CN110109240B - Dual-channel dual-wavelength phase microscopy imaging system and method in non-orthogonal basis - Google Patents
Dual-channel dual-wavelength phase microscopy imaging system and method in non-orthogonal basis Download PDFInfo
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Abstract
The invention provides a dual-channel dual-wavelength phase microscopic imaging system and method under a non-orthogonal basis, which comprises a light source, a 2f mirror, a beam splitting aperture diaphragm, a beam expanding collimating mirror, a sample platform, an objective lens, a first channel, a second channel and a computer, wherein the sample platform is provided with a first lens, a second lens and a third lens; the first channel and the second channel are arranged in parallel; the first channel comprises a first diaphragm, a first collimating mirror, a first grating, a first Fourier lens, a first spatial light modulator, a second Fourier lens and a first CCD camera; the second channel has the same structure as the first channel; a light beam emitted by a light source is divided into two non-orthogonal parallel light beams after sequentially passing through a 2f lens, a beam splitting aperture diaphragm and a beam expanding collimator, then sequentially passes through a sample platform and an objective lens, respectively enters a first channel and a second channel to obtain an interference fringe pattern, and is transmitted to a computer for pattern processing to obtain the three-dimensional form of a sample. The invention can carry out phase microscopic one-time synchronous imaging on dual-wavelength and dual-path light, can obtain the spatial phase distribution of a phase body according to the interference microscopic image, can obtain the three-dimensional shape of a sample by utilizing a reconstruction algorithm, and is particularly suitable for transient microscopic imaging of biological cell shapes.
Description
Technical Field
The invention belongs to the technical field of phase microscopic imaging, and particularly relates to a dual-channel dual-wavelength phase microscopic imaging system and method under a non-orthogonal basis.
Background
Optical microscopy is a vital tool in the fields of biology and medicine, because most cells and tissues are "transparent" under the microscope, and people traditionally have "visible" the cells or tissues by staining them. A series of technological innovations in recent years have broken through past limitations of phase microscopy, which has led to the emergence of quantitative microscopic imaging techniques (QPI). QPI operates on unlabeled samples and is therefore a complement to the established fluorescence microscope, with low phototoxicity and no photobleaching. Since the image represents a quantitative mapping of the path length delay introduced by the sample, QPI provides an objective morphological and kinetic measure that is not affected by the contrast agent. Quantitative microscopic imaging techniques have evolved rapidly over the past 10-15 years, improving phase sensitivity, stability and speed, and have become a valuable method of studying cells and tissues.
There are also some deficiencies in the prior art. Diffractive Phase Microscopy (DPM) with high stability of the common geometric optical path feature was proposed as Popescu equals 2006. The core idea of the technology is that a phase grating and a special spatial optical filter are utilized, 0-order diffraction fields and + 1-order diffraction fields containing sample image information can be separated and respectively used as a reference field and a sample field, and stable off-axis interference images can be formed on a CCD through the same devices. The method has the problem of low quality of the final image. Patent technology US2014085715a1(Diffraction phase microscopy with white light) selects white light to replace the original light source in the DPM technology to generate a corresponding white light Diffraction phase microscopy (wDPM) technique which has higher spatial phase sensitivity than the original technique, and in the wDPM technique, they adopt a space-time filtering method (essentially average operation) and have optical path sensitivity of the order of sub-angstrom. The method measures the relation between the phase of the discoid red blood cells and the dynamic phase of the hela cells as long as 18 hours and the change of the dry mass of the hela cells along with the time by using the technology, so that the growth change of the hela cells can be quantified. However, the image acquisition speed of the method cannot meet the requirements of some measurement scenes at present. Kim equals 2014 and applies dual wavelength technique to DPM under the technique, throws light on through a combination laser source that can produce two chromatic light constantly in succession, and different wavelength components are reflected to the different diffraction orders of grating, and dual wavelength interference mode can be distinguished through obvious fringe carrier frequency. Because it is a co-path system, it can provide sub-nanometer time stability, and because of its single shot nature, the acquisition time is in the order of milliseconds. In the method, interference fringes acquired by the CCD are superposed by two interference fringes with different frequencies corresponding to two wavelengths, and interference is caused to information extraction.
Disclosure of Invention
The invention aims to provide a dual-channel dual-wavelength phase microscopic imaging system and method under a non-orthogonal basis, aiming at the problems in the prior art, the system and method can carry out phase microscopic one-time synchronous imaging on dual-wavelength and dual-channel light, can obtain the spatial phase distribution of a phase body according to an interference microscopic image, and carry out pattern processing by using a computer to obtain the three-dimensional form of a sample, and are particularly suitable for transient microscopic imaging of biological cell forms.
