Polarization-selective dual-wavelength achromatic integrated fluorescence imaging device and method

Abstract

The present invention discloses a polarization-selective dual-wavelength achromatic integrated fluorescence imaging device and method, which includes an image sensor and a polarizing circular polarization film located on the surface of the image sensor, as well as a multilayer dielectric film, a meta-lens, and a light source for respectively providing incident light of two wavelengths to realize side waveguide illumination; the multilayer dielectric film includes a Bragg reflector and two layers of central cavity for filtering the two wavelengths of light; the phase of the nanostructure unit of the meta-lens modulates the orthogonal polarization state of the outgoing light when the first wavelength incident light is incident; and modulates the same polarization state of the outgoing light when the second wavelength incident light is incident; imaging detection of two-color fluorescence samples is realized by switching the wavelength and polarization state of the incident light; the meta-lens of the present invention can realize efficient dual-wavelength fluorescence imaging corresponding to both wavelengths, is simple to operate, has high integration, and is widely applicable to various two-color fluorescence imaging.

Description

Technical Field

[0001] The present invention relates to a fluorescence imaging device and method, in particular to a polarization-selective dual-wavelength integrated fluorescence imaging device and method. Background Art

[0002] Current traditional fluorescence microscopy systems are not only large and complex, but also extremely expensive. Simplifying the system, reducing costs, and ensuring adequate imaging performance are of particular concern. Currently, to miniaturize fluorescence imaging systems, most solutions are based on miniaturized lens systems and light sources (LEDs or LDs), while optimizing the illumination and receiving light paths, such as using oblique illumination or waveguide illumination to effectively avoid the reception of excitation light. Some integrated fluorescence systems also directly use mobile phones as imaging tools. However, existing integrated fluorescence systems are mostly based on traditional refractive lenses, so they are relatively thick compared to meta-lenses and only support monochrome fluorescence imaging.

[0003] In fact, many fluorescence imaging applications, such as fluorescence co-localization technology, require the detection of at least two colors of fluorescence signals to analyze the interrelationships between the structures of biological cell samples stained with fluorescent dyes. The meta-lenses in existing fluorescence integrated systems are designed for a single wavelength and can only perform single-pass filtering, making them inapplicable to multi-color fluorescence imaging. To achieve multi-color fluorescence imaging, it is often necessary to replace components in the system, such as filters, lenses, and light sources. As a result, the system stability is relatively poor, and it is impossible to observe different fluorescence signals in the same area of ​​the sample almost simultaneously. In addition, the corresponding multi-wavelength excitation light source is also relatively large, which runs counter to the goal of high integration. Summary of the Invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a highly integrated fluorescence imaging device that can achieve achromatic imaging detection of fluorescence at two wavelengths without replacing the meta-lens; the second purpose of the present invention is to provide a method for achieving achromatic imaging of fluorescence at two wavelengths.

[0005] Technical solution: The polarization-selective dual-wavelength achromatic integrated fluorescence imaging device of the present invention includes an image sensor and an analyzer circular polarization film located on the surface of the image sensor, as well as a multilayer dielectric film, a meta-lens, and a light source;

[0006] The multilayer dielectric film includes a Bragg reflector and two layers of central cavities, which are used to filter light of two wavelengths; the multilayer dielectric film is located between the meta-lens and the analyzer circular polarization film;

[0007] The phase of the nanostructure unit of the meta-lens modulates the orthogonal polarization state of the outgoing light when incident light of a first wavelength is incident; and modulates the same polarization state of the outgoing light when incident light of a second wavelength is incident;

[0008] The light source is used to provide incident light of two wavelengths to realize side waveguide illumination.

[0009] Furthermore, when the incident light of the first wavelength is incident, the orthogonal polarization state of the outgoing light is modulated, and the first phase delay of the nanostructure unit of the meta-lens is in is the propagation phase at the first wavelength λ1, θ is the rotation angle of the nanostructure unit;

[0010] When the second wavelength incident light is incident, the outgoing light is modulated in the same polarization state, and the second phase delay of the nanostructure unit of the meta-lens is in is the propagation phase at the second wavelength λ2.

