CN111474141A - Interference microscopic imaging method and interference microscope - Google Patents
Interference microscopic imaging method and interference microscope Download PDFInfo
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- CN111474141A CN111474141A CN202010431543.4A CN202010431543A CN111474141A CN 111474141 A CN111474141 A CN 111474141A CN 202010431543 A CN202010431543 A CN 202010431543A CN 111474141 A CN111474141 A CN 111474141A
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- 238000003384 imaging method Methods 0.000 title claims abstract description 16
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- 239000000523 sample Substances 0.000 claims description 85
- 238000010586 diagram Methods 0.000 claims description 8
- 238000000386 microscopy Methods 0.000 claims description 7
- 238000002310 reflectometry Methods 0.000 claims description 6
- 230000003595 spectral effect Effects 0.000 claims description 5
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/41—Refractivity; Phase-affecting properties, e.g. optical path length
- G01N21/45—Refractivity; Phase-affecting properties, e.g. optical path length using interferometric methods; using Schlieren methods
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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Abstract
The invention provides an interference microscopic imaging method and an interference microscope, in particular to an interference microscope, which comprises a light source generating device, a compensation interference cavity, a detection arm and a signal acquisition and processing unit, wherein the light source generating device is used for generating a light source signal; the compensating interference cavity receives and reflects the light beam generated by the light source generating device to form a reflected light beam which enters 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; the detection unit is provided with a second microscope objective which is used for focusing light on a sample and modulating the structural information of the sample into light rays to return. According to the interference microscope, the first microscope objective is inserted into the compensation interference cavity, so that the reference light and the sample light pass through the completely same microscope objective only in different sequences, the optical distances of the sample light and the reference light are ensured to be completely equal, and the optical distances of some points on an interference surface caused by inconsistency of the sample light and the reference light passing through an optical device are avoided.
Description
Technical Field
The invention belongs to an interference microscopic imaging technology, and particularly relates to an interference microscopic imaging method and an interference microscope.
Background
The interference microscope is a microscope adopting coherent light interference technology, and can see the structural distribution of transparent sample tissues compared with a traditional microscope. It uses interference principle to convert the phase difference distribution into intensity distribution for visual observation. The low coherence of the broad spectrum light source also enables optical sectioning that images only a sample slice within a specific optical path range, with the slice thickness depending on the temporal coherence length of the light source. The microscope adopting the low coherence interference technology is already applied to the fields of integrated circuit microscopic detection, clinical pathological diagnosis and the like, and has the advantages of non-contact and nondestructive detection.
The current low coherence interference microscope (patent 201634161. X) mostly adopts a Michelson interference structure, and a microscope is inserted between a reference arm and a sample arm, wherein the structure belongs to a light splitting path structure, when the two arms are far apart, different phase changes are formed under environmental vibration, which seriously affects normal interference pattern acquisition, so that a high-quality sample structure diagram cannot be obtained.
The interference system adopting the common optical path structure has the advantages that the phase change caused by external interference can not change the phase difference because the reference light and the sample light are transmitted in the same channel, so that the interference pattern is stable, and the interference system has the anti-interference structural advantage. In addition to the common optical path design using the Mirau objective lens, there are also some common optical path designs using two interferometers connected in series (e.g., Benoita laGuillaume E, Martins F, Boccara C, et al. high-resolution hand-driven vertical optical microscope based on full-field optical coherence tomography [ J ]. Journal of biological optics), which either do not make the objective lens numerical aperture too large or do not have complete symmetry between the reference optical path and the sample optical path, resulting in additional optical path differences.
In order to overcome some defects of the current interference microscope, the invention provides a completely symmetrical light path design, wherein reference light and sample light pass through the same path in different orders, so that the additional optical path difference caused by the system structure is eliminated, and meanwhile, the numerical aperture is not limited and is insensitive to environmental vibration.
Disclosure of Invention
In view of the above, an objective of the present invention is to provide an interference microscope, which, by inserting a first microscope objective into a compensation interference cavity, ensures that optical paths of a sample light and a reference light are completely equal, and avoids some point of unequal optical paths on an interference surface caused by inconsistency of passing through an optical device.
In order to achieve the purpose, the technical scheme of the invention is as follows:
an interference microscope comprises a light source generating device, a compensation interference cavity, a detection arm and a signal acquisition and processing unit;
the compensating interference cavity receives and reflects the light beam generated by the light source generating device to form a reflected light beam which enters 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; the detection unit is provided with a second microscope objective which is used for focusing light on a sample and modulating the structural information of the sample into light rays to return.
Further, the light source generating device includes a light emitting device having a spectral width and a first lens.
