single-mode fiber 2, it is collimated by the collimating lens 3, so that the spot size meets the system field of view size requirements. The collimated light passes through the first After reflection by the mirror 4, it enters the first half-wave plate 5. The optical axis of the first half-wave plate 5 is rotated to change the polarization direction of the excitation light, so that the horizontal and vertical polarizations of the excitation light each account for half of the laser energy. After the polarization beam splitter, it is divided into two light paths with symmetrical optical paths. The horizontally polarized light is transmitted through the polarization beam splitter 6, and the vertically polarized light is reflected through the polarization beam splitter 6;

(2) As shown in Figure 2a and Figure 2b, the reflected vertically polarized light in one of the symmetrical optical paths passes through the electro-optical modulator 7 to change the optical path (phase) of the reflected optical path relative to the transmitted horizontally polarized light, thereby making all The fringes generated by the interference of the two beams of light are translated to achieve phase shifting, and then pass through the second reflecting mirror 8 and the third reflecting mirror 9 to adjust the direction of the light so that it is on the incident surface that is perpendicular to the xy plane and passes through the y axis. The light is incident on the first acousto-optic deflector 29 of the first acousto-optic deflection module 11 at a Bragg angle, causing the light to scan along the y direction. The scanning light then passes through the second lens 30 and the third lens 31 of the first acousto-optic deflection module. Then, the scanning fixed point is imaged onto the second acousto-optic deflector 32, and is incident on the second acousto-optic deflector 32 at a Bragg angle on the incident plane perpendicular to the xy plane and passing through the x-axis, causing light to be generated along the x direction. Scanning, by setting the carrier frequency loaded on the first acousto-optic deflector 29 and the second acousto-optic deflector 32, the light spot can be scanned at different positions in the xy plane. The first acousto-optic deflector 29 is placed in front of The second half-wave plate 10 is used to change the polarization direction of the incident light so that its polarization direction is consistent with the deflection direction of the first acousto-optic deflector 29. The scanning light emitted by the second acousto-optic deflector 32 passes through the first Convergence is performed after scanning the lens 12;

(3) Except that the transmitted horizontally polarized light in one of the symmetrical light paths does not pass through the electro-optical modulator 7, other devices are the same as those passed by the reflected vertically polarized light path, and also passes through the third acousto-optical light in the second acousto-optical deflection module 16 The deflector 33 and the fourth acousto-optic deflector 36 are loaded with carrier frequencies of different frequencies, so that the light spots can be scanned at different positions in the xy plane and converged through the second scanning lens 17;

(4) As shown in Figure 3, the two symmetrical lights pass through the beam combiner 18 for beam combination, and then pass through the deflection rotator 19 to change the polarization direction of the light to S at the entrance pupil surface of each objective lens 23. Polarized so that the subsequent interference fringes have the highest contrast, they are then relayed by the sixth lens 20 and the seventh lens 21 , and then passed through the dichroic mirror 22 to converge the two lights of the first scanning lens 12 and the second scanning lens 17 The point is imaged to a symmetrical position at the center of the entrance pupil surface of the objective lens 23, and is then collimated by the objective lens 23 into two beams of parallel light, each with a numerical aperture angle close to the objective lens 23. The parallel light interferes on the sample surface to form a certain direction. Interference fringe illumination sample 24;

(5) The fluorescence excited by the sample 24 is also received by the objective lens 23, is reflected by the dichroic mirror 22, filters out the excitation light and ambient stray light through the filter 25, and then passes through the field lens 26 and is imaged on the camera 27;

(6) Change the voltage loaded on the electro-optical modulator 7, thereby changing the stripe phase of the illumination sample 24, achieving a three-step phase shift, and recording the fluorescence image under corresponding stripe excitation each time; changing the voltage loaded on the first acousto-optic deflector 29 , the carrier frequency on the second acousto-optic polarizer 32, the third acousto-optic deflector 33 and the fourth acousto-optic deflector 36 can change the scanning direction of the incident light, thereby changing the direction of the illumination stripes, thereby obtaining three-directional Illuminating stripes, corresponding fluorescence images were recorded in each direction, and 9 images were finally obtained.

(7) The computer 28 outputs a control signal to the electro-optic modulator 7 to change the output voltage, thereby adjusting the phase of the corresponding light beam; at the same time, the computer 28 outputs a control signal to the first acousto-optic deflector 29 and the first acousto-optic deflection module 11 The second acousto-optic deflector 32, as well as the third acousto-optic deflector 33 and the fourth acousto-optic deflector 36 of the second acousto-optic deflection module, change its scanning angle, thereby controlling its scanning position of the light beam; in addition, the computer 28 Images captured by camera 27 are also stored and processed.

The above are only examples of the preferred implementation of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention. within.