[0011] Furthermore, the phase distribution of the metalens satisfies:

[0012]

[0013]

[0014] in, is the phase distribution of the metalens at the first wavelength λ1, is the phase distribution of the metalens at the second wavelength λ2, x and y are the position coordinates of each nanostructure unit, f1 is the focal length set at the first wavelength λ1, f2 is the focal length set at the second wavelength λ2, and f1=f2.

[0015] Furthermore, a polarizing circular polarization film is provided between the sample and the meta-lens, and the polarizing circular polarization film is replaced to switch the polarization state of the incident light of two wavelengths provided by the light source.

[0016] Furthermore, the light source includes two light bars with different wavelengths, which are respectively placed at both ends of the sample, for coupling light into the sample from the side, performing waveguide illumination on the sample and stimulating fluorescence signals.

[0017] Furthermore, the nanostructure unit of the metalens is a structure that is mirror-symmetrical or has high-order selective symmetry, including an elliptical shape, a rectangular shape, a double rectangular shape, a cross shape, and an H shape.

[0018] In the polarization-selective dual-wavelength integrated fluorescence imaging method described in the present invention, a light source provides incident light of two wavelengths. The incident light is modulated into corresponding polarization states and then filtered by a multilayer dielectric film. The outgoing light after passing through the meta-lens is then received by the image sensor for fluorescence imaging.

[0019] The multilayer dielectric film includes a Bragg reflector and two layers of central cavities for filtering light of two wavelengths;

[0020] The phase of the nanostructure unit of the meta-lens modulates the orthogonal polarization state of the outgoing light when the incident light of the first wavelength is incident; and modulates the same polarization state of the outgoing light when the incident light of the second wavelength is incident.

[0021] Furthermore, when the incident light of the first wavelength is incident, the first phase delay of the nanostructure unit of the meta-lens is in is the propagation phase at the first wavelength λ1, θ is the rotation angle of the nanostructure unit; when the second wavelength incident light is incident, the second phase delay of the nanostructure unit of the meta-lens is in is the propagation phase at the second wavelength λ2.

[0022] Furthermore, the phase distribution of the metalens satisfies:

[0023]

[0024]

[0025] in, is the phase distribution of the metalens at the first wavelength λ1, is the phase distribution of the metalens at the second wavelength λ2, x and y are the position coordinates of each nanostructure unit, f1 is the focal length set at the first wavelength λ1, f2 is the focal length set at the second wavelength λ2, and f1=f2.

[0026] Furthermore, the incident light is modulated into the corresponding polarization state by a method in which the incident light passes through a circular polarizing film and is modulated into the corresponding polarization state.

[0027] Beneficial effects: Compared with the prior art, the advantages of the present invention are:

[0028] (1) Through the dual-wavelength-dependent meta-lens that can independently control the phase of both the co-polarization and orthogonal polarization, and the multi-layer dielectric film with dual-channel filtering, the meta-lens can achieve efficient dual-wavelength achromatic fluorescence imaging at both wavelengths;

[0029] (2) By switching the wavelength and polarization state of the illumination light source, the detection of two fluorescence signals with different wavelengths can be achieved, effectively distinguishing different fluorescence signals with simple operation;

[0030] (3) By integrating a polarization-dependent dual-wavelength achromatic meta-lens and an analyzer circular polarization film onto a CMOS image sensor and combining it with a double-sided illumination light source, a highly integrated dual-color fluorescence imaging device is realized;

[0031] (4) Through corresponding wavelength design, the fluorescence imaging device of the present invention can be applied to various dual-color fluorescence imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the fluorescence imaging device of the present invention.

[0033] Figure 2 Schematic diagram of the working principle of the dual-wavelength achromatic meta-lens in an embodiment of the present invention.

[0034] Figure 3 The focal plane spot diagram and the light field distribution diagram along the axial propagation obtained by simulation calculation of the dual-wavelength achromatic meta-lens in an embodiment of the present invention at two different wavelengths.

[0035] Figure 4 This is an optical micrograph of a dual-wavelength achromatic meta-lens according to an embodiment of the present invention.

[0036] Figure 5 Graph showing the focusing experiment results of the dual-wavelength achromatic meta-lens in an embodiment of the present invention.

[0037] Figure 6 This is a biological imaging result diagram of the dual-wavelength achromatic meta-lens in an embodiment of the present invention.

