CN111272066A - Dual-mode optical microscopic imaging device based on incident light polarization control - Google Patents
Dual-mode optical microscopic imaging device based on incident light polarization control Download PDFInfo
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- CN111272066A CN111272066A CN202010110734.0A CN202010110734A CN111272066A CN 111272066 A CN111272066 A CN 111272066A CN 202010110734 A CN202010110734 A CN 202010110734A CN 111272066 A CN111272066 A CN 111272066A
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Abstract
The invention discloses a dual-mode optical microscopic imaging device based on incident light polarization control, wherein incident light is converted into left-handed circularly polarized light and right-handed circularly polarized light through the combination of a polarizer and a filter to irradiate a sample to be detected, transmitted light passing through the sample passes through a microscope objective and a relay lens and then enters a microstructure spatial filter, the microstructure spatial filter applies two different additional phases to the two orthogonal left-handed circularly polarized light and right-handed circularly polarized light and reverses the circular polarization state of the two orthogonal left-handed circularly polarized light and right-handed circularly polarized light, and the transmitted light passing through the microstructure spatial filter passes through an imaging lens and a crossed circular polarizer to be imaged on an image acquisition. The microscopic imaging device can realize the real-time switching of the common bright field imaging and the phase difference imaging modes without adding active equipment such as an electric control device and the like, thereby being capable of quickly obtaining the information of the overall appearance, the edge form and the like of the amplitude type and phase type imaging objects, and having very important scientific significance and practical value.
Description
Technical Field
The invention belongs to the field of micro-nano optics, and particularly relates to a micro-imaging device capable of controlling an imaging mode by utilizing the polarization state of incident light.
Background
The main purpose of optical microscopes is to magnify tiny objects for human eye observation. Since birth, it has become a new means for human to gain insight into the world, and is widely used in many fields such as biomedicine, materials science, and physical chemistry. Because of its importance, improvements in microscopy have never been stopped. Even so far, it is still a very active research area.
The common optical microscope mainly takes a bright field imaging mode as a main mode. However, many biological samples (tissues, cells, bacteria, etc.) do not contain natural pigments, so they can be considered phase type objects rather than amplitude type objects, and the light difference of the samples is very small, so they cannot be effectively identified using conventional imaging modes. Marking and dyeing a sample is a conventional means, and the dyed sample changes the brightness and color of light to obtain a large contrast ratio, but causes problems of deformation of the sample and death of a living body.
To solve these problems, phase contrast microscopy was proposed in 1957. Phase contrast microscopy differs from the usual bright-dark field imaging techniques and mainly images the edge morphology of objects under observation, so it has important applications in scientific research and medical testing of phasic objects, such as for examination of parasites in blood and observation of certain living cells, for identification of various cells and casts in fresh urine, in particular the classification of hyaline casts, leukocytes, tubular epithelial cells and red blood cell morphology, for rapid identification of bacteria, spirochetes, fungal spores, etc. Because the common imaging mode and the phase difference imaging mode can respectively reflect the shape information of different dimensions of the amplitude type object and the phase type object, the method has very important scientific significance and practical value by integrating the common imaging mode and the phase difference imaging mode in a set of microscopic imaging device and realizing the dynamic mode switching.
Object of the Invention
The invention aims to provide a microscopic imaging device which can control a microscopic system to realize two different imaging modes of common bright field imaging and phase difference imaging by only changing two orthogonal polarization states of incident light, such as an orthogonal linear polarization state, an orthogonal circular polarization state and an orthogonal elliptical polarization state, and can control the imaging modes by utilizing the polarization states of the incident light without active control modes of an external electric field or a magnetic field and the like.