Claims (10)

1. A structured illumination microscopic imaging device based on acousto-optic deflection scanning, including a beam splitting light path for generating interference illumination fringes, an excitation light path for combining beam paths, and an imaging detection light path, which is characterized by: The excitation light path passes through a laser, a single-mode polarization-maintaining fiber, a collimating lens, a first reflecting mirror, a first half-wave plate, and a polarizing beam splitter in sequence, and the polarizing beam splitter divides the light into two symmetrical optical paths; One of the two symmetrical optical paths passes through the electro-optical modulator, the second reflective mirror, the third reflective mirror, the second half-wave plate, the first acoustic-optical deflector, the second lens, the third lens, the second acoustic-optical deflector in sequence. The optical deflector, the first scanning lens; the other path passes through the fourth reflector, the fifth reflector, the third half-wave plate, the third acousto-optic deflector, the fourth lens, the fifth lens, the fourth acousto-optic deflector, second scanning lens; The split light paths are then combined by a beam combiner, and pass through a polarization rotator, a sixth lens, a seventh lens, a dichroic mirror, an objective lens, and a sample in sequence; The imaging detection light path passes through the sample, objective lens, dichroic mirror, filter, field lens, and camera in sequence; Also included: computers. 2. The structured light microscopy imaging device based on acousto-optic deflection scanning as claimed in claim 1, characterized in that: the first acousto-optic deflector, the second lens, the third lens, the second acousto-optic deflector Constitute the first 4f system. 3. The structured light microscopy imaging device based on acousto-optic deflection scanning as claimed in claim 1, wherein the first acousto-optic deflector and the second acousto-optic deflector are placed perpendicularly to each other. 4. The structured light microscopy imaging device based on acousto-optic deflection scanning as claimed in claim 1, characterized in that: the third acousto-optic deflector, the fourth lens, the fifth lens and the fourth acousto-optic deflector constitute the third acousto-optic deflector. Two 4f system. 5. The structured light microscopy imaging device based on acousto-optic deflection scanning as claimed in claim 1, wherein the third acousto-optic deflector and the fourth acousto-optic deflector are placed perpendicularly to each other. 6. The structured illumination microscopic imaging device based on acousto-optic deflection scanning as claimed in claim 1, wherein the center of the second acousto-optic deflector is located at the front focal plane of the first scanning lens. 7. The structured illumination microscopic imaging device based on acousto-optic deflection scanning as claimed in claim 1, wherein the center of the fourth acousto-optic deflector is located at the front focal plane of the second scanning lens. 8. The structured illumination microscopic imaging device based on acousto-optic deflection scanning as claimed in claim 1, characterized in that: the common focal plane of the first scanning lens and the second scanning lens is in contact with the sixth lens, the seventh lens, and the objective lens. The entrance pupil surface forms the third 4f system. 9. A structured illumination microscopic imaging method based on acousto-optic deflection scanning, which is characterized by: Includes the following steps: (1) The laser emits laser light that passes through the polarization-maintaining single-mode fiber and then is collimated by the collimating lens. The collimated light enters the first half-wave plate after being reflected by the first reflector, and rotates the optical axis of the first half-wave plate. To change the polarization direction of the excitation light, so that the horizontal and vertical polarization of the excitation light each account for half of the laser energy, and then pass through the polarizing beam splitter to split the light into two symmetrical optical paths. The horizontally polarized light is transmitted through the polarizing beam splitter, and the vertically polarized light is reflected through the polarizing beam splitter. polarizing beam splitter; (2) The vertically polarized light passes through the electro-optical modulator, and then passes through the second reflective mirror and the third reflective mirror, so that it is incident on the first acousto-optical deflector at the Bragg angle on the incident plane that is perpendicular to the xy plane and passes through the y axis. on, the light is scanned along the y direction. After the scanning light passes through the second lens and the third lens, the scanning fixed point is imaged onto the second acousto-optic deflector, and is imaged on the vertical xy plane and passes through the incident surface of the x axis. is incident on the second acousto-optic deflector at a Bragg angle, causing the light to scan along the x-direction. By setting the carrier frequency loaded on the first acousto-optic deflector and the second acousto-optic deflector, the first acousto-optic deflector The second half-wave plate placed in front of the device is used to change the polarization direction of the incident light so that its polarization direction is consistent with the deflection direction of the first acousto-optic deflector. The scanning light emitted by the second acousto-optic deflector passes through the first scanning lens. Then gather; (3) The horizontally polarized light is loaded with carrier frequencies of different frequencies on the third acousto-optical deflector and the fourth acousto-optical deflector to achieve scanning of light spots at different positions in the xy plane, and is converged through the second scanning lens ; (4) The two paths of light pass through the beam combiner to combine, then pass through the deflection rotator to change the polarization direction of the light, then pass through the sixth lens and the seventh lens to relay, and pass through the dichroic mirror to scan the first The two light spots converged by the lens and the second scanning lens are imaged at a symmetrical position at the center of the entrance pupil surface of the objective lens, and are then collimated by the objective lens into two beams of parallel light, each with an angle close to the numerical aperture of the objective lens. The parallel light is reflected on the sample Interference occurs on the surface to form interference fringes in a certain direction to illuminate the sample; (5) The fluorescence excited by the sample is also received by the objective lens, is reflected by the dichroic mirror, filters out the excitation light and ambient stray light through a filter, and then passes through the field lens and is imaged on the camera. 10. The structured illumination microscopy imaging method based on acousto-optic deflection scanning as claimed in claim 9, characterized in that: By changing the voltage loaded on the electro-optical modulator, the stripe phase of the illumination sample is changed to achieve a three-step phase shift, and the fluorescence image under corresponding stripe excitation is recorded each time; changing the first acousto-optic deflector and the second acousto-optic polarization The carrier frequency on the deflector, the third acousto-optic deflector and the fourth acousto-optic deflector can change the scanning direction of the incident light, thereby changing the direction of the illumination stripes, thereby obtaining illumination stripes in three directions, and recording the corresponding Fluorescence images, and finally 9 images were obtained.