CN111474140A - Double-channel orthogonal phase microscopic imaging sampling system - Google Patents
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Abstract
The invention provides a two-channel orthogonal phase microscopic imaging sampling system which comprises a pentaprism, a laser emission device, a first image acquisition device and a second image acquisition device, wherein a hollow channel is arranged in the pentaprism; the first light beam horizontally enters the sample in the hollow channel after entering the pentaprism, part of the first light beam horizontally transmits out to form object light of a horizontal channel, and the other part of the first light beam forms reference light of a vertical channel; after the second light beam enters the pentaprism, part of the second light beam is horizontally transmitted out through a semi-transparent and semi-reflective prism surface of the pentaprism to form reference light of a horizontal channel, and the other part of the second light beam forms object light of a vertical channel; the first image acquisition device is used for acquiring interference amplification images of object light and reference light of a horizontal channel, and the second image acquisition device is used for acquiring interference amplification images of object light and reference light of a vertical channel. The invention realizes double light sources with the same mass and can improve the matching property between channels.
Description
Technical Field
The invention relates to the technical field of optical three-dimensional morphological imaging or the field of cell three-dimensional morphological microscopic imaging, in particular to a two-channel orthogonal phase microscopic imaging sampling system.
Background
There are many microscale phase-contrast objects (i.e. transparent to visible light) in scientific research and engineering applications that require 3D morphological microscopic imaging, where in particular cell morphology detection is of great scientific research and clinical significance. Since cells are phasic bodies and have dimensions on the order of microns, which are active, they are particularly dependent on the level of phase microscopy imaging techniques. For example: common diseases such as leukemia, local anemia, retinopathy and the like can be known from morphological analysis results of white blood cells; diabetes, sepsis, malaria, arteriosclerosis, myocardial infarction, cerebral infarction, schizophrenia, zinc deficiency, and the like can be judged based on the morphology of erythrocytes. Furthermore, precise, rapid imaging of 3D morphology of cellular substructures is important in the study of the cytokinetic behavior. Therefore, the dual-channel phase microscopic imaging technology has extremely important application significance in the detection of the biological cell substructure morphology.
The quantitative phase microscopy is an important lossless optical microscopy, is an application for generating phase shift characteristics by interaction of light and phase objects such as cells, and is rapidly developed in recent years, Yamaguchi and the like introduce a phase shift technology into a digital holographic technology in 1997, phase information is obtained from four holographic images through four-step phase shift operation, Kemper and the like at the university of germany and Minster propose a reconstruction method based on non-diffraction and a digital holographic microscopy based on a Michelson optical path respectively, Popescu at the university of Illinois in America subject group successively proposes a Fourier Phase Microscopy (FPM), a Diffraction Phase Microscopy (DPM) and some extension technologies using white light as a light source, and the like, in particular, the subject group proposed in 2011 a spatial light interference microscopy (S L IM) which is equivalent to the resolution precision of an atomic force microscope and has an ultra-fast processing speed, the U.S. Doeck group proposes interference and a micro phase microscopy as a spatial light interference microscopy technology (S L IM) which is equivalent to the resolution precision of an atomic force microscope and has an ultra-fast processing speed, and only a plurality of cell phase information can be obtained by a combination of a reflection technology, a cell phase information, a cell-phase microscopy system, a cell-phase microscopy system, and a cell-phase-imaging technology (cell-phase microscopy system which can not only obtain a cell-phase information, and a cell-phase-reflection technology (cell-reflection technology, and a cell-reflection technology which can be combined with a cell-reflection technology, and a cell-reflection technology, a cell-reflection technology which can not only can be combined with a cell-reflection technology, a cell-reflection technology (cell-reflection technology, a cell-reflection technology which can not only can be combined with a cell-cell.