The light source generating device comprises a light-emitting device with a spectral width and a first lens; wherein,
the compensating interference cavity comprises a first beam splitter, a first reflector, a second reflector and a first microobjective; first micro objective is in between first beam splitter and the second mirror, and the second mirror is in first micro objective's focal plane department, and first micro objective main optical axis is 40-50 jiaos with first beam splitter surface, just first speculum warp first beam splitter becomes the virtual image with the second mirror is parallel to each other, first speculum with the second mirror is in first beam splitter both sides respectively.
Further, the detection arm comprises a second lens, a second beam splitter, a third beam splitter and a second microscope objective; the third beam splitter is perpendicular to the main optical axis of the second microscope objective, and the second beam splitter forms an angle of 40-50 degrees with the main optical axis of the second microscope objective and forms an angle of 80-100 degrees with the first beam splitter in the compensation interference cavity.
Furthermore, the rear end faces of the second beam splitter and the third beam splitter are both semi-permeable membranes with reflectivity of 5% -15%, and the front end face is an anti-reflection film.
Further, the signal collecting and processing unit comprises a photoelectric sensor and a signal processor, the signal processor is electrically connected with the photoelectric sensor, and the sample light reaches the photoelectric sensor through the second lens.
Further, the photoelectric sensor comprises an array type photoelectric sensor and an area array type photoelectric sensor.
Further, the photoelectric sensor array surface is conjugated with the sample through a second lens and a second microscope objective in the detection arm, and is also conjugated with a second reflector through a second lens and a first microscope objective in the compensation interference cavity in the detection arm.
In view of the above, the second objective of the present invention is to provide an interference microscopy imaging method, which ensures that the optical paths of the sample light and the reference light are completely equal by adjusting to make the reference light and the sample light pass through the identical microscope objectives in different orders, so as to avoid the different optical paths of some points on the interference surface caused by the inconsistency of the reference light and the sample light passing through the optical device, and finally obtain a high-precision three-dimensional structure diagram inside the sample.
In order to achieve the purpose, the technical scheme of the invention is as follows:
an interference microscopy imaging method comprising the steps of:
receiving a light beam generated by a light source generating device, wherein the light beam is divided into two paths of light beams by a first beam splitter, one path of light beam is reflected by a first reflector and then transmitted by the first beam splitter, and the other path of light beam is reflected by a second reflector by a first objective lens;
the two beams reach the second beam splitter, are reflected to a third beam splitter by the second beam splitter, then a part of the beams are reflected by the third beam splitter to become reference light, the other part of the beams are transmitted by the third beam splitter, and the transmitted beams are focused on a sample by a second microscope objective and are modulated by the sample to become sample light which is reflected to the second microscope objective;
the sample light and the reference light reach the front side of the photoelectric sensor through a second beam splitter and a second lens;
and a signal processor collects the electric signal from the photoelectric sensor to obtain the structural information of the sample section.
Further, the sample light carries result information of the sample.
Further, the step of allowing the sample light and the reference light to reach the front surface of the photosensor through the second beam splitter and the second lens specifically includes:
adjusting the position of the first reflector to make the optical path difference between the sample light and the reference light within the range of the coherence length of the light source;
the sample light and the reference light reach the surface of the photoelectric sensor to form interference fringes.
Further, the method also comprises the following steps:
and moving the position of the sample, and acquiring the interference fringes for multiple times by the signal processor to form an internal three-dimensional structure diagram of the sample.
Advantageous effects
The invention provides an interference microscope, which inserts a first microscope objective lens in a compensation interference cavity, so that reference light and sample light pass through the same optical elements in different orders, wherein the sample light firstly passes through the reflector, then passes through the second microscope objective in the detection arm and then reaches the sample, the reference light firstly passes through the second microscope objective in the detection arm, then passes through the reflector and then is reflected by the beam splitter, two identical microscope objectives can ensure that the optical distances of the two paths of light are completely equal, thereby avoiding that not every point on an interference surface has equal optical distance due to inconsistency of passing through optical devices, meanwhile, the invention also provides an interference microscopic imaging method, in the method, the reference light and the sample light pass through the identical microscope objectives only in different sequences, so that the optical paths of the sample light and the reference light are completely equal, and a high-precision internal three-dimensional structure diagram of the sample is obtained.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It is obvious that the drawings in the following description are some embodiments of the invention, and that for a person skilled in the art, other drawings can be derived from them without inventive exercise.
FIG. 1 is a schematic structural diagram of an interference microscope according to an embodiment of the present invention;
FIG. 2 is a schematic structural diagram of an interference microscope according to another embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The examples are given for the purpose of better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.