The technical scheme of the invention is as follows: the dual-channel dual-wavelength phase microscopic imaging system under the non-orthogonal basis comprises a light source, a 2f mirror, a beam splitting aperture diaphragm, a beam expanding collimating lens, a sample platform, an objective lens, a first channel, a second channel and a computer; the light source, the 2f lens, the beam splitting aperture diaphragm, the beam expanding collimator, the sample platform and the objective lens are sequentially arranged, and the centers of the light source, the 2f lens, the beam expanding collimator, the sample platform and the objective lens are positioned on the same optical axis;
the first channel and the second channel are arranged in parallel, and the first channel comprises a first diaphragm, a first collimating mirror, a first grating and a first 4f system; the second channel comprises a second diaphragm, a second collimating mirror, a second grating and a second 4f system;
a light beam emitted by the light source is divided into two non-orthogonal collimated light beams after passing through the 2f lens, the beam splitting aperture diaphragm and the beam expanding collimator lens, and the two non-orthogonal collimated light beams simultaneously sequentially pass through the sample platform and the objective lens and then respectively enter the first channel and the second channel; the light beam entering the first channel sequentially passes through the first diaphragm and the first collimating mirror, diffraction is generated through the first grating, and the diffracted light beam is subjected to microscopic imaging through the first 4f system; the light beam entering the second channel sequentially passes through the second diaphragm and the second collimating mirror, diffraction is generated through the second grating, and the diffracted light beam is subjected to microscopic imaging through the second 4f system;
the first 4f system obtains an optical zero-order light and a first-order light interference fringe pattern with a first wavelength, and an optical zero-order light and a first-order light interference fringe pattern with a second wavelength; the second 4f system obtains an optical zero-order light and first-order light interference fringe pattern with the first wavelength, and an optical zero-order light and first-order light interference fringe pattern with the second wavelength;
the computer is respectively connected with the first 4f system and the second 4f system.
In the above scheme, the light source is a white light source, and the white light source emits low-coherence space light.
In the above scheme, the first 4f system includes a first fourier lens, a first spatial light modulator, a second fourier lens, and a first CCD camera, and centers of the first fourier lens, the first spatial light modulator, the second fourier lens, and the first CCD camera are all on the same optical axis;
the second 4f system comprises a third Fourier lens, a second spatial light modulator, a fourth Fourier lens and a second CCD camera, and the centers of the third Fourier lens, the second spatial light modulator, the fourth Fourier lens and the second CCD camera are all located on the same optical axis.
In the above scheme, the sample platform is made of a transparent material.
In the above scheme, the first spatial light modulator filters the multiple-order diffracted light, selects the first wavelength zero-order light and the first-order light, and the second wavelength zero-order light and the first-order light, modulates the first wavelength zero-order light and the second wavelength zero-order light into 45-degree polarization with the horizontal direction, modulates the first wavelength light into vertical polarization, modulates the second wavelength light into horizontal polarization, and uses the zero-order light as reference light and the first-order light as sample light;
the second spatial light modulator is identical in structure to the first spatial light modulator.
In the scheme, a first polarization analysis array is arranged on the first CCD camera and comprises a plurality of micro-polarizers in different polarization directions, and the micro-polarizers are arranged in order; the first CCD camera extracts an interference fringe pattern corresponding to the first wavelength light wave and an interference fringe pattern corresponding to the second wavelength light wave;
and a second polarization analysis array is integrated on a photosensitive chip of the second CCD camera, and the second CCD camera extracts an interference fringe pattern corresponding to the first wavelength light wave and an interference fringe pattern corresponding to the second wavelength light wave.