In 2007, Choi and Fang-Yen, et al, by means of a phase-shifting laser interferometer, and a method of rotating the angle of an illumination light source, achieve tomographic phase microscopy, but the method has certain limitations, such as being unsuitable for imaging relatively thick samples, because the physical thickness and phase shift of such samples cannot be approximately considered to be linear, Vishenyakov and L evin, by Russian Optical physics measurement research institute, combine phase microscopy with computer tomography, for measuring the three-dimensional distribution of the refractive index of transparent biological samples, such as cells, in 2006, Charri and Marian, based on digital holographic phase imaging techniques, indirectly changing the direction of illumination of a light source by rotating the sample, obtaining projection data in multiple directions, and performing complete projection reconstruction by using filtered back projection reconstruction, obtaining the three-dimensional distribution of the refractive index of cells, in 2007, Kagawa and Yasowa, and Israwa, and obtaining a high-resolution image by using a single tomographic imaging system, which is not limited by rotating the Optical interferometer, and which the refractive index of a sample is difficult to obtain a high-resolution by rotating a tomographic image, and a high-resolution by using a rotating Optical microscope, such a rotating tomographic imaging system, which is difficult to obtain a high-resolution by using a rotating a tomographic imaging system, such a high-resolution of a tomographic image obtained by rotating a tomographic imaging system, which is difficult to obtain a high-resolution image, such a high-resolution image, and a high-resolution image obtained by rotating a high-resolution image obtained by using a high-resolution image obtained by rotating a tomographic imaging system, such a high-Optical tomography image obtained by rotating a sample, such as a high-Optical microscope, which is not limited by rotating a high-Optical microscope, which is difficult to obtain a high-Optical microscope, and a high-Optical microscope.
In order to realize 3D morphological imaging of cells, some patent application technologies appear in China, and the patent discloses that double-angle phase imaging can be quickly realized by utilizing two total reflection mirrors, so that the method has high stability; the patent discloses that based on the Mach-Zehnder interference principle, the optical fiber coupler and the collimator are utilized to realize dual-wavelength coaxial phase-shift interference microscopic imaging, and 5 dual-wavelength coaxial phase-shift interference images can be obtained by changing the phase shift value of reference light through PZT. The sampling speed is high, but the fiber coupling efficiency influences the quality of interference imaging. The patent technologies have the defects of complex optical path and low stability, so that the developed instrument has high cost and complex maintenance.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a dual-channel orthogonal phase microscopic imaging sampling system, which solves three defects of the traditional dual-optical-path orthogonal phase microscopic imaging optical path, and one of the defects is that due to unbalance of devices, a dual optical source can generate different system errors in two orthogonal channels, so that the form reconstruction accuracy is influenced; the other is that the traditional imaging is usually static slide sample loading, a flow channel method is adopted, and the normal incidence of double light beams to a sampling channel is ensured, so that the imaging sampling speed can be effectively improved, the third is that the traditional orthogonal double-channel phase microscopic imaging light path structure is complex, devices are multiple, the maintenance cost is high, and the problem can be effectively solved by using a pentaprism as the light path structure under the modulation of the light path.
The present invention achieves the above-described object by the following technical means.
A double-channel orthogonal phase microscopic imaging sampling system comprises a pentaprism, a laser emission device, a first image acquisition device and a second image acquisition device, wherein a hollow channel is arranged in the pentaprism and used for placing a sample; the laser emitting device is used for generating parallel light beams; the method comprises the steps that a first light beam horizontally enters a sample in a hollow channel after entering a pentaprism, part of the first light beam is horizontally transmitted out through a semi-transparent and semi-reflective prism surface of the pentaprism and used for forming object light of a horizontal channel, and the other part of the first light beam vertically exits from the pentaprism after being reflected twice and refracted once and is used for forming reference light of a vertical channel; after the second light beam enters the pentaprism, part of the second light beam is horizontally transmitted out through a semi-transparent and semi-reflective prism surface of the pentaprism and is used for forming reference light of a horizontal channel, the other part of the second light beam is vertically emitted into a sample in the hollow channel after being reflected twice, and the other part of the second light beam is vertically emitted out of the pentaprism and is used for forming object light of a vertical channel; the first image acquisition device is used for acquiring interference images of object light and reference light of a horizontal channel, and the second image acquisition device is used for acquiring interference images of object light and reference light of a vertical channel.
Further, the first prism surface, the second prism surface and the fourth prism surface of the pentaprism are transmission surfaces, the third prism surface of the pentaprism is a semi-transmission semi-reflection surface, and the fifth prism surface of the pentaprism is a total reverse surface;
the first light beam and the second light beam which are incident on the first prism surface are parallel to pass through a hollow channel and then are emitted from the third prism surface, and the first light beam irradiates a sample through the hollow channel; one part of the first light beam and the second light beam are transmitted into the first image acquisition device through the third prism surface, and the other part of the first light beam and the second light beam are transmitted into the second image acquisition device through the third prism surface, the fifth prism surface and the second prism surface in sequence; another part of the second light beam irradiates the sample through the hollow channel; the first light beam and the second light beam passing through the hollow channel are perpendicular to the other parts of the first light beam and the second light beam respectively.
Further, a part of the second light beam emitted from the pentaprism passes through a grating filter device to form reference light of a horizontal channel; and the other part of the first light beam emitted from the pentaprism passes through a grating filter device to form reference light of a vertical channel.
