Interference microscopic imaging method and interference microscope

Abstract

The present invention provides an interference microscopic imaging method and an interference microscope, specifically, an interference microscope, comprising a light source generating device, a compensating interference cavity, a detection arm, and a signal acquisition and processing unit; the compensating interference cavity receives the light beam generated by the light source generating device and reflects it to become a reflected light beam entering the detection arm; the detection arm focuses the reflected light beam to form sample light, and sends the sample light to the signal acquisition and processing unit; wherein the detection unit is provided with a second microscope objective lens, and the second microscope objective lens is used to focus the light on the sample and modulate the structural information of the sample into light to return. The interference microscope, by inserting the first microscope objective lens in the compensating interference cavity, makes the reference light and the sample light pass through the same microscope objective lens but in a different order, thereby ensuring that the optical path of the sample light and the reference light is completely equal, and avoiding the unequal optical path of some points on the interference surface caused by the inconsistency of passing through the optical device.

Description

Technical Field

[0001] The invention belongs to an interference microscopic imaging technology, and in particular relates to an interference microscopic imaging method and an interference microscope. Background Art

[0002] Interference microscope is a microscope that uses coherent light interference technology. Compared with traditional microscopes, it can see the structural distribution of transparent sample tissues. It uses the interference principle to convert the phase difference distribution into intensity distribution for naked eye observation. The low coherence of the wide spectrum light source can also achieve optical sectioning, which only images the sample slices within a specific optical path range. The thickness of the slice depends on the temporal coherence length of the light source. Microscopes using low coherence interference technology have been applied to integrated circuit microscopic detection, clinical pathology diagnosis and other fields, and have the advantages of non-contact and non-destructive detection.

[0003] The current low-coherence interference microscope (patent 201680034161.X) mostly adopts the Michelson interference structure, inserting the microscope between the reference arm and the sample arm. This structure belongs to the split light path structure. When the two arms are far apart, different phase changes will be formed under environmental vibration, which seriously affects the normal interference pattern acquisition, and thus it is impossible to obtain a high-quality sample structure diagram. In practice, an air-floating platform is often added to filter out environmental interference, so it is bulky and inconvenient to use, which limits its actual practical scenarios. There is also a common light path interference system using Mirau objective lens, but when the numerical aperture is large, the beam splitter inside it will cause a large aberration (Stanley Siu-chor Chim, GS Kino. Correlation microscope [J]. Opt. Lett., 1990, 15 (10): 579-581.), which brings great difficulties to the structural design of Mirau objective lens.

[0004] The interference system with a common optical path structure has the advantage of anti-interference structure because the reference light and the sample light are transmitted in the same channel. The phase change caused by external interference will not change the phase difference, so the interference pattern is stable. In addition to the common optical path design using Mirau objective lens mentioned above, there are also some common optical path designs using two interferometers in series (such as Benoita laGuillaume E, Martins F, Boccara C, et al. High-resolution handheld rigidendomicroscope based on full-field optical coherence tomography [J]. Journal of biomedical optics). In these designs, either the numerical aperture of the objective lens cannot be made too large, or the reference optical path and the sample optical path cannot be completely symmetrical, resulting in additional optical path difference.

[0005] In order to improve some defects of current interference microscopes, the present invention proposes a completely symmetrical optical path design, in which the reference light and the sample light pass through exactly the same path, but in a different order, eliminating the additional optical path difference caused by the system structure. At the same time, the numerical aperture is not limited and is insensitive to environmental vibrations. Summary of the invention

[0006] In view of this, one of the objects of the present invention is to provide an interference microscope, which ensures that the optical paths of sample light and reference light are completely equal by inserting a first microscope objective lens into a compensating interference cavity, thereby avoiding unequal optical paths at some points on the interference surface caused by inconsistencies in passing through optical devices.