In order to achieve the purpose, the invention adopts the following technical scheme: a dual-mode optical microscopy imaging device based on incident light polarization control comprises: the device comprises a light source, a polarizer, a filter, a microscope objective, a relay lens, a microstructure spatial filter, an imaging lens, a crossed circular polarizer and an image acquisition device, wherein the polarizer, the filter, the microscope objective, the relay lens, the microstructure spatial filter, the imaging lens, the crossed circular polarizer and the image acquisition device are sequentially far away from the light source and are arranged on an emergent light path of the; the sample to be measured is placed between the filter and the microscope objective, incident light is converted into left-handed circularly polarized light and right-handed circularly polarized light through the combination of the polarizer and the filter to irradiate the sample to be measured, transmitted light passing through the sample passes through the microscope objective and the relay and then enters the microstructure spatial filter, the microstructure spatial filter applies two different additional phases to the two orthogonal left-handed circularly polarized light and right-handed circularly polarized light and reverses the circular polarization state of the two orthogonal left-handed circularly polarized light and right-handed circularly polarized light, and the transmitted light passing through the microstructure spatial filter is imaged on the image acquisition device after passing through the imaging lens and the crossed.
Further, the microstructure spatial filter comprises a transparent substrate and a microstructure pillar array arranged on the surface of the substrate, wherein the period and the height of the microstructure pillar array are close to the wavelength size, and the following conditions are met:
wherein:
theta is the rotation angle of the microstructure for additional phase of the microstructure's major and minor axes.
Furthermore, the microstructure adopts titanium oxide, hafnium oxide, silicon nitride, aluminum, silver or gold material.
Further, the transparent substrate is transparent quartz.
Further, the microstructure spatial filter is prepared by the following method:
(1) spin-coating a layer of PMMA on the surface of a transparent substrate, and coating 10nm of aluminum on the PMMA layer through thermal evaporation;
(2) completing electron beam lithography operation under the accelerating voltage of 125 kilovolts, developing for 120 seconds by using ethyl acetate, and then growing a layer of microstructure with the thickness of 150nm on the surface of the transparent substrate by using an atomic layer deposition technology, wherein the growth temperature is 90 ℃;
(3) using Cl2And BCl3The mixed gas carries out inductively coupled plasma reactive ion etching on the microstructure until the microstructure reaches the PMMA layer;
(4) and (3) placing the transparent substrate under ultraviolet irradiation, and then soaking the transparent substrate by using n-methyl-2-pyrrolidone to remove the remaining PMMA, thereby obtaining the microstructure column array on the surface of the transparent substrate, namely the microstructure spatial filter.
Furthermore, the number of the relay lenses is 1-2.
Further, the image acquisition device is an imaging CCD.
The invention mainly aims at the defect that the traditional microscopic imaging technology can not dynamically switch between the common bright field imaging mode and the phase difference imaging mode, and utilizes a micro-structure space filter consisting of sub-wavelength structures to form a microscopic imaging system to realize the control of the imaging mode by utilizing two orthogonal polarization states. The micro-imaging device can realize the real-time switching of the common bright field imaging and the phase difference imaging modes without adding active equipment such as an electric control device, and the like, thereby being capable of quickly obtaining the information of the overall appearance, the edge form and the like of an amplitude type imaging object and a phase type imaging object, and being capable of carrying out wide-wavelength imaging on incident light with different frequencies from ultraviolet to infrared, thereby having very important scientific significance and practical value.
Drawings
FIG. 1 is a schematic optical path diagram of a dual-mode optical microscopy imaging device according to an embodiment.
FIG. 2 is an example imaged object and imaging results: a. an imaging object composed of three light-transmitting slits; b. a spatial filtering function consisting of constant phases and a calculated common bright field imaging result; c. a spatial filtering function consisting of helical phases and a computed phase difference imaging result.
FIG. 3 is a conversion of polarized light by the microstructured spatial filter of an embodiment: a. for left-handed circularly polarized light incidence, the microstructure spatial filter performs constant phase filtering operation; b. for right-handed circularly polarized light incidence, the microstructured spatial filter performs a helical phase filtering operation.
Fig. 4 is a schematic structural diagram of a microstructure spatial filter according to an embodiment, wherein the right side is a side view and a top view of a microstructure unit.
