Microscopic holographic data acquisition device and method based on optical scanning

Abstract

The invention discloses a microscopic holographic data acquisition device and method based on optical scanning. A first annular scanning light beam of a heterodyne scanning light beam generation device is sequentially provided with a first diaphragm and a first photoelectric detector; a first lens, a first beam splitter, a microscope objective and a piezoelectric displacement table are sequentially arranged on the second annular scanning light beam, and the first lens and the microscope objective are confocal; an object to be scanned is arranged on the piezoelectric displacement table; the second photoelectric detector is arranged in the direction of reflected light of the first beam splitter from the microscope objective; the first photoelectric detector and the second photoelectric detector are respectively connected with the electrical processing part, and the electrical processing part is used for converting optical information acquired by the optical recording part into electrical signals and collecting, transmitting and processing the electrical signals. According to the invention, the recording process is more concise, the shooting time of single recording is shorter, the photobleaching effect increased when the fluorescent sample is recorded for a long time is avoided, the system cost is also reduced, and the imaging quality is better.

Description

Technical Field

[0001] The present invention relates to the field of holographic microscopy, and in particular to a microscopic holographic data acquisition device and method based on optical scanning. Background Art

[0002] In today's era of rapid technological development, microscopic imaging technology, as an indispensable and important tool in the field of life sciences and medicine, plays a vital role in revealing the internal microscopic structure and function of organisms. With the continuous advancement of science and technology, people's requirements for microscopic imaging technology are getting higher and higher, hoping to achieve higher resolution and more comprehensive imaging to better understand the mechanisms and laws of life activities. In recent years, three-dimensional imaging technologies such as laser scanning and confocal microscopy have been widely used, but these three-dimensional microscopy technologies require very complex processes, long time and high costs. Against this background, digital holography based on optical scanning came into being. As a new imaging method, it has huge potential and room for development.

[0003] However, one of the disadvantages of traditional digital holography is that the object light and the reference light are coherent, so both the hologram and the reconstructed image contain severe speckle noise. Digital holography based on optical scanning is a special type of digital holography that uses a heterodyne scanning beam generating device to generate a time-varying annular scanning beam to scan the three-dimensional object being measured, and then uses a single-pixel photodetector to collect the information of the object. This allows the system to obtain the three-dimensional information of the object without being limited by the image resolution of the photodetector, that is, the number of pixels. The theoretically achievable resolution is much higher than that of traditional digital holography. Therefore, more microscopic digital holograms can be captured. In addition, the incoherent imaging method also solves the problem of severe speckle noise in traditional digital holography.

[0004] In a holographic device based on optical scanning, a converging lens is used to converge the object information onto a photodetector. However, the existence of the converging lens causes a partial loss of high-frequency information of the object, which reduces the numerical aperture NA of the system, thereby affecting the resolution accuracy of the system.

[0005] Compared with the digital holographic system built in free space, the use of a multi-axis cage structure for the optical path construction of the system is highly integrated. Since the height of the cage rods is fixed and the original slide frames are coaxial with the system optical axis, this structure has very high stability and accuracy compared to the optical path construction in free space, and also reduces the difficulty of optical path calibration. The highly stable optical recording part is also easy to carry, allowing the system to go out of the laboratory. Summary of the invention

[0006] The object of the present invention is to provide a micro-holographic data acquisition device and method based on optical scanning.

[0007] In order to achieve the above invention object, the technical solution adopted by the present invention is as follows:

[0008] A microscopic holographic data acquisition device based on optical scanning comprises: an optical recording part, an electrical processing part;

[0009] The optical recording part includes, in sequence along the optical path: a heterodyne scanning beam generating device, a first lens, a first beam splitter, a microscope objective, an object, a piezoelectric displacement stage, a first diaphragm, a first photodetector, and a second photodetector; the heterodyne scanning beam generating device emits two time-varying annular scanning beams with the same heterodyne signal, and the first diaphragm is placed between the first annular scanning beam and the first photodetector; the first lens is placed behind the second annular scanning beam, the first beam splitter is placed between the first lens and the microscope objective, and the first lens and the microscope objective are confocal; the object to be scanned is placed on the piezoelectric displacement stage, and the piezoelectric displacement stage is placed behind the microscope objective; the second photodetector is placed in the direction of the reflected light from the microscope objective by the first beam splitter.

[0010] The electrical processing part includes: a bandpass filter, a phase-locked amplifier, a data acquisition card, and a computer.

[0011] The electrical processing part is connected to the optical recording part through a first photodetector and a second photodetector. The electrical processing part is used to convert the optical information obtained by the optical recording part into an electrical signal, and collect, transmit and process the electrical signal; the output end of the first photodetector is connected to the reference signal end of the phase-locked amplifier; the output end of the second photodetector is connected to a bandpass filter; the bandpass filter is directly connected to the input signal end of the phase-locked amplifier; the output signal end of the phase-locked amplifier is closely connected to a data acquisition card; and the data acquisition card is connected to a computer.