[0007] To achieve the above object, the technical solution of the present invention is:

[0008] An interference microscope comprises a light source generating device, a compensating interference cavity, a detection arm, and a signal acquisition and processing unit;

[0009] The compensation interference cavity receives the light beam generated by the light source generating device and reflects it to become a reflected light beam entering the detection arm;

[0010] The detection arm focuses the reflected light beam to form sample light, and sends the sample light to the signal acquisition and processing unit; wherein the detection unit is provided with a second microscope objective lens, and the second microscope objective lens is used to focus the light on the sample and modulate the structural information of the sample into light to be returned.

[0011] Furthermore, the light source generating device includes a light emitting device with a spectrum width and a first lens.

[0012] The light source generating device comprises a light emitting device with a spectrum width and a first lens; wherein,

[0013] The compensating interference cavity includes a first beam splitter, a first reflector, a second reflector, and a first microscope objective lens; the first microscope objective lens is located between the first beam splitter and the second reflector, the second reflector is located at the focal plane of the first microscope objective lens, the main optical axis of the first microscope objective lens forms an angle of 40°-50° with the surface of the first beam splitter, and the virtual image formed by the first reflector through the first beam splitter is parallel to the second reflector, and the first reflector and the second reflector are respectively located on both sides of the first beam splitter.

[0014] Furthermore, the detection arm includes a second lens, a second beam splitter, a third beam splitter and a second microscope objective lens; wherein the third beam splitter is perpendicular to the main optical axis of the second microscope objective lens, the second beam splitter is at an angle of 40°-50° to the main optical axis of the second microscope objective lens and is at an angle of 80°-100° to the first beam splitter in the compensating interference cavity.

[0015] Furthermore, the rear end faces of the second beam splitter and the third beam splitter are both semi-transparent films with a reflectivity of 5%-15%, and the front end faces are anti-reflection films.

[0016] Furthermore, the signal acquisition and processing unit includes a photoelectric sensor and a signal processor, the signal processor is electrically connected to the photoelectric sensor, and the sample light reaches the photoelectric sensor through the second lens.

[0017] Furthermore, the photoelectric sensor includes an array photoelectric sensor and a planar array photoelectric sensor.

[0018] Furthermore, the photoelectric sensor array is conjugated with the sample through the second lens and the second microscope objective lens in the detection arm, and is also conjugated with the second reflector through the second lens in the detection arm and the first microscope objective lens in the compensation interference cavity.

[0019] In view of this, a second object of the present invention is to provide an interference microscopy imaging method, which ensures that the optical path lengths of the sample light and the reference light are completely equal by adjusting the reference light and the sample light to pass through exactly the same microscope objective lens but in a different order, thereby avoiding unequal optical path lengths at some points on the interference surface due to inconsistencies in passing through optical devices, and ultimately obtaining a high-precision three-dimensional structure image of the sample's interior.

[0020] To achieve the above object, the technical solution of the present invention is:

[0021] An interference microscopy imaging method comprises the following steps:

[0022] Receive a light beam generated by a light source generating device, the light beam is divided into two light beams by a first beam splitter, one of which is reflected by a first reflector and then transmitted by the first beam splitter, and the other is reflected by a second reflector after passing through a first objective lens;

[0023] The two beams reach the second beam splitter, are reflected by the second beam splitter to the third beam splitter, and then a part of the beam is reflected by the third beam splitter to become reference light, and the other part is transmitted by the third beam splitter, and the transmitted beam is focused on the sample by the second microscope objective lens and modulated by the sample to become sample light and reflected back to the second microscope objective lens;

[0024] The sample light and the reference light reach the front of the photoelectric sensor through the second beam splitter and the second lens;

[0025] The signal processor collects the electrical signal from the photoelectric sensor to obtain the structural information of the sample section.

[0026] Furthermore, the sample light carries result information of the sample.

[0027] Furthermore, the step of allowing the sample light and the reference light to reach the front of the photoelectric sensor through the second beam splitter and the second lens specifically includes:

[0028] Adjusting the position of the first reflector so that the optical path difference between the sample light and the reference light is within the range of the light source coherence length;

[0029] The sample light and the reference light arrive at the surface of the photoelectric sensor to form interference fringes.