[0038] Figure 7 This is a transmission spectrum diagram of the multilayer dielectric film for dual-channel filtering in an embodiment of the present invention.

[0039] Figure 8 Schematic diagram of a multilayer dielectric film for dual-channel filtering in an embodiment of the present invention. DETAILED DESCRIPTION

[0040] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0041] like Figure 1 As shown, the polarization-selective dual-wavelength achromatic integrated fluorescence imaging device described in the present invention includes a CMOS image sensor 1, a polarizing circular polarization film 2, a quartz substrate 3, a multilayer dielectric film 4, a meta-lens 5, a polarizing circular polarization film 6, a light source 7 and a sample 8; sample 8 is a fluorescent sample.

[0042] The multilayer dielectric film 4 and the meta-lens 5 are located between the polarizing circular polarizing film 6 and the analyzing circular polarizing film 2 . The polarizing circular polarizing film 6 can be replaced to switch the polarization state. The analyzing circular polarizing film 2 and the meta-lens 5 are integrated on the surface of the CMOS sensor 1 .

[0043] The multilayer dielectric film 4 is designed to achieve the function of dual-wavelength bandpass filtering, corresponding to the two central wavelengths of fluorescence imaging, namely wavelength 1 and wavelength 2. The multilayer dielectric film 4 is designed based on the principle of Bragg reflection waveguide, and is composed of a distributed Bragg reflector and a central cavity introduced therein; the distributed Bragg reflector is made of two materials with different nanometer thicknesses and refractive indices arranged in a periodic manner, which can cause Bragg scattering when light propagates therein, forming a highly reflective film at certain frequencies. The central cavity generally uses a low-refractive-index material to produce defects within the frequency range of high reflection, thereby achieving the function of overall filtering. The specific design process can control the size and number of the defect center frequency by changing the position, thickness and number of the central cavity. For the design process of the multilayer dielectric film 4 for dual-channel filtering: first, a periodic Bragg reflector is constructed to form a high reflection effect in a certain frequency range, and then two layers of central cavities are introduced to achieve the filtering effect of the two center frequencies.

[0044] Metalens 5 uses a wavelength-dependent design scheme for simultaneous and independent phase control of both the same polarization and the orthogonal polarization. This allows different phase designs to be achieved by changing the circular polarization state and wavelength of the incident light when the outgoing light has the same polarization state. The polarization multiplexing design method is based on the principle of joint control of geometric phase and propagation phase. By selecting appropriate nanounit structures and rotation angles, different intensity and phase distributions at different wavelengths can be achieved. The nanounit structure can include elliptical, rectangular, double rectangular, cross, H-shaped, and other structures that meet mirror symmetry or high-order selective symmetry to ensure that the two orthogonal circularly polarized lights have no phase delay.

[0045] Specifically, for a single nanostructure unit, the relationship between the outgoing field and the incident field can be expressed as: where t x , t y is the complex amplitude obtained after the incident linear polarization light along the x and y directions, and θ is the rotation angle of a single structure. If the selected nanostructure only satisfies the orthogonal polarization control, then and If the selected nanostructure unit only satisfies the same polarization phase control, we can get and If wavelength 1 is controlled by orthogonal polarization, the phase of wavelength 1 satisfies The corresponding wavelength 2 is controlled by the same polarization, and the phase satisfies where φRR ,φ RL ,φ LR ,φ LL To ensure the feasibility of the above design, it is necessary to find a structure that satisfies the conditions that the orthogonal polarization conversion efficiency is close to 1 at wavelength 1 and the same polarization conversion efficiency is close to 1 at wavelength 2.

[0046] Light source 7 is constructed by embedding two LED light strips with different wavelengths, each corresponding to the wavelengths of the excitation light sources for the two fluorescent lights. These two LED light strips are fixed to either end of the glass slide of sample 8, coupling light from the sides of the slide into the planar waveguide formed by the slide for propagation. Once the light reaches sample 8, fluorescence is stimulated, and the illumination light source is confined by total internal reflection within the slide, effectively isolating the excitation and emission light for fluorescence imaging.

