A near-infrared multi-channel synchronous microscopy imaging system

Abstract

The present invention relates to a near-infrared multi-channel synchronous microscopic imaging system, and relates to the field of microscopic optical imaging technology, comprising: a light-shielding structure and a multi-channel imaging unit installed in the light-shielding structure, a barrel lens, a switchable dichroic filter unit, a switchable objective lens unit, a multi-channel excitation unit and an operating stage for object translation; the multi-channel imaging unit comprises: a camera group and a first dichroic mirror group; the multi-channel excitation unit comprises: an optical waveguide lens group and a second dichroic mirror group. The imaging system in the present invention can realize imaging and detection of multiple components at the same time and position.

Description

Technical Field

[0001] The invention relates to the technical field of microscopic optical imaging, and in particular to a near-infrared multi-channel synchronous microscopic imaging system. Background Art

[0002] In the field of life sciences, optical imaging has the advantages of real-time feedback, simple operation, and non-invasiveness. Early optical imaging used fluorescent probes that emitted light in the range of 400nm to 900nm, but because biological tissues have high absorption and scattering of photons in this band, the imaging resolution and penetration depth were low. In recent years, scientists have discovered that photons in the range of 1000nm to 1700nm have low scattering, absorption, and autofluorescence in biological tissues. Fluorescent materials based on emission in this band can achieve deep tissue, high-resolution, and high signal-to-noise ratio optical imaging. At present, wide-field microscopy has been developed to a relatively complete level, and can achieve high-resolution and dynamic real-time observation of living biological samples. However, the existing near-infrared wide-field microscopy systems on the market can only achieve single excitation / single fluorescence imaging functions, but cannot achieve simultaneous imaging and detection of multiple components at the same time and position. Summary of the invention

[0003] The purpose of the present invention is to provide a near-infrared multi-channel synchronous microscopic imaging system, which can realize the imaging and detection of multiple components at the same time and position.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] A near-infrared multi-channel synchronous microscopic imaging system, the imaging system comprising:

[0006] A light-shielding structure and a multi-channel imaging unit, a barrel lens, a switchable dichroic filter unit, a switchable objective lens unit, a multi-channel excitation unit and an operating object translation stage installed in the light-shielding structure;

[0007] The multi-channel imaging unit comprises: a camera group and a first dichroic mirror group;

[0008] The multi-channel excitation unit comprises: an optical waveguide lens group and a second dichroic mirror group;

[0009] The optical waveguide lens group is used to adjust the divergence angle of the excitation light and the diameter of the light source. After the several beams of excitation light are combined through the second dichroic mirror, they reach the switchable dichroic filter unit, and then are reflected by the third dichroic mirror in the switchable dichroic filter unit, and converge to the plane of the operating stage through the objective lens in the switchable objective lens unit. The excitation light is aligned with the position to be measured under the XY two-axis motion adjustment of the operating stage, and the Z axis is adjusted to complete the focusing. The fluorescence generated by the excited fluorescent probe at the imaging position is collimated into parallel light through the switchable objective lens unit, and is transmitted through the dichroic mirror in the switchable dichroic filter unit and filtered out non-fluorescent interference by the filter, and then converged to the focal plane of the camera group through the lens barrel lens to complete the imaging process.

[0010] Optionally, the light-shielding structure is a box.

[0011] Optionally, the camera includes multiple near-infrared cameras.

[0012] Optionally, the first dichroic mirror group includes a plurality of first dichroic mirrors, and the second dichroic mirror group includes a plurality of second dichroic mirrors.

[0013] Optionally, the operating stage includes three-axis combined motion, including three dimensions: X, Y and Z.

[0014] Optionally, the switchable objective lens unit is a 10, 25 or 50x near-infrared objective lens.

[0015] Optionally, the switchable dichroic mirror filter unit includes a third dichroic mirror and a long-pass filter.

[0016] Optionally, the cut-off wavelength of the third dichroic mirror is 1000nm to 1500nm, and the cut-off wavelength of the filter is 1000nm to 1500nm.

[0017] According to the specific embodiments provided by the present invention, the present invention discloses the following technical effects:

[0018] The present invention discloses a near-infrared multi-channel synchronous microscopic imaging system, comprising: a light-shielding structure and a multi-channel imaging unit installed in the light-shielding structure, a lens barrel, a switchable dichroic filter unit, a switchable objective lens unit, a multi-channel excitation unit and an operating object translation stage; the multi-channel imaging unit comprises: a camera group and a first dichroic mirror group; the multi-channel excitation unit comprises: an optical waveguide lens group and a second dichroic mirror group; the optical waveguide lens group is used to adjust the divergence angle of the excitation light and the diameter of the light source, and after several beams of excitation light are combined through the second dichroic mirror, they reach the switchable dichroic filter unit and then pass through the switchable dichroic mirror. The light is reflected by the third dichroic mirror in the dichroic filter unit and converged to the plane of the operating stage through the objective lens in the switchable objective lens unit. The operating stage is used to align the excitation light with the position to be measured under the XY two-axis motion adjustment, and the Z axis is adjusted to complete the focusing. The fluorescence generated by the excited fluorescent probe at the imaging position is collimated into parallel light through the switchable objective lens unit, and is transmitted through the dichroic mirror in the switchable dichroic filter unit and filtered out by the filter to filter out non-fluorescent interference, and then converged to the focal plane of the camera group through the lens barrel lens to complete the imaging process, thereby realizing the imaging and detection of multiple components at the same time and position. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0020] Figure 1 This is a schematic diagram of the structure of a near-infrared multi-channel synchronous microscopic imaging system according to an embodiment of the present invention;