[0012] For the heterodyne scanning beam generating device, three different examples are proposed; using plane wave and spherical wave interference, and modulating the two lights at different frequencies to form a time-varying annular scanning beam; or using a spatial light modulator to quickly play different annular beam patterns to achieve the effect of a time-varying annular scanning beam.

[0013] The heterodyne scanning beam generating device example 1 comprises: a laser, a first reflector, a second reflector, an attenuation plate, a second beam splitter, a first acousto-optic frequency shifter, a third reflector, a second lens, a first pinhole, a third lens, a second acousto-optic frequency shifter, a fourth reflector, a fourth lens, a second pinhole, a fifth lens, a sixth lens, and a third beam splitter; the laser output light passes through the first reflector and the second reflector and then is adjusted in intensity by the attenuation plate, and is kept coaxial with the center of the second beam splitter; the first reflector is placed in the laser output direction; the first acousto-optic frequency shifter is placed in the direction of the reflected light of the second beam splitter and is placed between the third reflector and the second beam splitter; the third reflector is placed in the direction of the first-order diffraction light of the first acousto-optic frequency shifter, and is deflected at a certain angle compared with the direction of the zero-order light; the second lens is placed in the direction of the reflected light of the third reflector direction, and the second lens is confocal with the third lens, the first pinhole is placed at the focus of the second lens and the third lens, and the optical axis direction of the light reflected by the third reflector is consistent with the center of the second lens, the first pinhole and the third lens; the second acousto-optic frequency shifter is placed in the direction of the light transmitted by the second beam splitter; the fourth reflector is placed in the direction of the first-order diffraction light of the second acousto-optic frequency shifter, and is deflected at a certain angle compared with the direction of the zero-order light; the fourth lens is placed in the direction of the light reflected by the fourth reflector, and the fourth lens is confocal with the fifth lens, the second pinhole is placed at the focus of the fourth lens and the fifth lens, and the optical axis direction of the light reflected by the fourth reflector is consistent with the center of the fourth lens, the second pinhole and the fifth lens; the sixth lens is placed behind the fifth lens; the third beam splitter is placed at the coaxial intersection of the light transmitted by the sixth lens and the light transmitted by the third lens.

[0014] The second example of the heterodyne scanning beam generating device includes: a laser, a first reflector, a second reflector, an attenuation plate, a second beam splitter, a first acousto-optic frequency shifter, a third reflector, a second lens, a first pinhole, a third lens, a second acousto-optic frequency shifter, a fourth reflector, a fourth lens, a second pinhole, a fifth lens, a sixth lens, a third beam splitter; a fourth beam splitter; a fifth reflector; the laser output light is adjusted in intensity by the attenuation plate and then passes through the first reflector and the second reflector to keep coaxial with the center of the second beam splitter, the first reflector is placed in the laser output direction; the first acousto-optic frequency shifter is placed in the direction of the reflected light of the second beam splitter, the third reflector is placed in the vertical direction of the first-order diffraction light of the first acousto-optic frequency shifter, and is deflected at a certain angle compared to the direction of the zero-order light; the second lens is placed in the direction of the vertical reflected light of the third reflector after being deflected by the first acousto-optic frequency shifter, and the second lens and the third lens are confocal, and the first pinhole is placed between the second lens and The third lens is at the focus of the third lens, and the optical axis direction of the vertically reflected light of the third reflector after being transmitted by the first acousto-optic frequency shifter is consistent with the center of the second beam splitter, the second lens, the first pinhole and the third lens; the second acousto-optic frequency shifter is placed in the direction of the light transmitted by the second beam splitter; the fourth reflector is placed in the vertical direction of the first-order diffracted light of the second acousto-optic frequency shifter, and is deflected at a certain angle compared with the direction of the zero-order light; the third beam splitter is placed between the second beam splitter and the second acousto-optic frequency shifter; the fourth lens is placed in the direction of the light reflected by the third beam splitter through the second acousto-optic frequency shifter, and the fourth lens is confocal with the fifth lens, the second pinhole is placed at the focus of the fourth lens and the fifth lens, and the optical axis direction of the light reflected by the third beam splitter is consistent with the center of the fourth lens, the second pinhole and the fifth lens; the sixth lens is placed behind the fifth lens; the fifth reflector is placed behind the sixth lens, and the fourth beam splitter is placed at the coaxial intersection of the light reflected by the sixth lens through the fifth reflector and the light transmitted by the third lens.