[0047] When performing a focus characterization test on the metalens 5, the following components are also required: a light source 7, a polarizing circular polarizing film 6 (including both left-handed and right-handed circular polarizing films), a 50x objective lens, and a CCD. The polarizing circular polarizing film 6 is positioned between the light source 7 and the metalens 5; the objective lens is positioned between the metalens 5 and the CCD. The metalens 5 can produce focused light spots with the same focal length when incident on two different wavelengths of light with corresponding circular polarization states, thus satisfying dual-wavelength achromatism.

[0048] When performing imaging characterization testing on the metalens 5, the following components are also required: a light source 7, a polarizing circular polarizing film 6 (including a left-handed circular polarizing film and a right-handed circular polarizing film), an imaging object, and a CMOS image sensor 1. The polarizing circular polarizing film 6 is positioned between the light source 7 and the imaging object; and the metalens 5 is positioned between the imaging object and the CMOS image sensor 1. When the metalens 5 is illuminated by light of two different wavelengths and corresponding circular polarization states, imaging results at different wavelengths at the same focal length can be obtained, for the same imaging magnification.

[0049] In this embodiment, a polarization-dependent dual-wavelength achromatic meta-lens 5 is designed for two different wavelengths, corresponding to the same polarization and orthogonal polarization control, respectively, to determine the polarization state of the output light as right-handed circularly polarized light, such as Figure 2 shown.

[0050] To design the dual-wavelength achromatic metalens 5, it is first necessary to select an appropriate nanostructure unit structure such that the structure achieves a cross-polarization conversion efficiency close to 1 and a same-polarization conversion efficiency close to 1 at the two achromatic wavelengths. The nanostructure units in this embodiment are made of silicon nitride, have a period of 400 nm, and a height of 1.5 μm. The corresponding cross-sectional shapes of the nanostructure units include elliptical, rectangular, and double-rectangular. The quartz substrate 3, which serves as the substrate for the metalens 5, is 500 μm thick and measures 1 cm × 1 cm.

[0051] The phase of the nanounit structure satisfies:

[0052]

[0053]

[0054] in It represents the first phase delay that the nanometer unit structure can provide under different polarization control at the first wavelength λ1. represents the second phase delay that the nanostructure can provide under different polarization control at the first wavelength λ2; θ is the rotation angle of the nanostructure unit; is the propagation phase corresponding to λ1 and λ2.

[0055] The phase distribution of metalens 5 satisfies:

[0056]

[0057]

[0058] in represents the phase distribution of the metalens 5 under λ1 and λ2; x, y are the position coordinates of each nanostructure; in this embodiment, λ1 is set to 525nm and λ2 is set to 450nm; f1 and f2 are the focal lengths set under λ1 and λ2. Under the design goal of dual-wavelength achromatism, the focal lengths of different wavelengths are set to the same value.

[0059] In order to verify the accuracy and feasibility of the dual-wavelength achromatic meta-lens 5 in this embodiment, the meta-lens 5 in this embodiment is first simulated using simulation software. Since the simulation software (Lumerical FDTDSolutions) requires a lot of computing resources, the diameter of the meta-lens 5 is set to 4μm, and f1=f2=9μm in this simulation. The simulation results are shown in Figure 2. Figure 3 As shown, Figure 3 (a) shows the focal plane spot diagram corresponding to a wavelength of 525nm and the light field distribution diagram along the axial propagation. Figure 3 (b) shows the focal plane spot diagram corresponding to a wavelength of 450nm and the light field distribution diagram propagating along the axial direction.

[0060] Furthermore, the specific processing of the meta-lens 5 is achieved by electron beam exposure and dry etching, such as Figure 4 As shown, the meta-lens

[0061] The diameter is 400 μm, the focal length is 500 μm, and the corresponding two working wavelengths are 525 nm and 450 nm. The focus of the processed meta-lens 5 is characterized, and the focal spot diagram on the focal plane is as follows: Figure 5 As shown, it is in perfect accordance with the design. Figure 6 The following diagrams show the imaging results at two different wavelengths. The magnification of the two wavelengths is consistent, and the image details are well matched, so they can be used for dual-color fluorescence imaging.