Fig. 5 is a microscope picture of an example microstructured spatial filter: a. an optical microscope picture of the processed microstructure spatial filter; b. the processed microstructure spatial filter scans the electron microscope picture.
FIG. 6 shows experimental results of dual-mode optical microscopy imaging for the examples: a. under the incidence of the left-handed circularly polarized light, bright field imaging images of resolution test plates with the wavelengths of 480nm, 530nm, 580nm and 630nm respectively; b. and under the incidence of right-handed circularly polarized light, the wavelengths of the phase difference imaging images of the resolution test plate are 480nm, 530nm, 580nm and 630nm respectively.
Detailed Description
In order to make the technical solutions better understood by those skilled in the art, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only partial embodiments of the present application, but 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 application.
It should be noted that the terms "comprises" and "comprising," and any variations thereof, in the description and claims of this application and the above-described drawings, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
1. Dual mode microscopic imaging device principles
The optical path of the dual-mode microscopic imaging device system is schematically shown in FIG. 1, and the dual-mode microscopic imaging device system mainly comprises a polarizer, a wave plate, a microscope objective, a relay lens, a microstructure spatial filter, an imaging lens and an imaging CCD. Wherein the key core component is a microstructure spatial filter. Taking the example of controlling orthogonal circularly polarized light, any incident light can be converted to left-handed circularly polarized Light (LCP) and right-handed circularly polarized light (RCP) by a combination of polarizer and wave plate to illuminate the sample. The transmitted light passing through the sample is incident on the microstructure spatial filter surface after passing through the microscope objective and the relay lens. The microstructured spatial filter surface will impose two different additional phases for two orthogonal left-handed and right-handed circularly polarized light and will reverse the circular polarization it has. For left-handed circularly polarized light, the additional phase is an equiphase plane with an arbitrary constant, and the polarization state is converted into right-handed circularly polarized light. And for the right-handed circularly polarized light, the additional phase of the right-handed circularly polarized light is in a spiral phase distribution of 0-2 pi, and the polarization state of the right-handed circularly polarized light is converted into left-handed circularly polarized light. And the transmitted light passing through the microstructure spatial filter enters an imaging CCD after passing through an imaging lens and a crossed circular polarizer, so that imaging recording is completed. Taking a group of imaging objects comprising three light-transmitting slits as an example (fig. 2a), wherein the corresponding additional phase is left-handed circularly polarized light of an equiphase plane, the imaging mode is common bright field imaging, and the obtained image is the overall appearance of the three light-transmitting slits (fig. 2 b). And the right-handed circularly polarized light corresponding to the additional phase and showing a 0-2 pi spiral phase distribution can generate a pi phase difference relative to the frequency spectrums at two ends of the central zero frequency in any direction of the frequency spectrum plane of the incident light field, and the hilbert transform can be realized in any direction, so that the imaging mode is edge-enhanced phase difference imaging, and the obtained image is the edge morphology of three light-transmitting slits (fig. 2 c). Dynamic switching between the two imaging modes can be achieved by only changing the rotation angles of the polarizer and the wave plate to control the two orthogonal circular polarization states of the incident light.