[0015] Example 3 of the heterodyne scanning beam generating device comprises: a laser, a first reflector, a second reflector, an attenuation plate, a second beam splitter, a second lens, a first pinhole, a third lens, a sixth lens, a third beam splitter, a spatial light modulator, and a second aperture; the laser output light is adjusted in intensity by the attenuation plate and then passes through the first reflector and the second reflector to remain coaxial with the center of the second beam splitter, and the first reflector is placed in the laser output direction; the second lens is placed in the direction of the light reflected by the second reflector, and the second lens is confocal with the third lens, the first pinhole is placed at the focal point of the second lens and the third lens, and the optical axis direction of the light reflected by the second reflector is consistent with the center of the second lens, the first pinhole, and the third lens; the second beam splitter is placed behind the third lens, and the spatial light modulator is placed in the direction of the light reflected by the second beam splitter; the sixth lens is placed in the direction of the light reflected by the spatial light modulator and transmitted through the second beam splitter; the second aperture is placed at the focal position of the sixth lens, and the third beam splitter is placed behind the second aperture.

[0016] The optical recording part needs to be compactly and firmly fixed on an optical platform that can be laid flat and hung vertically on the desktop; in order to make the optical path more stable, concise and specific, a multi-axis cage structure is used to build the optical path to realize an integrated optical microscope system. The construction concept of this structure is different from that of the optical path in free space. First, the fixed support frame needs to be fixed on the optical platform, and then the cage rod is passed through the pores of the base at the required height, and then the sliding frames of the original components such as the lens required in the system are inserted into the cage rod in turn, and finally the second fixed support frame is fixed at the other end of the cage rod. Since the height of the cage rod is fixed and the original sliding frames are coaxial with the optical axis of the system, the optical path construction of this structure has very high stability and accuracy compared with the free space, and also reduces the difficulty of optical path calibration.

[0017] Another object of the present invention is to provide a micro-holographic data acquisition method based on optical scanning, comprising the following steps:

[0018] Step 1: The heterodyne scanning beam generating device emits two time-varying annular scanning beams with the same heterodyne signal, wherein the first annular scanning beam passes through the first aperture to obtain a light spot with a single difference frequency variation, which is received by the first photodetector and converted into a difference frequency electrical signal, which is connected to the reference signal end of the phase-locked amplifier via a coaxial cable, and the second annular scanning beam is used to scan the object.

[0019] Step 2: The scanning light beam compresses the diameter of the optical path through the first lens so that the diameter of the light spot can just fill the aperture of the microscope objective lens; then the diameter of the optical path is further compressed through the microscope objective lens, and after two steps of compression, the diameter of the time-varying annular scanning light beam is compressed to a width that cannot be distinguished by the naked eye; the piezoelectric displacement stage is moved by the scanning controller to realize the two-dimensional scanning of the object to be scanned (a tiny object to be imaged or a biological specimen) placed on the piezoelectric displacement stage by the scanning light beam, and the reflected light carrying the three-dimensional information of the object obtained by the scanning is magnified by the microscope objective lens again, and is received by the second photodetector through the reflection surface of the third beam splitter;

[0020] In step 2, the expression of the intensity S of the time-varying annular scanning beam formed by the heterodyne scanning beam generating device when it hits the surface of the object is:

[0021]

[0022] Wherein, A and B represent the amplitudes of the plane wave and the spherical wave respectively, j is an imaginary unit, w0 is the inherent temporal frequency of the laser emitted by the laser, Ω1 is the frequency shift of the first acousto-optic frequency shifter in the heterodyne scanning beam generating device, Ω2 is the frequency shift of the second acousto-optic frequency shifter in the heterodyne scanning beam generating device, Ω2>Ω1, t is the time variable, k0 is the wave number, x and y represent the coordinates of the wavefront plane, and z is the distance from the first convergence point of the spherical wave between the microscope objective and the object in the propagation direction of the spherical wave to the object.

[0023] Step 3: The second photodetector converts the light intensity information carrying the three-dimensional information of the object into a current signal. The current signal is connected to a bandpass filter and then filtered out by a phase-locked amplifier to remove the DC current and non-difference frequency signals to obtain a difference frequency current signal. The signal is then transmitted to a data acquisition card. The computer can observe the current signal of the object to be scanned on the moving piezoelectric displacement stage in real time through the data acquisition card, and obtain a three-dimensional hologram of the object to be scanned after the scan is completed.

[0024] In step 3, the second photodetector converts the light intensity information carrying the three-dimensional information of the object into the current i(t) as follows:

[0025] i(t)∝∫|S(x,y,t;z)Γ(x,y,t;z)| 2 dxdy]

[0026] After passing through the bandpass filter, the current signal i is obtained. Ω for:

[0027]

[0028] Wherein, Re[] represents the real part; D represents the two-dimensional area of ​​the second photodetector; x and y represent the coordinates of the wavefront plane, z is the distance from the first convergence point of the spherical wave between the microscope objective and the object in the propagation direction of the spherical wave to the object; t is the time variable, k0 is the wave number; Ω=Ω2-Ω1; Γ(x, y; z) represents the object function located at the spatial coordinates (x, y; z).

[0029] Step 4: Based on the hologram reconstruction procedure, the obtained hologram is reconstructed to obtain the three-dimensional information of the high-resolution biological specimen.