[0062] In this embodiment, a dual-channel filtering multilayer dielectric film 4 is designed for two different wavelengths. Corresponding to the design of the meta-lens 5 in this embodiment, λ1 is 525nm and λ2 is 450nm. Figure 7 The figure shows the transmission spectrum of the multilayer dielectric film 4. The materials of the distributed Bragg reflector designed in this embodiment are silicon nitride (n=2) and silicon oxide (n=1.48), and the central cavity material introduced is silicon oxide. First, a high reflective mirror film with a wavelength of 350nm-600nm needs to be constructed. Five different groups of Bragg reflectors (DBRs) are selected. Each group of reflectors consists of five layers of silicon nitride (DBRs) with different thicknesses. H ) and silicon oxide (D L ) film composition, as shown in the table below; in order to achieve the filtering effect at 525nm and 450nm, a certain thickness of silicon oxide is introduced between the 3rd and 4th groups and the 2nd and 3rd groups. The thickness is determined by the specific parameter scanning calculation process. Figure 8 The thicknesses of the thin film interlayers shown are 88 nm and 68 nm, corresponding to filtering at 525 nm and 450 nm, respectively.

[0063] thickness Group 1 Group 2 Group 3 Group 4 Group 5 <![CDATA[D H / nm]]> 44 51 60 70 81 <![CDATA[D L / nm]]> 63 73 84 96 111

[0064] The light source 7 in this embodiment includes LED lighting beads with wavelengths of 470 nm and 365 nm, respectively, for exciting fluorescent signals near 525 nm and 450 nm.

[0065] The working process of the polarization-selective dual-wavelength integrated fluorescence imaging device described in the present invention is as follows: the first incident light emitted by the blue light LED (470nm) in the light source 7 enters the sample 8 slide from the side and emits total internal reflection. When the light is transmitted to the sample 8 area, it excites the sample 8 to emit fluorescence. The emitted fluorescence passes through the polarizing circular polarizing film 6 and changes to the corresponding designed polarization state. It passes through the dual-wavelength achromatic meta-lens 5 and the dual-channel filtered multi-layer dielectric film 4, and then passes through the fixed analyzer circular polarizing film 2, and is finally received by the CMOS image sensor 1; the polarizing circular polarizing film 6 is replaced; the second incident light emitted by the ultraviolet LED (365nm) on the other side enters the sample 8 slide from the side, and then excites fluorescence. The emitted fluorescence passes through the polarizing circular polarizing film 6, and passes through the subsequent meta-lens 5, the multi-layer dielectric film 4, and the analyzer circular polarizing film 2 in sequence, and is received by the CMOS image sensor 1.

[0066] Based on the same inventive concept, the polarization-selective dual-wavelength achromatic integrated fluorescence imaging method described in the present invention comprises a light source 7 providing incident light of two wavelengths, which is modulated into corresponding polarization states and then filtered by a multilayer dielectric film 4. The outgoing light, after passing through a meta-lens 5, is then received by a CMOS image sensor 1 for fluorescence imaging.

[0067] The multilayer dielectric film 4 includes a Bragg reflector and two layers of central cavities, and is used to filter light of two wavelengths;

[0068] The phase of the nanostructure unit of the meta-lens 5 modulates the orthogonal polarization state of the outgoing light when incident light of a first wavelength is incident; and modulates the same polarization state of the outgoing light when incident light of a second wavelength is incident.

[0069] When the incident light of the first wavelength is incident, the first phase delay of the nanostructure unit of the meta-lens 5 is in is the propagation phase at the first wavelength λ1, θ is the rotation angle of the nanostructure unit; when the second wavelength incident light is incident, the second phase delay of the nanostructure unit of the meta-lens 5 is in is the propagation phase at the second wavelength λ2.

[0070] The phase distribution of the metalens 5 satisfies:

[0071]

[0072]

[0073] in, is the phase distribution of the metalens (5) at the first wavelength λ1, is the phase distribution of the meta-lens (5) at the second wavelength λ2, x and y are the position coordinates of each nanostructure unit, f1 is the focal length set at the first wavelength λ1, f2 is the focal length set at the second wavelength λ2, and f1=f2.

[0074] The incident light is modulated into the corresponding polarization state in the following manner: the incident light passes through the circular polarizing film 6 and is modulated into the corresponding polarization state.