2. Design of micro-structured spatial filter
As a key core component of a dual-mode microscopic imaging apparatus, a microstructured spatial filter must have two sets of completely independent additional phase responses to incident light exhibiting two orthogonal polarizations. The microstructured spatial filter we designed consists of a series of subwavelength scale microstructures. Each microstructure can be regarded as a sub-wavelength scale waveguide that can perform wavefront modulation on incident light. Again using a set of orthogonal circularly polarized light as an example, left-handed light
And rotation to the right
Respectively incident on the surfaces of the microstructure spatial filters. If the device is to perform two additional phase filtering effects (M) independently1The additional phase is an equiphase plane distribution, M2The additional phase is a 0-2 pi helical phase distribution), then it must satisfy the following transformation:
LCP:|L>→M1|R>(1)
RCP:|R>→M2|L>(2)
for left-handed Gaussian beam incidence, the microstructured spatial filter performs M1In operation, the outgoing light is a right-handed gaussian beam, as shown in fig. 3 a. For right-handed Gaussian beam incidence, the microstructured spatial filter performs M2In operation, the outgoing light is a left-handed beam with orbital angular momentum of order 1, as shown in FIG. 3 b. In this case, the microstructural spatial filter can be written in the form of a jones matrix, as follows:
the hermitian symmetric form of the matrix ensures that it can be diagonalized. Eigenvalues and eigenvectors require that the device should have a birefringent response with additional phase introduced by the major and minor axes of each microstructure
And the rotation angle θ can be written as the following analytic expression:
according to the formulas (4) - (6), a set of appropriate microstructures can be found out through calculation, so that the phase shift and the variation range of the orientation angle of the microstructures cover 0-2 pi, and a corresponding microstructure spatial filter can be designed. In the ultraviolet to infrared frequency band, the non-metal material which can be selected to form the microstructure mainly comprises titanium oxide, hafnium oxide, silicon nitride and the like, the metal material comprises aluminum, silver, gold and the like, and the non-metal material is superior to the metal material in efficiency due to low absorption loss.
Examples
Taking visible light frequency range as an example, titanium oxide TiO with low loss is selected2As a constituent material of the microstructure spatial filter. FIG. 4 is a schematic diagram of a microstructure spatial filter made of TiO2The rectangular nano-columns are arranged in a tetragonal lattice mode. The period of the nanopillars was 450nm and the height was 600 nm. Each nanorod is effectively a small half-wave plate with a phase shift along the symmetry axis of the plate
And
can adjust the in-plane dimension DxAnd DyThe geometric phase can be controlled by the in-plane rotation angle of the nano-column. In LCP and RCP form, depending on the appropriate combination of nanopillar propagation phase and geometric phaseTwo groups of mutually independent phase distributions are obtained on incident light in the state.
The processing and preparation method of the microstructure spatial filter comprises the following steps: first, a 600nm thick layer of PMMA was spin coated on top of a quartz substrate. Subsequently, the sample was coated with 10nm of aluminum by thermal evaporation to avoid charging effects in the electron beam lithography step. The electron beam lithography operation was performed at an accelerating voltage of 125 kv and developed using ethyl acetate for 120 seconds. Then growing a layer of TiO 150nm thick in the pattern by using atomic layer deposition technology2The growth temperature was set to 90 ℃ to ensure that the PMMA was not deformed. On the basis of this, Cl is used2And BCl3Performing inductively coupled plasma reactive ion etching (ICP-RIE) on the coated TiO2Etching is performed until the PMMA layer is reached. Finally, the sample was exposed to ultraviolet radiation and then soaked with n-methyl-2-pyrrolidone. This step removes the remaining PMMA, yielding a predesigned TiO2An array of pillars. Fig. 5 shows a machined microstructured spatial filter (2 mm diameter), optical and scanning electron micrographs showing higher finesse and lower surface roughness.
A schematic of a dual mode optical microscopy imaging apparatus is shown in fig. 1. The sample is placed on a target plane and irradiated by the supercontinuum laser connected with an acousto-optic tunable filter system. The complete conversion of the polarization state of the incident light from linear polarization to circular polarization is achieved by using a linear polarizer and a quarter-wave plate. A 20-fold microscope objective is used to magnify biological sample images. The back focal plane of the objective lens is extended backward by using a relay system composed of two lenses. The super-surface spatial filter is placed at the exact position of the fourier plane of the objective lens. In order to improve the imaging quality, a crossed circular polarizer is used between the imaging lens and the CCD to eliminate background light with the same rotation direction as that of the input light beam. To demonstrate the actual functioning of the imaging system, a standard resolution test plate (1951USAF) was used as the imaging target and illuminated by supercontinuum lasers of different wavelengths. For left-handed polarized incident light with visible wavelengths of 480nm, 530nm, 580nm, and 630nm, FIG. 6a shows the first resolution test plateBright field images of four sets of samples corresponding to a constant phase function M implemented by a microstructured spatial filter1The imaging effect of (1). When the polarization state of the incident light is changed from left-handed to right-handed, FIG. 6b shows an edge-enhanced phase difference image of the same target test area, which corresponds to the spiral phase function M implemented by the micro-structured spatial filter2The imaging effect of (1). The contrast of the image of all sample edges was enhanced regardless of the direction and wavelength of the incident light, indicating that such edge-enhanced phase-contrast imaging is isotropic and works well over a wide wavelength range.