[0030] The gain effect of the present invention is:

[0031] The present invention removes the common convergence lens in front of the photodetector. The lensless imaging technology used for digital holography has significant advantages in cost, portability, imaging field of view, three-dimensional reconstruction capability, digital processing flexibility and depth of field. These advantages make lensless imaging have broad application prospects in the fields of microbial detection, medical research, instant detection, etc.

[0032] Compared with the currently common three-dimensional imaging technologies such as laser scanning and confocal microscopy, the microscopic holographic data acquisition technology based on optical scanning can capture the three-dimensional information of the specimen with just one shot, so the recording process of the holographic device is simpler and the single recording shooting time is shorter. It can avoid the increased photobleaching effect when recording fluorescent samples for a long time, and the system cost is also greatly reduced.

[0033] Compared with traditional digital holographic microscopy, the number of recorded pixels and the size of the CCD limit the resolution of digital holography. In addition, the inherent speckle noise of coherent illumination also has a great impact on the imaging effect. Since the microscopic holographic data acquisition device based on optical scanning uses a two-dimensional light beam to scan a three-dimensional object to obtain the three-dimensional information of the object, the resolution is not affected by the number of recorded pixels and the size of the CCD. Incoherent imaging reconstruction no longer has speckle noise and has better imaging quality.

[0034] Compared with the digital holographic system built in free space, the multi-axis cage structure is used for optical path construction, which is highly integrated. Since the height of the cage rod is fixed and the original slide frame is coaxial with the system optical axis, this structure has very high stability and accuracy compared with the optical path construction in free space, and also reduces the difficulty of optical path calibration. The highly stable optical recording part is also easy to carry, so that the system can go out of the laboratory. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the optical path diagram of the overall structure of the present invention.

[0036] Figure 2 It is a schematic diagram of the overall implementation process of the method of the present invention.

[0037] Figure 3 This is a light path diagram of Example 1 of the heterodyne scanning light beam generating device of the present invention.

[0038] Figure 4 This is the optical path diagram of Example 2 of the heterodyne scanning light beam generating device of the present invention.

[0039] Figure 5 This is the optical path diagram of Example 3 of the heterodyne scanning light beam generating device of the present invention.

[0040] In the figure: a heterodyne scanning beam generating device 3, a first lens 4, a first beam splitter 5, a microscope objective 6, an object 7, a piezoelectric displacement stage 8, a first aperture 9, a first photodetector 10, a second photodetector 11, a bandpass filter 12, a phase-locked amplifier 13, a data acquisition card 14, a computer 15, a laser 16, a first reflector 17, a second reflector 18, an attenuation plate 19, a second beam splitter 20, a first acousto-optic frequency shifter 21, a third reflector 22, a second lens 23, a first pinhole 24, a third lens 25, a second acousto-optic frequency shifter 26, a fourth reflector 27, a fourth lens 28, a second pinhole 29, a fifth lens 30, a sixth lens 31, a third beam splitter 32, a fourth beam splitter 33, a fifth reflector 34, a spatial light modulator 35, and a second aperture 36. DETAILED DESCRIPTION

[0041] The present invention will be further described below in conjunction with the accompanying drawings and specific implementation methods.

[0042] Example 1

[0043] A microscopic holographic data acquisition device based on optical scanning, such as Figure 1 shown.

[0044] The reference numerals in the accompanying drawings respectively represent: a heterodyne scanning beam generating device 3, a first lens 4, a first beam splitter 5, a microscope objective 6, an object 7, a piezoelectric displacement stage 8, a first aperture 9, a first photodetector 10, a second photodetector 11, a bandpass filter 12, a phase-locked amplifier 13, a data acquisition card 14, and a computer 15.

[0045] The overall implementation process diagram of the method system device of the present invention is as follows Figure 2 As shown, the optical recording part 1 needs to be compactly and firmly fixed on an optical platform that can be placed flat and also hung vertically on a desktop; the information obtained by the optical recording part 1 through scanning is transmitted to the electrical processing part 2 through a data line for processing.

[0046] For the optical recording part 1, in order to make the optical path more stable, simple and specific, a multi-axis cage structure is used to build the optical path to realize an integrated optical microscope system. The construction concept of this structure is different from that of the optical path construction in free space. First, it is necessary to fix the fixed support frame on the optical platform, and then pass the cage rod through the hole of the base at the required height, and then insert the sliding frame of the lens and other original components required in the system into the cage rod in turn, and finally fix the second fixed support frame at the other end of the cage rod. Since the height of the cage rod is fixed and the original sliding frame is coaxial with the optical axis of the system, this structure has very high stability and accuracy compared with the optical path construction in free space, and also reduces the difficulty of optical path calibration. On the basis of the cage structure, combined with Figure 1As shown, the heterodyne scanning beam generating device 3 emits two time-varying annular scanning beams with the same heterodyne signal, the first aperture 9 is placed between the first annular scanning beam and the first photodetector 10; the first lens 4 is placed behind the second annular scanning beam, the first beam splitter 5 is placed between the first lens 4 and the microscope objective 6, and the first lens 4 is confocal with the microscope objective 6; the object 7 to be scanned is placed on the piezoelectric displacement stage 8, and the piezoelectric displacement stage 8 is placed behind the microscope objective 6; the second photodetector 11 is placed in the direction of the reflected light from the microscope objective 6 by the first beam splitter 5.