Claims

1. A polarization-selective dual-wavelength achromatic integrated fluorescence imaging device, comprising an image sensor (1) and an analyzer circular polarization film (2) located on the surface of the image sensor (1), characterized in that: It also includes a multilayer dielectric film (4), a meta-lens (5), a light source (7) and a polarizing circular polarization film (6); The multilayer dielectric film (4) includes a Bragg reflector and two layers of central cavities, and is used for filtering light of two wavelengths; the multilayer dielectric film (4) is located between the meta-lens (5) and the analyzer circular polarization film (2); The phase of the nanostructure unit of the meta-lens (5) modulates the orthogonal polarization state of the outgoing light when incident light of a first wavelength is incident; and modulates the same polarization state of the outgoing light when incident light of a second wavelength is incident; The light source (7) is used to provide incident light of two wavelengths respectively; A polarizing circular polarization film (6) is provided between the sample (8) and the meta-lens (5), and the polarizing circular polarization film (6) is replaced to switch the polarization state of the incident light of two wavelengths provided by the light source (7).

2. The polarization-selective dual-wavelength achromatic integrated fluorescence imaging device according to claim 1, characterized in that: When the incident light of the first wavelength is incident, the orthogonal polarization state of the outgoing light is modulated, and the first phase delay of the nanostructure unit of the meta-lens (5) is in is the propagation phase at the first wavelength λ1, θ is the rotation angle of the nanostructure unit; When the second wavelength incident light is incident, the outgoing light is modulated in the same polarization state, and the second phase delay of the nanostructure unit of the meta-lens (5) is in is the propagation phase at the second wavelength λ2.

3. The polarization-selective dual-wavelength achromatic integrated fluorescence imaging device according to claim 1, characterized in that: The phase distribution of the meta-lens (5) satisfies: in, is the phase distribution of the metalens (5) at the first wavelength λ1, is the phase distribution of the meta-lens (5) at the second wavelength λ2, x and y are the position coordinates of each nanostructure unit, f1 is the focal length set at the first wavelength λ1, f2 is the focal length set at the second wavelength λ2, and f1=f2.

4. The polarization-selective dual-wavelength achromatic integrated fluorescence imaging device according to claim 1, characterized in that: The light source (7) includes two light bars with different wavelengths, which are respectively placed at both ends of the sample (8) and are used to couple light from the side of the sample (8) to perform waveguide illumination on the sample (8) and stimulate fluorescence signals.

5. The polarization-selective dual-wavelength achromatic integrated fluorescence imaging device according to claim 1, characterized in that: The nanostructure unit of the meta-lens (5) is a structure with mirror symmetry or high-order selective symmetry, including elliptical, rectangular, double rectangular, cross and H-shaped.

6. A polarization-selective dual-wavelength achromatic integrated fluorescence imaging method, characterized in that: The light source (7) provides incident light of two wavelengths respectively. The incident light is modulated into corresponding polarization states and then filtered by a multilayer dielectric film (4). Then, the outgoing light after passing through a meta-lens (5) is received by an image sensor (1) for fluorescence imaging. The multilayer dielectric film (4) includes a Bragg reflector and two layers of central cavities for filtering the two wavelengths of light. The phase of the nanostructure unit of the meta-lens (5) modulates the orthogonal polarization state of the outgoing light when incident light of a first wavelength is incident; and modulates the same polarization state of the outgoing light when incident light of a second wavelength is incident; the method of modulating the incident light into the corresponding polarization state is that the incident light is modulated into the corresponding polarization state by passing through a polarizing circular polarization film (6).

7. The polarization-selective dual-wavelength achromatic integrated fluorescence imaging method according to claim 6, characterized in that: When the incident light of the first wavelength is incident, the first phase delay of the nanostructure unit of the meta-lens (5) is in is the propagation phase at the first wavelength λ1, θ is the rotation angle of the nanostructure unit; when the second wavelength incident light is incident, the second phase delay of the nanostructure unit of the meta-lens (5) is in is the propagation phase at the second wavelength λ2.

8. The polarization-selective dual-wavelength achromatic integrated fluorescence imaging method according to claim 6, characterized in that: The phase distribution of the meta-lens (5) satisfies: in, is the phase distribution of the metalens (5) at the first wavelength λ1, is the phase distribution of the meta-lens (5) at the second wavelength λ2, x and y are the position coordinates of each nanostructure unit, f1 is the focal length set at the first wavelength λ1, f2 is the focal length set at the second wavelength λ2, and f1=f2.

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