[0021] Figure 2 This is a schematic diagram of synchronous dual-channel emission imaging of living capillary elastic fibers and blood based on a near-infrared multi-channel synchronous microscopic imaging system according to an embodiment of the present invention;

[0022] Figure 3 This is a schematic diagram of synchronous dual-channel excitation imaging of living capillary elastic fibers and blood based on a near-infrared multi-channel synchronous microscopic imaging system according to an embodiment of the present invention.

[0023] Explanation of symbols:

[0024] Near-infrared camera-1, first dichroic mirror-2, tube lens-3, switchable dichroic filter unit-4, switchable objective lens unit-5, second dichroic mirror-6, optical waveguide lens group-7, operating stage-8 and light-shielding structure 9. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] The purpose of the present invention is to provide a near-infrared multi-channel synchronous microscopic imaging system, which can realize the imaging and detection of multiple components at the same time and position.

[0027] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0028] Figure 1 FIG. 1 is a schematic diagram of the structure of a near-infrared multi-channel synchronous microscopic imaging system according to an embodiment of the present invention. Figure 1 As shown, the system comprises:

[0029] A light-shielding structure 9 and a multi-channel imaging unit, a barrel lens, a switchable dichroic filter unit, a switchable objective lens unit, a multi-channel excitation unit and an operating stage for carrying objects installed in the light-shielding structure 9;

[0030] The multi-channel imaging unit comprises: a camera group and a first dichroic mirror group;

[0031] The multi-channel excitation unit comprises: an optical waveguide lens group and a second dichroic mirror group;

[0032] The optical waveguide lens group is used to adjust the divergence angle of the excitation light and the diameter of the light source. After the several beams of excitation light are combined through the second dichroic mirror, they reach the switchable dichroic filter unit, and then are reflected by the third dichroic mirror in the switchable dichroic filter unit, and converge to the plane of the operating stage through the objective lens in the switchable objective lens unit. The excitation light is aligned with the position to be measured under the XY two-axis motion adjustment of the operating stage, and the Z axis is adjusted to complete the focusing. The fluorescence generated by the excited fluorescent probe at the imaging position is collimated into parallel light through the switchable objective lens unit, and is transmitted through the dichroic mirror in the switchable dichroic filter unit and filtered out non-fluorescent interference by the filter, and then converged to the focal plane of the camera group through the lens barrel lens to complete the imaging process.

[0033] The light-shielding structure 9 is a box body, and the light-shielding structure 9 covers the entire component to prevent the interference of ambient light on the imaging quality.

[0034] In the above imaging process, a second dichroic mirror group is arranged in front of the optical waveguide lens group 7, and the second dichroic mirror 6 in the second dichroic mirror group can switch at least three channels of the excitation light to achieve multi-channel timing control excitation. Similarly, a first dichroic mirror group is arranged in front of the near-infrared imaging camera 1, and the first dichroic mirror 2 in the first dichroic mirror group can complete synchronous fluorescence imaging of at least three different bands at the same time and at the same position.

[0035] The switchable dichroic mirror and filter unit 4 can preset up to 8 groups of dichroic mirror and filter combinations with different wavelengths according to the excitation / emission properties of the fluorescent probe.

[0036] The above-mentioned near-infrared multi-channel synchronous microscopy imaging system includes bright field and dark field observations taken by a near-infrared imaging camera. The bright field uses a near-infrared laser as the illumination light source to observe the sample through scattered and reflected light; the dark field is to perform imaging analysis after adding a fluorescent probe and a corresponding filter combination.

[0037] The near-infrared multi-channel synchronous microscopic imaging system includes a switchable objective lens unit 5 with a maximum of 6 channels, and objective lenses with different magnifications can be switched according to needs.

[0038] The near-infrared multi-channel synchronous microscopic imaging system includes three-axis combined motion, including three dimensions of X, Y and Z. The Z-axis motion has two adjustment modes, coarse adjustment and fine adjustment, which can achieve accurate imaging of different parts of the living body.

[0039] The camera includes a plurality of near-infrared cameras 1 , and in this embodiment, there are three near-infrared cameras.

[0040] The first dichroic mirror group includes a plurality of first dichroic mirrors, and the second dichroic mirror group includes a plurality of second dichroic mirrors. In this embodiment, there are two first dichroic mirrors and two second dichroic mirrors.

[0041] The near-infrared multi-channel synchronous microscopic imaging system formed by the above-mentioned device, the multi-channel emission imaging operation method includes the following steps:

[0042] The fluorescent probes used are Er-doped nanoparticles and Ho-doped nanoparticles, with an excitation wavelength of 980 nm and emission wavelengths of 1525 nm and 1180 nm, respectively.