[0047] For the heterodyne scanning beam generating device, plane wave and spherical wave interference are used, and the two lights are modulated at different frequencies to form a time-varying annular scanning beam; or a spatial light modulator is used to quickly play different annular beam patterns to achieve the effect of a time-varying annular scanning beam.

[0048] like Figure 1 As shown, the electrical processing part 2 is connected to the optical recording part 1 through the first photodetector 10 and the second photodetector 11. The electrical processing part 2 is used to convert the optical information obtained by the optical recording part 1 into an electrical signal, and collect, transmit and process the electrical signal; the output end of the first photodetector 10 is connected to the reference signal end of the phase-locked amplifier 13; the output end of the second photodetector 11 is connected to the bandpass filter 12; the bandpass filter 12 is directly connected to the input signal end of the phase-locked amplifier 13; the output signal end of the phase-locked amplifier 13 is closely connected to the data acquisition card 14; the data acquisition card 14 is connected to the computer 15.

[0049] Example 2

[0050] A micro-holographic data acquisition technology based on optical scanning, specifically comprising the following steps:

[0051] Step 1: The heterodyne scanning beam generating device 3 emits two time-varying annular scanning beams with the same heterodyne signal, wherein the first annular scanning beam passes through the first aperture to obtain a light spot with a single difference frequency variation, which is received by the first photodetector and converted into a difference frequency electrical signal, which is connected to the reference signal end of the phase-locked amplifier via a coaxial cable, and the second annular scanning beam is used to scan the object.

[0052] Step 2: The scanning light beam compresses the diameter of the optical path through the first lens 4 so that the diameter of the light spot can just fill the light aperture of the microscope objective 6, and then continues to further compress the diameter of the optical path through the microscope objective 6. After two steps of compression, the diameter of the annular scanning light beam is compressed to a width that cannot be distinguished by the naked eye; the piezoelectric displacement stage 8 is moved by the scanning controller to realize two-dimensional scanning of the object 7 to be scanned placed on the piezoelectric displacement stage 8 by the scanning light beam, and the reflected light carrying the three-dimensional information of the object obtained by the scanning is magnified by the microscope objective 6 again, and is received by the second photodetector 11 through the reflection surface of the first beam splitter 5.

[0053] Step 3: The second photodetector 11 converts the light intensity information carrying the three-dimensional information of the object into a current signal. The current signal is connected to the bandpass filter 12 and then filtered out by the phase-locked amplifier 13 to obtain a difference frequency current signal. The signal is then transmitted to the data acquisition card 14. The computer 15 can observe the current signal of the object to be scanned 7 on the moving piezoelectric displacement stage 8 in real time through the data acquisition card 14, and obtain a three-dimensional hologram of the object to be scanned 7 after the scanning is completed.

[0054] Step 4: Based on the hologram reconstruction procedure, the obtained hologram is reconstructed to obtain the three-dimensional information of the high-resolution biological specimen.

[0055] Example 3

[0056] A first embodiment of the heterodyne scanning beam generating device 3 is as follows: Figure 3 shown.

[0057] The reference numerals in the accompanying drawings respectively represent: laser 16, first reflector 17, second reflector 18, attenuation plate 19, second beam splitter 20, first acousto-optic frequency shifter 21, third reflector 22, second lens 23, first pinhole 24, third lens 25, second acousto-optic frequency shifter 26, fourth reflector 27, fourth lens 28, second pinhole 29, fifth lens 30, sixth lens 31, third beam splitter 32;

[0058] The parallel light emitted by the laser 16 is collimated by the first reflector 17 and the second reflector 18, and then the light intensity is adjusted by the attenuation plate 19, and then it is divided into two paths by the second beam splitter 20. The reflected light path passes through the first acousto-optic frequency shifter 21 and the third reflector 22, and then is expanded by the second lens 23, the first pinhole 24, and the third lens 25 to generate a plane wave; the transmitted light path passes through the second acousto-optic frequency shifter 26 and the fourth reflector 27, and then is expanded by the fourth lens 28, the second pinhole 29, and the fifth lens 30 to generate a plane wave, and then passes through the sixth lens 31 to generate a spherical wave; the two light paths converge at the third beam splitter 32 to interfere and form a time-varying annular scanning beam, and the time-varying annular scanning beam on one side of the third beam splitter 32 is used to scan the object, and the other side is used as a reference signal.