While the invention has been described with respect to the preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. A dual-mode optical microscopy imaging device based on incident light polarization control is characterized by comprising: the device comprises a light source, a polarizer, a filter, a microscope objective, a relay lens, a microstructure spatial filter, an imaging lens, a crossed circular polarizer and an image acquisition device, wherein the polarizer, the filter, the microscope objective, the relay lens, the microstructure spatial filter, the imaging lens, the crossed circular polarizer and the image acquisition device are sequentially far away from the light source and are arranged on an emergent light path of the; the sample to be measured is placed between the filter and the microscope objective, incident light is converted into left-handed circularly polarized light and right-handed circularly polarized light through the combination of the polarizer and the filter to irradiate the sample to be measured, transmitted light passing through the sample passes through the microscope objective and the relay and then enters the microstructure spatial filter, the microstructure spatial filter applies two different additional phases to the two orthogonal left-handed circularly polarized light and right-handed circularly polarized light and reverses the circular polarization state of the two orthogonal left-handed circularly polarized light and right-handed circularly polarized light, and the transmitted light passing through the microstructure spatial filter is imaged on the image acquisition device after passing through the imaging lens and the crossed.
2. The dual-mode optical microscopy imaging setup based on incident light polarization control of claim 1, wherein: the microstructure spatial filter comprises a transparent substrate and a microstructure column array arranged on the surface of the substrate, wherein the period and the height of the microstructure column array are close to the wavelength size and meet the following conditions:
wherein:
theta is the rotation angle of the microstructure for additional phase of the microstructure's major and minor axes.
3. The dual-mode optical microscopy imaging device based on incident light polarization control of claim 2, wherein: the microstructure adopts titanium oxide, hafnium oxide, silicon nitride, aluminum, silver or gold materials.
4. The dual-mode optical microscopy imaging device based on incident light polarization control of claim 2, wherein: the transparent substrate is transparent quartz.
5. The dual-mode optical microscopy imaging device based on incident light polarization control of claim 2, wherein: the microstructure spatial filter is prepared by the following method:
(1) spin-coating a layer of PMMA on the surface of a transparent substrate, and coating 10nm of aluminum on the PMMA layer through thermal evaporation;
(2) completing electron beam lithography operation under the accelerating voltage of 125 kilovolts, developing for 120 seconds by using ethyl acetate, and then growing a layer of microstructure with the thickness of hundreds of nanometers on the surface of the transparent substrate by using an atomic layer deposition technology or other vacuum coating technologies, wherein the growth temperature is 90 ℃;
(3) using Cl2And BCl3The mixed gas carries out inductively coupled plasma reactive ion etching on the microstructure until the microstructure reaches the PMMA layer;
(4) and (3) placing the transparent substrate under ultraviolet irradiation, and then soaking the transparent substrate by using n-methyl-2-pyrrolidone to remove the remaining PMMA, thereby obtaining the microstructure column array on the surface of the transparent substrate, namely the microstructure spatial filter.
6. The dual-mode optical microscopy imaging setup based on incident light polarization control of claim 1, wherein: the number of the relay lenses is 1-2.
7. The dual-mode optical microscopy imaging setup based on incident light polarization control of claim 1, wherein: the image acquisition device is an imaging CCD.
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