Further, the grating filtering device comprises a grating and a filter plate; part of the second light beam or the other part of the first light beam sequentially passes through the grating and the filter plate, and the grating is used for enabling part of the second light beam or the other part of the first light beam to generate diffraction; the filter is used for screening zero-order light of the diffracted part of the second light beam or the other part of the first light beam.
Further, the object light and the reference light of the horizontal channel are emitted into the first image acquisition device through a light combination amplification device, and the object light and the reference light of the vertical channel are emitted into the second image acquisition device through the light combination amplification device; the light combination amplifying device is used for combining the reference light and the object light and then amplifying the combined light.
Further, the light combination amplifying device comprises a light combination lens and a lens assembly; the light combining mirror is used for converging the reference light and the object light to generate an interference light beam, the lens assembly is used for amplifying the interference light beam, and the interference light beam is emitted into the first image acquisition device or the second image acquisition device.
The system further comprises a control system, wherein the control system carries out sample three-dimensional morphological reconstruction according to the patterns acquired by the first image acquisition device and the second image acquisition device by a maximum entropy analysis reconstruction method and is used for acquiring sample morphological images.
The invention has the beneficial effects that:
1. according to the two-channel orthogonal phase microscopic imaging sampling system, the double light sources with the same mass are realized through the light splitting sheet, and the matching performance among the channels can be improved.
2. The two-channel orthogonal phase microscopic imaging sampling system realizes the interference imaging of orthogonal double light paths by utilizing the transmission and reflection characteristics of the surface of the pentaprism, and has the advantages of simple light path and devices, low cost and high interference imaging efficiency.
3. According to the two-channel orthogonal phase microscopic imaging sampling system, the tetragonal sampling channel is arranged in the pentaprism body, so that flow type sampling can be realized, normal incidence is ensured, and the sampling imaging speed and the calculation efficiency can be effectively improved.
4. The dual-channel orthogonal phase microscopic imaging sampling system disclosed by the invention can effectively solve the problem of near-field amplification caused by pentaprism duty by using a post-amplification method and utilizing far-field imaging.
Drawings
Fig. 1 is a schematic diagram of a two-channel quadrature-phase microscopic imaging sampling system according to embodiment 1 of the present invention.
Fig. 2 is a schematic diagram of a two-channel quadrature-phase microscopic imaging sampling system according to embodiment 2 of the present invention.
In the figure:
1-a laser; 2-a spectroscope; 3-a pentaprism; 4-a hollow channel; 5-a first grating; 6-a first filter segment; 7-a first light combiner; 8-a first lens; 9-a second lens; 10-a first CCD camera; 11-a second grating; 12-a second filter segment; 13-a second light combining mirror; 14-a third lens; 15-a fourth lens; 16-second CCD camera.
Detailed Description
The invention will be further described with reference to the following figures and specific examples, but the scope of the invention is not limited thereto.
As shown in fig. 1, the two-channel orthogonal phase microscopic imaging sampling system of the present invention comprises a pentaprism 3, a laser emitting device, a first image collecting device and a second image collecting device, wherein a hollow channel 4 is arranged inside the pentaprism 3 for placing a sample; the hollow channel 4 is a cuboid, which can be square or rectangular. The hollow channel 4 is a square hollow channel at the position of the pentaprism 3 where the two light beams are orthogonal, and is used for passing through a phase body sample, so that flow type sampling is realized, two light beams in the horizontal direction and the vertical direction are both normal incidence, secondary refraction is avoided, and sampling imaging speed and calculation efficiency are improved.
The laser emitting device is used for generating parallel light beams; the first light beam horizontally enters the sample in the hollow channel 4 after entering the pentaprism 3, part of the first light beam is horizontally transmitted out through the semi-transparent and semi-reflective prism surface of the pentaprism 3 to form object light of a horizontal channel, and the other part of the first light beam vertically exits from the pentaprism 3 after twice reflection and once refraction to form reference light of a vertical channel; after the second light beam enters the pentaprism 3, a part of the second light beam is horizontally transmitted out through a semi-transparent and semi-reflective prism surface of the pentaprism 3 and is used for forming reference light of a horizontal channel, the other part of the second light beam is reflected twice and then vertically enters a sample in the hollow channel 4, and the other part of the second light beam is vertically emitted out of the pentaprism 3 and is used for forming object light of a vertical channel; part of the second light beam emitted from the pentaprism 3 passes through a grating filter device to form reference light of a horizontal channel; another part of the first light beam emitted from the pentaprism 3 passes through a grating filter device to form reference light of a vertical channel. The first image acquisition device is used for acquiring interference images of object light and reference light of a horizontal channel, and the second image acquisition device is used for acquiring interference images of object light and reference light of a vertical channel. The object light and the reference light of the horizontal channel are emitted into the first image acquisition device through a light combination amplification device, and the object light and the reference light of the vertical channel are emitted into the second image acquisition device through the light combination amplification device; the light combination amplifying device is used for combining the reference light and the object light and then amplifying the combined light.