[0059] Furthermore, in this embodiment, the intensity S(x, y, t; z) of the time-varying annular scanning beam formed by the heterodyne scanning beam generating device when it hits the surface of the object is obtained by the interference of the spherical wave and the plane wave.

[0060] Example 4

[0061] A second embodiment of the heterodyne scanning beam generating device 3 is as follows: Figure 4 shown.

[0062] The numbers in the accompanying drawings respectively represent: laser 16, first reflector 17, second reflector 18, attenuation plate 19, second beam splitter 20, first acousto-optic frequency shifter 21, third reflector 22, second lens 23, first pinhole 24, third lens 25, second acousto-optic frequency shifter 26, fourth reflector 27, fourth lens 28, second pinhole 29, fifth lens 30, sixth lens 31, third beam splitter 32; fourth beam splitter 33; fifth reflector 34.

[0063] The parallel light emitted by the laser 16 is adjusted in intensity by the attenuation plate 19, collimated by the first reflector 17 and the second reflector 18, and then divided into two paths by the second beam splitter 20. The reflected light path passes through the first acousto-optic frequency shifter 21 and the third reflector 22, returns to the original path and passes through the first acousto-optic frequency shifter 21 again, and then transmits through the second beam splitter 20, and is expanded by the second lens 23, the first pinhole 24, and the third lens 25 to generate a plane wave; the transmitted light path is transmitted through the third beam splitter 32, and then passes through the second acousto-optic frequency shifter 26, the fourth reflector 27, and the second acousto-optic frequency shifter 28. After the projection mirror 27, it returns to the original path and passes through the second acousto-optic frequency shifter 26 again, and then reflects through the third beam splitter 32, and is expanded by the fourth lens 28, the second pinhole 29, and the fifth lens 30 to generate a plane wave, and then passes through the sixth lens 31 to generate a spherical wave, and then is reflected by the fifth reflector 34 vertically into the fourth beam splitter 33; the two light paths converge at the fourth beam splitter 33 and interfere to form a time-varying annular scanning beam. The time-varying annular scanning beam on one side of the fourth beam splitter 33 is used to scan the object, and the other side is used as a reference signal.

[0064] Furthermore, in this embodiment, the intensity S(x, y, t; z) of the time-varying annular scanning beam formed by the heterodyne scanning beam generating device when it hits the surface of the object is obtained by the interference of the spherical wave and the plane wave.

[0065] Example 5

[0066] The third embodiment of the heterodyne scanning beam generating device 3 is as follows: Figure 5 shown.

[0067] The numbers in the accompanying drawings respectively represent: laser 16, first reflector 17, second reflector 18, attenuation plate 19, second beam splitter 20, second lens 23, first pinhole 24, third lens 25, sixth lens 31, third beam splitter 32, spatial light modulator 35, second aperture 36.

[0068] The parallel light emitted by the laser 16 is adjusted in intensity by the attenuation plate 19 and then collimated by the first reflector 17 and the second reflector 18, and then expanded by the second lens 23, the first pinhole 24, and the third lens 25 to generate a plane wave; the plane wave is reflected by the second beam splitter 20 and enters the spatial light modulator 35, and the reflected light of the spatial light modulator 35 irradiated by the plane wave is a fast playback of different annular beam patterns, so as to achieve the effect of a time-varying annular scanning beam; the scanning beam is reflected by the spatial light modulator 35 and transmitted through the second beam splitter 20, and then through the sixth lens 31, and the second aperture 36 is located at the focus of the sixth lens 31, so as to achieve the effect of filtering out stray light; after the third beam splitter 32 is placed on the second aperture 36, the time-varying annular scanning beam on one side of the third beam splitter 32 is used to scan the object, and the other side is used as a reference signal.

[0069] Furthermore, in this embodiment, the intensity S(x, y; z) of the time-varying annular scanning beam formed by the heterodyne scanning beam generating device when it hits the surface of the object is directly generated by a computer, rather than being obtained through the interference of spherical waves and plane waves.

Claims

1. A microscopic holographic data acquisition device based on optical scanning, characterized in that: include: Optical recording part (1), electrical processing part (2); The optical recording part (1) comprises: a heterodyne scanning beam generating device (3), a first lens (4), a first beam splitter (5), a microscope objective lens (6), an object (7), a piezoelectric displacement stage (8), a first aperture (9), a first photodetector (10), and a second photodetector (11); The heterodyne scanning beam generating device (3) emits two time-varying annular scanning beams having the same heterodyne signal, wherein a first aperture (9) and a first photodetector (10) are sequentially arranged on the first annular scanning beam; a first lens (4), a first beam splitter (5), a microscope objective lens (6) and a piezoelectric displacement stage (8) are sequentially arranged on the second annular scanning beam, and the first lens (4) and the microscope objective lens (6) are confocal; an object (7) to be scanned is placed on the piezoelectric displacement stage (8); and a second photodetector (11) is placed in the direction of the reflected light from the microscope objective lens (6) of the first beam splitter (5); The first photodetector (10) and the second photodetector (11) of the optical recording part (1) are respectively connected to the electrical processing part (2), and the electrical processing part (2) is used to convert the optical information obtained by the optical recording part (1) into an electrical signal, and collect, transmit and process the electrical signal; the electrical processing part (2) includes a bandpass filter (12), a phase-locked amplifier (13), a data acquisition card (14), and a computer (15).