Example 1
Referring to fig. 1, a schematic structural diagram of an embodiment of an interference microscope of the present embodiment is shown, specifically, an interference microscope includes:
the device comprises a light source generating device, a compensation interference cavity, a detection arm and a signal acquisition and processing unit; wherein,
the light source generating device comprises a light emitting device S with a spectral width and a first lens L1, wherein the light emitting device S adopts an led light source in the embodiment, the central wavelength lambda 0 is 850nm, the spectral width delta lambda is 30nm, and the light source becomes quasi-parallel light after being collimated by the first lens L1;
the compensating interference cavity comprises a first beam splitter BS1, a first reflector M1, a second reflector M2, a first microscope objective MS1, a first microscope objective MS1 is positioned between the first beam splitter BS1 and the second reflector M2, a second reflector M2 is positioned at the focal plane of the first microscope objective MS1, the main optical axis of the first microscope objective MS1 forms an angle of 45 degrees with the surface of the first beam splitter BS1, in other embodiments, the main optical axis of the first microscope objective MS1 forms an angle with the surface of the first beam splitter BS1 within a range of 40 degrees to 50 degrees, a 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 respectively positioned at two sides of the first beam splitter BS1, the first reflector M1 is further fixedly connected with piezoceramic PZT 1 and the first reflector M2 are all reflection mirrors PZT 969;
the detection arm comprises a second lens L2, a second beam splitter BS2, a third beam splitter BS3 and a second microscope objective MS2, wherein the rear end faces of the second beam splitter BS2 and the third beam splitter BS3 are semi-transparent films, the reflectivity is 5% -15%, the front end face is an antireflection film, the third beam splitter BS3 is perpendicular to the main optical axis of the second microscope objective MS2, and the second beam splitter BS2 and the main optical axis of the second microscope objective MS2 form an angle of 45 degrees and form an angle of 80-100 degrees with the first beam splitter BS1 in the compensation interference cavity.
The signal acquisition and processing unit comprises an array type photoelectric sensor CAM and a signal processor PS, wherein the front surface of the photoelectric sensor CAM is conjugated with the Sample through a second lens L2 and a micro-objective MS2 in a detection arm, and is also conjugated with a second mirror M2 through a second lens L2 in the detection arm and a first micro-objective MS1 in a compensation interference cavity, so that the Sample and the second mirror M2 are imaged on the surface of the photoelectric sensor, the second mirror M2 and the Sample can be ensured to be imaged on the front surface of the photoelectric sensor CAM through the same optical path by adjusting the position of the first mirror M1 relative to a first beam splitter BS1, and the light beams participating in imaging are from the same light beam and form interference fringes on the sensor CAM, the signal processor PS is electrically connected with the sensor CAM, and the signal processor PS is also connected with the electric ceramic PZT.
Referring to the arrows in fig. 1, one interference microscopy imaging method in this embodiment is:
the wide-spectrum light source emitted by the light-emitting device S is collimated by the first lens L1 and then is divided into two beams by the first beam splitter BS1 in the compensation interference cavity, wherein one beam is reflected by the first reflector M1 and then is transmitted out by the first beam splitter BS1, and the other beam is reflected by the first microscope objective MS1 and then is reflected by the second reflector M2 and then is reflected by the first beam splitter BS 1.
Two beams of light from the compensating interference cavity enter the detection arm and are reflected by the second beam splitter BS2 in the detection arm to the third beam splitter BS3, then are reflected by the third beam splitter BS3 with a reflectivity of 10% and a transmissivity of 90% (in a specific embodiment, the reflectivity of the third beam splitter BS3 is set to be 10%), the transmitted light is focused on the sample by the second microscope objective MS2 and is modulated by the sample and then reflected back to the second microscope objective MS2 and is reflected by the third microscope objective BS3 at the third beam splitter BS3 to form four beams (the four beams include two beams of light reflected by the third beam splitter BS3 with a reflectivity of 10% and two beams reflected by the sample by the second microscope objective MS 2), the four beams of light reach the front of the sensor CAM through the same path of the second beam splitter BS2 and the second lens L), wherein the two beams of light carry sample information as sample information (the sample light is reflected by the second microscope objective BS2, the sample light reflection light is reflected by the sample light reflection structure reflection mirror M, the sample reflection light reflection mirror, the reflection light reflection mirror reaches the front of the sample map of the sample CAM, the sample map can be adjusted by the sample map, the sample map can be obtained by the sample map, the sample light reflection mirror, the sample map can be obtained by the sample map, the map can be obtained by the map, the map can be obtained by the map after the sample map, the map can be obtained by the map after the map, the map can be obtained by the map, the map can be obtained by the map, the map obtained by the map, the map obtained by the map after the map obtained by the sample light reflection mirror after the map.