[0030] Furthermore, the method further comprises the steps of:

[0031] The position of the sample is moved, and the signal processor collects the interference fringes multiple times to form a three-dimensional internal structure diagram of the sample.

[0032] Beneficial Effects

[0033] The invention provides an interference microscope. The first microscope objective lens is inserted into a compensation interference cavity, so that the reference light and the sample light pass through the completely identical optical elements but in different orders, wherein the sample light first passes through a reflector and then passes through a second microscope objective lens in a detection arm and then reaches the sample, and the reference light first passes through the second microscope objective lens in the detection arm and then passes through a reflector and then is reflected by a beam splitter. The use of two completely identical microscope objective lenses can ensure that the optical paths of the two paths of light are completely equal, thereby avoiding the problem that not every point on the interference surface has the same optical path due to the inconsistency of passing through optical devices. At the same time, the invention also provides an interference microscopic imaging method, wherein the reference light and the sample light are both passed through the completely identical microscope objective lens but in different orders, thereby ensuring that the optical paths of the sample light and the reference light are completely equal, thereby obtaining a high-precision three-dimensional structure diagram of the internal structure of the sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the prior art descriptions. Obviously, the drawings described below are some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor.

[0035] Figure 1 It is a schematic structural diagram of an embodiment of an interference microscope of the present invention;

[0036] Figure 2 The figure is a schematic structural diagram of another embodiment of an interference microscope of the present invention. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0039] Example 1

[0040] refer to Figure 1 , which is a schematic diagram of the structure of an interference microscope according to an embodiment of the present invention, specifically, an interference microscope comprises:

[0041] Light source generating device, compensating interference cavity, detection arm, signal acquisition and processing unit; wherein,

[0042] The light source generating device includes a light emitting device S with a spectrum width and a first lens L1. In this embodiment, the light emitting device S adopts an LED light source with a central wavelength λ0=850nm and a spectrum width Δλ=30nm. The light source becomes quasi-parallel light after being collimated by the first lens L1.

[0043] The compensating interference cavity comprises a first beam splitter BS1, a first reflector M1, a second reflector M2, and a first microscope objective lens MS1. The first microscope objective lens MS1 is located between the first beam splitter BS1 and the second reflector M2, and the second reflector M2 is located at the focal plane of the first microscope objective lens MS1. The main optical axis of the first microscope objective lens MS1 forms an angle of 45° with the surface of the first beam splitter BS1. In other embodiments, the angle between the main optical axis of the first microscope objective lens MS1 and the surface of the first beam splitter BS1 can be in the range of 40°-50°, and the virtual image formed by the first reflector M1 through the first beam splitter BS1 is parallel to the second reflector M2. The first reflector M1 and the second reflector M2 are located on both sides of the first beam splitter BS1, respectively. The first reflector M1 is also fixedly connected to the piezoelectric ceramic PZT. In this embodiment, the first reflector M1 and the second reflector M2 are both total reflectors.

[0044] The detection arm includes a second lens L2, a second beam splitter BS2, a third beam splitter BS3, and a second microscope objective lens MS2. The rear end faces of the second beam splitter BS2 and the third beam splitter BS3 are semi-transparent films with a reflectivity of 5%-15%, and the front end faces are anti-reflection films. The third beam splitter BS3 is perpendicular to the main optical axis of the second microscope objective lens MS2. The second beam splitter BS2 is at an angle of 45° to the main optical axis of the second microscope objective lens MS2 and is at an angle of 80°-100° to the first beam splitter BS1 in the compensation interference cavity.

[0045] The signal acquisition and processing unit includes an array photoelectric sensor CAM and a signal processor PS. The array surface of the photoelectric sensor CAM is conjugated with the sample Sample through the second lens L2 and the microscope objective lens MS2 in the detection arm, and is also conjugated with the second reflector M2 through the second lens L2 in the detection arm and the first microscope objective lens MS1 in the compensation interference cavity. Therefore, the sample and the second reflector M2 are both imaged on the surface of the photoelectric sensor. By adjusting the position of the first reflector M1 relative to the first beam splitter BS1, it can be ensured that the second reflector surface M2 and the sample Sample are imaged on the array surface of the photoelectric sensor CAM through the same optical path. Since the light beams involved in the imaging are all from the same light beam, they will form interference fringes on the array surface of the sensor CAM. The signal processor PS is electrically connected to the sensor CAM, and the signal processor PS is also connected to the electric ceramic PZT.