2. The optical scanning-based micro-holographic data acquisition device according to claim 1, characterized in that: The output end of the first photodetector (10) is connected to the reference signal end of the phase-locked amplifier (13); the output end of the second photodetector (11) is connected to the bandpass filter (12); the bandpass filter (12) is directly connected to the input signal end of the phase-locked amplifier (13); the output signal end of the phase-locked amplifier (13) is closely connected to the data acquisition card (14); and the data acquisition card (14) is connected to the computer (15).

3. The optical scanning-based micro-holographic data acquisition device according to claim 1, characterized in that: The heterodyne scanning beam generating device (3) comprises: a laser (16), a first reflector (17), a second reflector (18), an attenuation plate (19), a second beam splitter (20), a first acousto-optic frequency shifter (21), a third reflector (22), a second lens (23), a first pinhole (24), a third lens (25), a second acousto-optic frequency shifter (26), a fourth reflector (27), a fourth lens (28), a second pinhole (29), a fifth lens (30), a sixth lens (31), and a third beam splitter (32); The light emitted by the laser (16) passes through a first reflector (17) and a second reflector (18) and then passes through an attenuation plate (19) to adjust the light intensity, and is kept coaxial with the center of the second beam splitter (20). The first reflector (17) is placed in the emission direction of the laser (16); the first acousto-optic frequency shifter (21) is placed in the direction of the light reflected by the second beam splitter (20), and is placed between the third reflector (22) and the second beam splitter (20); the third reflector (22) is placed in the direction of the first-order diffracted light of the first acousto-optic frequency shifter (21), and is angularly deflected compared with the direction of the zero-order light; the second lens (23) is placed in the direction of the light reflected by the third reflector (22), and the second lens (23) and the third lens (25) are cofocal at the same time, and the first pinhole (24) is placed at the focus of the second lens (23) and the third lens (25), and the optical axis direction of the light reflected by the third reflector (22) is aligned with the second lens (23) and the third lens (25). The centers of the first pinhole (24) and the third lens (25) are kept consistent; the second acousto-optic frequency shifter (26) is placed in the direction of the transmitted light of the second beam splitter (20); the fourth reflector (27) is placed in the direction of the first-order diffracted light of the second acousto-optic frequency shifter (26), and is deflected at an angle compared with the direction of the zero-order light; the fourth lens (28) is placed in the direction of the reflected light of the fourth reflector (27), and the fourth lens (28) and the fifth lens (30) are cofocal, the second pinhole (29) is placed at the focus of the fourth lens (28) and the fifth lens (30), and the optical axis direction of the reflected light of the fourth reflector (27) is kept consistent with the centers of the fourth lens (28), the second pinhole (29) and the fifth lens (30); the sixth lens (31) is placed behind the fifth lens (30); and the third beam splitter (32) is placed at the coaxial intersection of the transmitted light of the sixth lens (31) and the transmitted light of the third lens (25).

4. The optical scanning-based micro-holographic data acquisition device according to claim 1, characterized in that: The heterodyne scanning beam generating device (3) comprises: a laser (16), a first reflector (17), a second reflector (18), an attenuation plate (19), a second beam splitter (20), a first acousto-optic frequency shifter (21), a third reflector (22), a second lens (23), a first pinhole (24), a third lens (25), a second acousto-optic frequency shifter (26), a fourth reflector (27), a fourth lens (28), a second pinhole (29), a fifth lens (30), a sixth lens (31), a third beam splitter (32); a fourth beam splitter (33); and a fifth reflector (34); The light emitted from the laser (16) is adjusted in intensity by an attenuation plate (19) and then passes through a first reflector (17) and a second reflector (18), and is kept coaxial with the center of a second beam splitter (20). The first reflector (17) is arranged in the emission direction of the laser (16); a first acousto-optic frequency shifter (21) is arranged in the direction of the reflected light of the second beam splitter (20); a third reflector (22) is arranged in a direction perpendicular to the first order diffracted light of the first acousto-optic frequency shifter (21), and is deflected at an angle compared to the direction of the zero order light; a second lens (23) is arranged in the first direction of the diffracted light of the first acousto-optic frequency shifter (21). The vertically reflected light of the three reflectors (22) is transmitted through the first acousto-optic frequency shifter (21) and deflected in the direction thereof; the second lens (23) and the third lens (25) are co-focal, the first pinhole (24) is placed at the focal point of the second lens (23) and the third lens (25), and the optical axis direction of the vertically reflected light of the third reflector (22) after being transmitted through the first acousto-optic frequency shifter (21) is consistent with the center of the two-beam splitter (20), the second lens (23), the first pinhole (24) and the third lens (25); the second The acousto-optic frequency shifter (26) is arranged in the direction of the transmitted light of the second beam splitter (20); the fourth reflector (27) is arranged in the vertical direction of the first-order diffracted light of the second acousto-optic frequency shifter (26), and is deflected at an angle compared with the direction of the zero-order light; the third beam splitter (32) is arranged between the second beam splitter (20) and the second acousto-optic frequency shifter (26); the fourth lens (28) is arranged in the direction of the reflected light of the third beam splitter (32) through the second acousto-optic frequency shifter (26), and the fourth lens (28) is co-focal with the fifth lens (30), and the second lens (28) is co-focal with the fifth lens (30). The hole (29) is placed at the focal point of the fourth lens (28) and the fifth lens (30), and the optical axis direction of the reflected light of the third beam splitter (32) is consistent with the center of the fourth lens (28), the second pinhole (29), and the fifth lens (30); the sixth lens (31) is placed behind the fifth lens (30); the fifth reflector (34) is placed behind the sixth lens (31), and the fourth beam splitter (33) is placed at the coaxial intersection of the light reflected by the sixth lens (31) through the fifth reflector (34) and the light transmitted by the third lens (25).