Embodiment 1 as shown in fig. 1, the two-channel orthogonal phase microscopic imaging sampling system of the present invention includes a laser 1, a spectroscope 2 and a pentaprism 3, wherein a light beam emitted by the laser 1 passes through the spectroscope 2 to form two light beams with parallel and same performance characteristics; the first prism surface, the second prism surface and the fourth prism surface of the pentaprism 3 are transmission surfaces, the third prism surface of the pentaprism 3 is a semi-transparent semi-reflecting surface, and the fifth prism surface of the pentaprism 3 is a total reverse surface; the first light beam and the second light beam which are incident on the first prism surface are emitted from the third prism surface in parallel through the hollow channel 4, and the first light beam irradiates a sample through the hollow channel 4; one part of the first light beam and the second light beam are transmitted into the first image acquisition device through the third prism surface, and the other part of the first light beam and the second light beam are transmitted into the second image acquisition device through the third prism surface, the fifth prism surface and the second prism surface in sequence; another part of the second light beam irradiates the sample through the hollow channel 4; the first and second beams passing through the hollow channel 4 are perpendicular to the other portions of the first and second beams, respectively.
The first image acquisition device acquires horizontal channel information: the first light beam emitted from the spectroscope 2 enters the pentaprism 3 and horizontally passes through the sample channel 4 to irradiate a sample, and then after passing through the third prism surface of the pentaprism, part of the first light beam is horizontally emitted to form object light of a horizontal channel; after the second light beam passes through a third prism surface of the pentaprism, part of the second light beam is horizontally transmitted out, diffraction is generated through the first grating 5, and after the diffracted light passes through the first filter 6, zero-order light is screened out to form reference light of a horizontal channel; the object light and the reference light of the horizontal channel converge to generate interference after passing through the first light combining mirror 7, the interference light beam passes through a first post-amplification lens group formed by a first lens 8 and a second lens 9 for post-amplification, and an interference image is imaged on a first CCD camera 10.
The second image acquisition device acquires vertical channel information: after the second light beam emitted from the spectroscope 2 passes through the third prism surface of the pentaprism 3, the other part of the second light beam vertically passes through the sample hollow channel 4 after being reflected twice and refracted once, and irradiates a sample to form object light of a vertical channel; after the first light beam horizontally passes through the sample channel 4 after passing through the pentaprism 3, the other part of the first light beam is reflected twice and refracted once and then vertically emitted from the second prism surface of the pentaprism 3, diffraction is generated through the second grating 11, and after the diffracted light passes through the second filter 12, zero-order light is screened out to form a reference light beam of a vertical channel. The object light and the reference light of the vertical channel are converged to generate interference after passing through the second light combining mirror 13, and the interference light beam is post-amplified through a second post-amplification lens group formed by a third lens 14 and a fourth lens 15 to image an interference image on a second CCD camera 16.
The CCD on the horizontal channel and the vertical channel transmits the captured interference microscopic pattern to a computer for pattern processing, phase recovery is firstly carried out to determine the three-dimensional morphological distribution of cells, the two-channel phase distribution is obtained, then the refractive index spatial distribution of a phase object needs to be reconstructed, a grid method is applied to divide the phase object to be reconstructed at equal intervals, and the established cubic grid is required to completely cover the phase object.
The control system firstly carries out phase recovery on the orthogonal interferogram according to the patterns acquired by the first image acquisition device and the second image acquisition device to acquire the phase distribution of two orthogonal channels; then based on orthogonal phase distribution, utilizing maximum entropy analysis reconstruction method to carry out sample three-dimensional form reconstruction and obtaining sample form image
As shown in fig. 2, which is an embodiment 2 of the present invention, five interior angles of the pentaprism 3 are respectively ∠ 1 of 90 °, ∠ 2 of 117 °, ∠ 3 of 117 °, ∠ 4 of 108 ° and ∠ 5 of 108 °, and attributes of five surfaces are that the BC surface is a coated total reverse surface, the DE surface is a coated semi-transparent semi-reflective surface, each accounting for 50%, and the AB surface, the AE surface and the CD are natural transmission surfaces.