[0046] refer to Figure 1 As indicated by the arrow in FIG. 1 , an interference microscopy imaging method in this embodiment is:

[0047] The wide spectrum light source emitted by the light emitting device S is collimated by the first lens L1 and then split into two light beams by the first beam splitter BS1 in the compensating interference cavity. One beam is reflected by the first reflector M1 and then transmitted through the first beam splitter BS1, and the other beam passes through the first microscope objective MS1 and then is reflected by the second reflector M2 and then emitted through the first beam splitter BS1.

[0048] After entering the detection arm, the two beams of light coming out of the compensation interference cavity are reflected by the second beam splitter BS2 in the detection arm to the third beam splitter BS3, and then are reflected by the third beam splitter BS3 with a reflectivity of 10% and a transmittance of 90% (the reflectivity of the third beam splitter BS3 in a specific embodiment is set to be 10%). The transmitted light is focused on the sample by the second microscope objective MS2 and modulated by the sample, then reflected back to the second microscope objective MS2 and combined with the third beam splitter BS3 with a transmittance of 10%. The reflected light converges at the third beam splitter BS3 to form four light beams (the four light beams include two light beams reflected by the third beam splitter BS3 at 10% and two light beams reflected by the second microscope MS2 through the sample). The four light beams then pass through the same path, the second beam splitter BS2 and the second lens L2 to reach the array surface of the sensor CAM. Among the four light beams, two of them carry the structural information of the sample and become sample light (the sample light reaches the sample through the second microscope MS2 and is reflected after being adjusted by the sample), and the other two do not have the structural information of the sample and become reference light (the reference light is the light reflected by the third beam splitter BS3). One of the sample light and the reference light both pass through the microscope once. By adjusting the position of the reference mirror (reflector M1) in the compensating interference cavity, it can be ensured that the optical path difference between the sample light and the reference light is within the coherence length of the light source, thereby forming interference fringes on the surface of the photoelectric sensor, and the other two light beams form background light superimposed on the interference fringes. The signal processor PS collects electrical signals from the photoelectric sensor. Each time an interference pattern is collected, the voltage loaded on the PZT is controlled to drive the first reflector M1 to perform a small displacement, and the displacement range shall not exceed one wavelength. The displacement of the first reflector M1 will change the optical path difference between the sample light and the reference light involved in the interference, thereby moving the interference fringes on the sensor CAM array surface. After collecting multiple interference images, the reflectivity of the section perpendicular to the optical axis in the sample can be restored through numerical fitting, and the structural information of the section can be obtained. By moving the sample to scan the internal section, the obtained tomographic image (section structure information diagram) can be displayed on the display MN, and finally a three-dimensional structure diagram of the sample is formed. The interference microscope in this embodiment can also be applied to tissue live detection.

[0049] An interference microscope in the present embodiment adopts a common optical path design, and the detection arm can be designed to be very long and used for real-time in vivo endoscopy detection without the need for tissue biopsy sampling; thanks to the first microscope objective lens MS1 inserted in the compensating interference cavity, the reference light and the sample light pass through exactly the same optical elements but in a different order, wherein the sample light first passes through the first reflector M1 and then through the second microscope objective lens MS2 before being reflected by the sample, and the reference light first passes through the microscope objective lens MS1 and then through the second reflector M2 and then is reflected by the third beam splitter BS3. The use of two exactly the same microscope objective lenses can ensure that the optical path lengths of the two light paths are completely equal, thereby avoiding the problem that not every point on the interference surface has equal optical path lengths due to inconsistencies in passing through optical devices.