5. The optical scanning-based micro-holographic data acquisition device according to claim 1, characterized in that: The heterodyne scanning beam generating device (3) comprises: a laser (16), a first reflector (17), a second reflector (18), an attenuation plate (19), a second beam splitter (20), a second lens (23), a first pinhole (24), a third lens (25), a sixth lens (31), a third beam splitter (32), a spatial light modulator (35), and a second aperture (36); The light emitted by the laser (16) is adjusted in intensity by an attenuation plate (19) and then passes through a first reflector (17), a second reflector (18), and is kept coaxial with the center of a second beam splitter (20). The first reflector (17) is placed in the direction of the laser (16) emission; the second lens (23) is placed in the direction of the light reflected by the second reflector (18), and the second lens (23) and the third lens (25) are cofocal at the same time. The first pinhole (24) is placed at the focal point of the second lens (23) and the third lens (25), and the second reflector (18 ) The optical axis direction of the reflected light is consistent with the center of the second lens (23), the first pinhole (24) and the third lens (25); the second beam splitter (20) is placed behind the third lens (25), and the spatial light modulator (35) is placed in the direction of the reflected light of the second beam splitter (20); the sixth lens (31) is placed in the direction of the reflected light of the spatial light modulator and transmits through the second beam splitter (20); the second diaphragm (36) is placed at the focal position of the sixth lens (31), and the third beam splitter (32) is placed behind the second diaphragm (36).

6. A micro-holographic data acquisition method based on optical scanning, characterized in that: A microscopic holographic data acquisition device based on optical scanning according to any one of claims 1 to 5 is used, comprising the following steps: Step 1: In the optical recording part (1), the heterodyne scanning beam generating device (3) emits two time-varying annular scanning beams having the same heterodyne signal, wherein the first annular scanning beam is used to scan the object, and the second annular scanning beam passes through the first aperture (9) to obtain a single light spot with a difference frequency variation, and the light spot is received by the first photodetector (10) and converted into a difference frequency electrical signal, which is connected to the reference signal terminal of the phase-locked amplifier (13) via a coaxial cable; Step 2: The scanning light beam compresses the diameter of the optical path through the first lens (4) so ​​that the diameter of the light spot just fills the aperture of the microscope objective lens (6), and then continues to further compress the diameter of the optical path through the microscope objective lens (6). After two steps of compression, the diameter of the annular scanning light beam is compressed to a width that cannot be distinguished by the naked eye; the piezoelectric displacement stage (8) is moved by the scanning controller to achieve two-dimensional scanning of the object to be scanned (7) placed on the piezoelectric displacement stage (8), and the reflected light carrying the three-dimensional information of the object obtained by the scanning is magnified by the microscope objective lens (6) again, and is received by the second photoelectric detector (11) through the reflection surface of the first beam splitter (5); Step 3: The second photodetector (11) converts the light intensity information carrying the three-dimensional information of the object into a current signal. The current signal is connected to a bandpass filter (12) and then filtered out by a phase-locked amplifier (13) to obtain a difference frequency current signal. The signal is then transmitted to a data acquisition card (14). The computer (15) observes the current signal of the object to be scanned (7) on the moving piezoelectric displacement stage (8) in real time through the data acquisition card (14), and obtains a three-dimensional hologram of the object to be scanned (7) after the scanning is completed. Step 4: Based on the hologram reconstruction procedure, the obtained hologram is reconstructed to obtain the three-dimensional information of the high-resolution biological specimen.

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