The microscopic imaging method of the dual-channel dual-wavelength phase microscopic imaging system under the non-orthogonal basis comprises the following steps:
white light sorting: light beams emitted by the light source pass through the 2f lens, the beam splitting aperture diaphragm and the beam expanding collimating lens to form two non-orthogonal parallel light beams;
non-orthogonal parallel light sampling: two non-orthogonal parallel light beams are irradiated on a sample of the sample platform, and two light beams carrying sample image field information are formed through an objective lens; the two light beams respectively enter a first channel and a second channel;
the double-wavelength diffraction light splitting modulation and imaging collection are carried out, namely, a light beam entering a first channel passes through a first diaphragm and a first collimating lens in sequence, after beam expansion and collimation, the light beam is converted into a parallel light beam again, diffraction is generated through a first grating, the light beam is separated into multi-level diffraction light through the first grating, and the multi-level diffraction light irradiates a first spatial light modulator after passing through a first Fourier lens; the first spatial light modulator filters the multi-level diffracted light, selects first-wavelength light wave zero-order light and first-order light, second-wavelength light wave zero-order light and first-order light, modulates the first-wavelength light and the second-wavelength light wave zero-order light into 45-degree polarization with the horizontal direction, modulates the first-order light wave of the first wavelength into vertical polarization, modulates the first-order light wave of the second wavelength into horizontal polarization, takes the zero-order light as reference light, and takes the first-order light as sample light; the reference light and the sample light pass through a second Fourier lens, interfere and generate a spatially modulated interference fringe pattern on the first CCD camera; the process in the second channel is the same as the process in the first channel, and a spatially modulated interference fringe pattern is generated on the second CCD camera;
pattern analysis: the first CCD camera and the second CCD camera acquire interference fringe patterns and transmit the interference fringe patterns to a computer for pattern processing.
Compared with the prior art, the invention has the beneficial effects that:
1. the light source is white light. Speckle causes spatial inhomogeneities in the quantitative phase image that blur details, and is due to reflections from various samples and slide surfaces or coherent superposition of unwanted scattered light fields formed by dust, optics defects. White light provides a coherence length in the order of 1 μm, and the superposition between different field components is coherent only if the optical path difference is in this order or less, thus effectively suppressing noise.
2. The two-channel imaging under the non-orthogonal basis can not bring extra phase difference to the two channels, so that the axial noise is reduced, and the axial space-time stability is improved.
3. The common-path off-axis method provides nearly identical optical paths for the imaging beam and the reference beam, since both are transmitted through the same components. On one hand, interference of low-coherence white light is guaranteed, on the other hand, axial microscopic noise is remarkably reduced, and instability (mechanical vibration or thermal change) of the system does not influence the obtained result.
And 4, integrating an analyzing array on the CCD chip, wherein the analyzing array selectively passes through the irradiated light waves in combination with the polarization modulation condition of the spatial modulator, and two interference fringes with different fringe frequencies corresponding to the light waves with the first wavelength and the second wavelength are respectively formed on the CCD. The incoherent superposition of interference light beams is avoided, errors are eliminated, and the effect of interference patterns is guaranteed.
5. Because of the dual-channel dual-wavelength imaging, four interference patterns can be obtained from single shooting controlled by a computer, on one hand, the bandwidth of the CCD is fully utilized, on the other hand, a quantitative phase diagram can be quickly recovered through a corresponding phase recovery algorithm, and the acquisition speed of cell information is greatly improved.
Drawings
Fig. 1 is a schematic diagram of an optical path according to an embodiment of the present invention.
The system comprises a white light source 1, a 2.2 f mirror, a 3 splitting aperture diaphragm, a 4 expanding collimator, a 5 sample platform, a 6 objective lens, a 7 first diaphragm, a 8 first collimator lens, a 9 first grating, a 10 first Fourier lens, a 11 first spatial modulator, a 12 second Fourier lens, a 13 first polarization analyzing array, a 14 first CCD camera, a 15 second diaphragm, a 16 second collimator lens, a 17 second grating, a 18 third Fourier lens, a 19 second spatial modulator, a 20 fourth Fourier lens, a 21 second polarization analyzing array and a 22 second CCD camera.
Detailed Description
In order to more clearly understand the technical features, objects, and effects of the present invention, embodiments of the present invention will now be described with reference to the accompanying drawings, in which the same reference numerals refer to the same or similar parts throughout the drawings. The drawings are intended to depict only the invention, and not to represent the actual construction or actual scale of the invention.