The present invention is not limited to the above-described embodiments, and any obvious improvements, substitutions or modifications can be made by those skilled in the art without departing from the spirit of the present invention.
Claims (7)
1. The double-channel orthogonal phase microscopic imaging sampling system is characterized by comprising a pentaprism (3), a laser emitting device, a first image acquisition device and a second image acquisition device, wherein a hollow channel (4) is arranged in the pentaprism (3) and used for placing a sample;
the laser emitting device is used for generating parallel light beams; the first light beam horizontally enters a sample in the hollow channel (4) after entering the pentaprism (3), part of the first light beam is horizontally transmitted out through a semi-transparent and semi-reflective prism surface of the pentaprism (3) for forming object light of a horizontal channel, and the other part of the first light beam vertically exits from the pentaprism (3) after being reflected and refracted for forming reference light of a vertical channel; after the second light beams enter the pentaprism (3), part of the second light beams are horizontally transmitted out through a semi-transparent and semi-reflective prism surface of the pentaprism (3) and are used for forming reference light of a horizontal channel, the other part of the second light beams are reflected and then vertically enter a sample in the hollow channel (4), and the other part of the second light beams are vertically emitted out of the pentaprism (3) and are used for forming object light of a vertical channel;
the first image acquisition device is used for acquiring interference images of object light and reference light of a horizontal channel, and the second image acquisition device is used for acquiring interference images of object light and reference light of a vertical channel.
2. The two-channel quadrature-phase microscopy imaging sampling system according to claim 1, wherein the first, second and fourth prism faces of the pentaprism (3) are transmissive faces, the third prism face of the pentaprism (3) is a transflective face, and the fifth prism face of the pentaprism (3) is an all-negative face;
the first light beam and the second light beam which are emitted into the first prism surface are emitted out of the third prism surface in parallel through a hollow channel (4), and the first light beam irradiates a sample through the hollow channel (4); one part of the first light beam and the second light beam are transmitted into the first image acquisition device through the third prism surface, and the other part of the first light beam and the second light beam are transmitted into the second image acquisition device through the third prism surface, the fifth prism surface and the second prism surface in sequence; another part of the second light beam irradiates the sample through the hollow channel (4); the first light beam and the second light beam passing through the hollow channel (4) are perpendicular to the other parts of the first light beam and the second light beam respectively.
3. The dual-channel quadrature phase microscopy imaging sampling system according to claim 1, characterized in that part of the second beam emerging from the pentaprism (3) passes through a grating filter arrangement to form a horizontal channel of reference light; and another part of the first light beam emitted from the pentaprism (3) passes through a grating filter device to form reference light of a vertical channel.
4. The two-channel quadrature phase microscopy imaging sampling system according to claim 3, characterized in that the grating filter arrangement comprises a grating (5, 11) and a filter plate (6, 12); part of the second light beam or the other part of the first light beam sequentially passes through a grating (5, 11) and a filter plate (6, 12), wherein the grating (5, 11) is used for diffracting the part of the second light beam or the other part of the first light beam; the filters (6, 12) are used for screening zero-order diffracted light of the diffracted part of the second light beam or the other part of the first light beam.
5. The dual-channel orthogonal phase microscopy imaging sampling system according to claim 1, wherein the object light and the reference light of the horizontal channel are emitted into the first image acquisition device through a light combination amplification device, and the object light and the reference light of the vertical channel are emitted into the second image acquisition device through the light combination amplification device; the light combination amplifying device is used for combining the reference light and the object light and then amplifying the combined light.
6. The dual-channel quadrature phase microscopy imaging sampling system according to claim 5, wherein the light combining and amplifying device comprises a light combining mirror (7, 13) and a lens assembly; the light-combining mirrors (7, 13) are used for converging the reference light and the object light to generate interference beams, the lens assembly is used for amplifying the interference beams, and the interference beams are emitted into the first image acquisition device or the second image acquisition device.
7. The dual-channel orthogonal phase microscopy imaging sampling system of claim 1, further comprising a control system for performing a three-dimensional morphological reconstruction of the sample by a maximum entropy tomographic reconstruction method based on the patterns acquired by the first image acquisition device and the second image acquisition device for obtaining morphological images of the sample.
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| CN202010208783.8A CN111474140A (en) | 2020-03-23 | 2020-03-23 | Double-channel orthogonal phase microscopic imaging sampling system |
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