[0050] Example 2

[0051] like Figure 2 As shown, this embodiment uses the same compensating interference cavity as in Embodiment 1, except that an image transmission unit cell lens rod or an image transmission fiber bundle (FB) is inserted into the detection arm. Both the reference light and the sample light are transmitted in the image transmission unit, and the optical path difference will not be changed, so that endoscopic interference imaging can be achieved. The specific imaging process is consistent with that in Embodiment 1 and will not be repeated here.

[0052] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. An interference microscope, characterized in that: It includes a light source generating device, a compensating interference cavity, a detection arm, and a signal acquisition and processing unit; The compensation interference cavity receives the light beam generated by the light source generating device and reflects it to become a reflected light beam entering the detection arm; The detection arm is used to focus the reflected light beam to form sample light, and send the sample light to the signal acquisition and processing unit; wherein the detection arm is provided with a second microscope objective lens, and the second microscope objective lens is used to focus the light on the sample and modulate the structural information of the sample into light to be returned; The light source generating device comprises a light emitting device with a spectrum width and a first lens, and the compensating interference cavity comprises a first beam splitter, a first reflector, a second reflector, and a first microscope objective lens; wherein, The first microscope objective is located between the first beam splitter and the second reflector, the second reflector is located at the focal plane of the first microscope objective, the main optical axis of the first microscope objective forms an angle of 40°-50° with the surface of the first beam splitter, and the virtual image of the first reflector formed by the first beam splitter is parallel to the second reflector, and the first reflector and the second reflector are located on both sides of the first beam splitter respectively; The detection arm comprises a second lens, a second beam splitter, a third beam splitter and a second microscope objective lens; wherein the third beam splitter is perpendicular to the main optical axis of the second microscope objective lens, the second beam splitter is at an angle of 40°-50° to the main optical axis of the second microscope objective lens and is at an angle of 80°-100° to the first beam splitter in the compensation interference cavity; The rear end faces of the second beam splitter and the third beam splitter are both semi-transparent films with a reflectivity of 5%-15%, and the front end faces are anti-reflection films.

2. An interference microscope according to claim 1, characterized in that: The signal acquisition and processing unit includes a photoelectric sensor and a signal processor. The signal processor is electrically connected to the photoelectric sensor, and the sample light reaches the photoelectric sensor through the second lens.

3. An interference microscope according to claim 2, characterized in that: The photoelectric sensor array is conjugated with the sample through the second lens and the second microscope objective lens in the detection arm, and is also conjugated with the second reflector through the second lens in the detection arm and the first microscope objective lens in the compensation interference cavity.

4. An interference microscopic imaging method based on the interference microscope according to claim 1, characterized in that: The following steps are involved: Receive a light beam generated by a light source generating device, the light beam is divided into two light beams by a first beam splitter, one of which is reflected by a first reflector and then transmitted by the first beam splitter, and the other is reflected by a second reflector after passing through a first objective lens; The two beams reach the second beam splitter, are reflected by the second beam splitter to the third beam splitter, and then a part of the beam is reflected by the third beam splitter to become reference light, and the other part is transmitted by the third beam splitter, and the transmitted beam is focused on the sample by the second microscope objective lens and modulated by the sample to become sample light and reflected back to the second microscope objective lens; The sample light and the reference light reach the front of the photoelectric sensor through the second beam splitter and the second lens; The signal processor collects the electrical signal from the photoelectric sensor to obtain the structural information of the sample section.

5. The interference microscopy imaging method according to claim 4, characterized in that: The sample light carries the result information of the sample.

6. The interference microscopy imaging method according to claim 5, characterized in that: The step of allowing the sample light and the reference light to reach the front of the photoelectric sensor through the second beam splitter and the second lens specifically includes: Adjusting the position of the first reflector so that the optical path difference between the sample light and the reference light is within the range of the light source coherence length; The sample light and the reference light arrive at the surface of the photoelectric sensor to form interference fringes.

7. An interference microscopy imaging method according to claim 6, characterized in that: Also includes the steps: The position of the sample is moved, and the signal processor collects the interference fringes multiple times to form a three-dimensional internal structure diagram of the sample.

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