Example 1
Fig. 1 is an embodiment of a dual-channel dual-wavelength phase microscopic imaging system under a non-orthogonal basis according to the present invention, where the dual-channel dual-wavelength phase microscopic imaging system under the non-orthogonal basis includes a light source 1, a 2f mirror 2, a spectroscopic aperture stop 3, a beam expanding collimator 4, a sample platform 5, an objective lens 6, a first channel, a second channel, and a computer; the light source 1, the 2f lens 2, the beam splitting aperture diaphragm 3, the beam expanding collimator 4, the sample platform 5 and the objective lens 6 are sequentially arranged, and the centers of the light source 1, the beam expanding collimator 4 and the sample platform are positioned on the same optical axis;
the first channel and the second channel are arranged in parallel, the first channel comprises a first diaphragm 7, a first collimating mirror 8, a first grating 9 and a first 4f system, and the centers of the first diaphragm 7, the first collimating mirror 8 and the first grating 9 are all positioned on the same optical axis; the second channel comprises a second diaphragm 15, a second collimating mirror 16, a second grating 17 and a second 4f system, and the centers of the second diaphragm 15, the second collimating mirror 16 and the second grating 17 are all positioned on the same optical axis;
the light source 1 is a white light source, the white light source emits low-coherence space light, the coherence length is short, noise can be well suppressed, and the final imaging quality is improved. The beam splitting aperture diaphragm 3 is positioned on the front focal plane of the beam expanding collimating lens 2, and the beam splitting aperture diaphragm 3 is in plane conjugation with the first diaphragm 7 and the second diaphragm 15 on the rear focal plane of the objective lens 6, so that the interference of white light is guaranteed. The sample platform 5 is made of transparent material. The beam splitting aperture diaphragm 3 is provided with two small holes, a light beam emitted by the light source 1 is divided into two non-orthogonal collimated light beams after passing through the 2f mirror 2, the beam splitting aperture diaphragm 3 and the beam expanding collimator 4, the two non-orthogonal collimated light beams irradiate onto a sample of the sample platform 5 from two different angles, and the two light beams carry sample image field information and enter a first channel and a second channel through the objective lens 6 respectively.
The light beam entering the first channel passes through the first diaphragm 7 and the first collimating mirror 8 in sequence, and is diffracted by the first grating 9, the first grating 9 separates the light beam into multi-order diffracted light, and the multi-order diffracted light comprises first-wavelength light zero-order light, first-order light, second-wavelength light zero-order light and first-order light. The multi-order diffraction light is subjected to microscopic imaging through a first 4f system; the light beam entering the second channel sequentially passes through a second diaphragm 15 and a second collimating mirror 16, diffraction is generated through a second grating 17, and the diffracted light is subjected to microscopic imaging through a second 4f system;
the first 4f system comprises a first Fourier lens 10, a first spatial light modulator 11, a second Fourier lens 12 and a first CCD camera 14, wherein the centers of the first Fourier lens 10, the first spatial light modulator 11, the second Fourier lens 12 and the first CCD camera 14 are all positioned on the same optical axis;
the second 4f system comprises a third fourier lens 18, a second spatial light modulator 19, a fourth fourier lens 20 and a second CCD camera 22, and the centers of the third fourier lens 18, the second spatial light modulator 19, the fourth fourier lens 10 and the second CCD camera 22 are all on the same optical axis.
The first spatial light modulator 11 is provided with three small holes, and 3 small holes are provided with different polaroids; the first spatial light modulator 11 filters the multi-level diffracted light, selects the first wavelength light wave zero-order light and the first level light, the second wavelength light wave zero-order light and the first level light, modulates the first wavelength light and the second wavelength light wave zero-order light into 45-degree polarization through polarizing films on 3 pores, modulates the first wavelength light wave first level light into vertical polarization, modulates the second wavelength light wave first level light into horizontal polarization, takes the zero-level light as reference light, and takes the first level light as sample light; the second spatial light modulator 19 is structured the same as the first spatial light modulator 11.
The first 4f system obtains an optical zero-order light and a first-order light interference fringe pattern with a first wavelength, and an optical zero-order light and a first-order light interference fringe pattern with a second wavelength; the second 4f system obtains an optical zero-order light and first-order light interference fringe pattern with the first wavelength, and an optical zero-order light and first-order light interference fringe pattern with the second wavelength;
a first polarization detection array 13 is arranged on a photosensitive chip of the first CCD camera 14, the first polarization detection array 13 comprises a plurality of micro-polarizing films with different polarization directions, and the micro-polarizing films are arranged in order to avoid incoherent superposition of two interference fringes; the first CCD camera 14 extracts an optical interference fringe pattern corresponding to a first wavelength and an optical interference fringe pattern corresponding to a second wavelength;
a second polarization detection array 21 is arranged on a photosensitive chip of the second CCD camera 22, the second polarization detection array 21 comprises a plurality of micro-polarizing films with different polarization directions, and the micro-polarizing films are arranged in order to avoid incoherent superposition of two interference fringes; the second CCD camera 22 extracts the optical interference fringe pattern corresponding to the first wavelength and the optical interference fringe pattern corresponding to the second wavelength.