The interference microscope in the embodiment adopts a common light path design, and the detection arm can be designed to be very long and used for in-vivo endoscopic real-time detection without tissue biopsy sampling; thanks to the first microscope objective MS1 inserted in the compensation interference cavity, the reference light and the sample light are only in different orders after passing through the completely same optical elements, wherein the sample light firstly passes through the first reflector M1, then passes through the second microscope objective MS2, and then is reflected by the sample, the reference light firstly passes through the microscope objective MS1, then passes through the second reflector M2, and then is reflected by the third beam splitter BS3, the two completely same microscope objectives can ensure that the optical paths of the two paths of light are completely equal, and the optical paths of not every point on the interference surface are not equal due to the inconsistency of the optical devices.
Example 2
As shown in FIG. 2, this embodiment uses the same compensating interference cavity as in embodiment 1, except that an image-transmitting cell, a Green's lens rod or an image-transmitting fiber bundle (F-B) is inserted into the probe arm. The reference light and the sample light are transmitted in the image transmission unit, and the optical path difference is not changed, so that the endoscopic interference imaging can be realized. The specific imaging process is the same as that of embodiment 1, and is not described herein again.
While the present invention has been described with reference to the embodiments shown in the drawings, the present invention is not limited to the embodiments, which are illustrative and not restrictive, and it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. An interference microscope is characterized by comprising a light source generating device, a compensation interference cavity, a detection arm and a signal acquisition and processing unit;
the compensating interference cavity receives and reflects the light beam generated by the light source generating device to form a reflected light beam which enters the detection arm;
the detection arm is used for focusing the reflected light beam to form sample light and sending the sample light to the signal acquisition and processing unit; the detection unit is provided with a second microscope objective which is used for focusing light on a sample and modulating the structural information of the sample into light rays to return.
2. The interference microscope of claim 1, wherein the light source generating means comprises a light emitting device having a spectral width and a first lens, and the compensating interference cavity comprises a first beam splitter, a first mirror, a second mirror, a first microscope objective; wherein,
first micro objective is in between first beam splitter and the second mirror, and the second mirror is in first micro objective's focal plane department, and first micro objective main optical axis is 40-50 jiaos with first beam splitter surface, just first speculum warp first beam splitter becomes the virtual image with the second mirror is parallel to each other, first speculum with the second mirror is in first beam splitter both sides respectively.
3. An interference microscope according to claim 2 wherein the probe arm comprises a second lens, a second beam splitter, a third beam splitter and a second microscope objective; the third beam splitter is perpendicular to the main optical axis of the second microscope objective, and the second beam splitter forms an angle of 40-50 degrees with the main optical axis of the second microscope objective and forms an angle of 80-100 degrees with the first beam splitter in the compensation interference cavity.
4. The interference microscope of claim 3, wherein the rear end faces of the second beam splitter and the third beam splitter are both semi-permeable membranes with reflectivity of 5% -15%, and the front end faces are anti-reflection films.
5. An interference microscope according to claim 4 wherein the signal acquisition and processing unit comprises a photosensor and a signal processor, the signal processor being electrically connected to the photosensor and the sample light passing through the second lens to the photosensor.
6. An interference microscope according to claim 5 wherein the photosensor front is conjugated to the sample through the second lens in the probe arm, the second microscope objective, and the first microscope objective in the complementary interference chamber is conjugated to the second mirror through the second lens in the probe arm.
7. An interference microscopy imaging method, comprising the steps of:
receiving a light beam generated by a light source generating device, wherein the light beam is divided into two paths of light beams by a first beam splitter, one path of light beam is reflected by a first reflector and then transmitted by the first beam splitter, and the other path of light beam is reflected by a second reflector by a first objective lens;
the two beams reach the second beam splitter, are reflected to a third beam splitter by the second beam splitter, then a part of the beams are reflected by the third beam splitter to become reference light, the other part of the beams are transmitted by the third beam splitter, and the transmitted beams are focused on a sample by a second microscope objective and are modulated by the sample to become sample light which is reflected to the second microscope objective;
the sample light and the reference light reach the front side of the photoelectric sensor through a second beam splitter and a second lens;
and a signal processor collects the electric signal from the photoelectric sensor to obtain the structural information of the sample section.
8. The interference microscopy imaging method of claim 7, wherein the sample light carries information about the result of the sample.
9. The interference microscopy imaging method according to claim 7, wherein the step of passing the sample light and the reference light through a second beam splitter and a second lens to reach the front surface of the photosensor specifically comprises:
adjusting the position of the first reflector to make the optical path difference between the sample light and the reference light within the range of the coherence length of the light source;
the sample light and the reference light reach the surface of the photoelectric sensor to form interference fringes.
10. The interference microscopy imaging method of claim 9, further comprising the step of:
and moving the position of the sample, and acquiring the interference fringes for multiple times by the signal processor to form an internal three-dimensional structure diagram of the sample.
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