The first CCD camera 14 extracts an optical interference fringe pattern corresponding to a first wavelength and an optical interference fringe pattern corresponding to a second wavelength; the amplitude, phase and polarization state of the light beam in the second channel are synchronously modulated using the second spatial light modulator 19, and the second CCD camera 22 extracts the light wave interference fringe pattern corresponding to the first wavelength and the light wave interference fringe pattern corresponding to the second wavelength. Therefore, the two-channel dual-wavelength off-axis synchronous interference under white light is realized, and 4 interference fringe patterns are obtained.
The computer is respectively connected with the first 4f system and the second 4f system, interference fringe patterns are collected and displayed on the computer through the first CCD camera 14 in the first 4f system and the second CCD camera 22 in the second 4f system, and the three-dimensional form of the sample is obtained through pattern processing of the computer.
Example 2
An imaging method of the non-orthogonal basis dual-channel dual-wavelength phase microscopy imaging system according to embodiment 1, comprising the steps of:
white light sorting: the light beam emitted by the light source 1 passes through the 2f mirror 2, the beam splitting aperture diaphragm 3 and the beam expanding collimating mirror 4 to form two non-orthogonal parallel light beams;
non-orthogonal parallel light sampling: two non-orthogonal parallel light beams irradiate a sample on the sample platform 5, and the two light beams carry sample image field information and respectively enter a first channel and a second channel through the objective lens 6;
the dual-wavelength diffraction light splitting modulation and imaging collection are carried out, namely, a light beam entering a first channel passes through a first diaphragm 7 and a first collimating mirror 8 in sequence, after beam expansion and collimation, the light beam is converted into parallel light beams again, diffraction is generated through a first grating 9, the first grating 9 separates the light beam into multi-order diffraction light, and the multi-order diffraction light comprises light zero-order light with a first wavelength, first-order light, light zero-order light with a second wavelength and first-order light; after passing through the first fourier lens 10, the multi-order diffracted light is irradiated to the first spatial light modulator 11; the first spatial light modulator 11 filters the multiple-level diffracted light, selects the first-wavelength light zero-order light and the first-level light, the second-wavelength light zero-order light and the first-level light, modulates the first-wavelength light and the second-wavelength light zero-order light into 45-degree polarization with the horizontal direction, modulates the first-wavelength light first-level light into vertical polarization, modulates the second-wavelength light first-level light into horizontal polarization, takes the zero-level light as reference light, and takes the first-level light as sample light; the reference light and the sample light pass through a second fourier lens 12, interfere and produce a spatially modulated interference fringe pattern on a first CCD camera 14; the process in the second channel is the same as in the first channel, producing a spatially modulated interference fringe pattern on the second CCD camera 22;
pattern analysis: the interference fringe patterns are acquired by the first CCD camera 14 and the second CCD camera 22 and transmitted to a computer for pattern processing.
It should be understood that although the present invention has been described in terms of various embodiments, not every embodiment includes only a single embodiment, and such description is for clarity purposes only, and those skilled in the art will recognize that the embodiments described herein can be combined as a whole to form other embodiments as would be understood by those skilled in the art.
The above detailed description is given for the purpose of illustrating a practical embodiment of the present invention and is not to be construed as limiting the scope of the present invention, and any equivalent embodiments or modifications thereof without departing from the technical spirit of the present invention are included in the scope of the present invention.
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| US11525991B2 (en) * | 2019-08-30 | 2022-12-13 | The Chinese University Of Hong Kong | Portable quantitative phase microscope for material metrology and biological imaging |
| CN110824688A (en) * | 2019-10-12 | 2020-02-21 | 江苏大学 | A fast reconstruction method of biological cell subsurface morphology based on phase imaging |
| CN110955039B (en) * | 2019-11-15 | 2022-10-14 | 上海安翰医疗技术有限公司 | Phase contrast microscopic imaging system and imaging method thereof |
| CN116297578A (en) * | 2021-12-20 | 2023-06-23 | 中国科学院深圳先进技术研究院 | X-ray Phase Quantitative Imaging Technology and Measurement Method |
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| KR101563810B1 (en) * | 2014-06-25 | 2015-10-28 | 한국과학기술원 | Common-path 3-dimension diffraction light microscopy and method for measuring deformabilty of sample |
| CN104089573A (en) * | 2014-07-03 | 2014-10-08 | 佛山市南海区欧谱曼迪科技有限责任公司 | Multi-channel white light common-channel interference microscopic chromatography system |
| CN106442413A (en) * | 2016-09-29 | 2017-02-22 | 江苏大学 | Two-beam non-orthogonal phase microscopic instant imaging system and method |
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