[0043] The surface of the fluorescent probe is modified with mPEG to achieve water phase transfer.

[0044] The Er probe is used to label arterial elastic fibers, and the Ho probe is dispersed in the blood.

[0045] The laser used in the multi-channel excitation unit is a 980nm laser.

[0046] The switchable dichroic filter unit 4 uses a 1100nm long-pass dichroic mirror, with a 1100nm long-pass filter placed on top.

[0047] The switchable objective lens unit 5 uses a 10x near-infrared objective lens.

[0048] The first dichroic mirror 2 in the multi-channel imaging unit is a long-pass dichroic mirror with a cut-off wavelength of 1300 nm.

[0049] The near-infrared camera 1 in the multi-channel imaging unit adopts an InGaAs camera, and the Er probe fluorescence is detected above the first dichroic mirror, and the other camera detects the Ho probe fluorescence.

[0050] The processed sample to be observed is placed on the operating translation stage 8, the instrument is turned on, the laser light source is turned on, and the initial focusing is completed by operating the translation stage 8 through the z-axis translation, and the imaging position is found through the x- and y-axis movements.

[0051] After completing the preliminary focusing, adjust the laser power density, observe the image captured by the near-infrared imaging camera 1 through the computer software, and perform precise focusing to obtain clear fluorescence imaging. The results are as follows: Figure 2 shown.

[0052] The multi-channel excitation imaging operation includes the following steps:

[0053] The fluorescent probes used are two Er-doped nanoparticles ① and ②. The excitation wavelength of probe ① is 808 nm, the excitation wavelength of probe ② is 980 nm, and the emission wavelengths of ① and ② are both 1525 nm.

[0054] The surfaces of probes ① and ② were modified with mPEG to achieve water phase transfer.

[0055] The 808nm excitation probe ① is used to label the arterial elastic fibers, and the 980nm excitation probe ② is dispersed in the blood.

[0056] The lasers in the multi-channel excitation unit are 980nm and 808nm lasers, and the second dichroic mirror 6 is a long-pass dichroic mirror with a cut-off wavelength of 900nm.

[0057] The switchable dichroic filter unit 4 uses a 1100nm long-pass dichroic mirror, with a 1100nm long-pass filter placed on top.

[0058] The switchable objective lens unit 5 uses a 25x near-infrared objective lens.

[0059] The near infrared camera 1 in the multi-channel imaging unit adopts an InGaAs camera.

[0060] Place the processed sample to be observed on the operating translation stage 8, turn on the instrument, turn on the laser light source respectively, complete the preliminary focusing by operating the translation stage 8 on the z-axis, and find the imaging position by moving on the x- and y-axis.

[0061] After completing the preliminary focusing, adjust the laser power density, observe the image captured by the near-infrared imaging camera 1 through the computer software, and perform precise focusing to obtain clear fluorescence imaging. The results are as follows: Figure 3 shown.

[0062] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0063] The principles and implementation methods of the present invention are described in this article using specific examples. The description of the above embodiments is only used to help understand the method and core idea of ​​the present invention. At the same time, for those skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A near-infrared multi-channel synchronous microscopic imaging system, characterized in that: The imaging system comprises: A light-shielding structure and a multi-channel imaging unit, a barrel lens, a switchable dichroic filter unit, a switchable objective lens unit, a multi-channel excitation unit and an operating object translation stage installed in the light-shielding structure; The multi-channel imaging unit comprises: a camera group and a first dichroic mirror group; the camera group comprises a plurality of near-infrared cameras, and the near-infrared imaging camera performs bright field and dark field observations, wherein the bright field uses near-infrared laser as an illumination light source, and observes the sample through scattered and reflected light; the dark field is performed by adding a fluorescent probe and a corresponding filter combination for imaging analysis; The multi-channel excitation unit comprises: an optical waveguide lens group and a second dichroic mirror group; The optical waveguide lens group is used to adjust the divergence angle of the excitation light and the diameter of the light source. After the several beams of excitation light are combined through the second dichroic mirror, they reach the switchable dichroic filter unit, and then are reflected by the third dichroic mirror in the switchable dichroic filter unit, and converge to the plane of the operating stage through the objective lens in the switchable objective lens unit. The excitation light is aligned with the position to be measured under the XY two-axis motion adjustment of the operating stage, and the Z axis is adjusted to complete the focusing. The fluorescence generated by the excited fluorescent probe at the imaging position is collimated into parallel light through the switchable objective lens unit, and is transmitted through the dichroic mirror in the switchable dichroic filter unit and filtered out non-fluorescent interference by the filter, and then converged to the focal plane of the camera group through the lens barrel lens to complete the imaging process.

2. The near-infrared multi-channel synchronous microscopic imaging system according to claim 1, characterized in that: The light-shielding structure is a box.

3. The near-infrared multi-channel synchronous microscopic imaging system according to claim 1, characterized in that: The first dichroic mirror group includes a plurality of first dichroic mirrors, and the second dichroic mirror group includes a plurality of second dichroic mirrors.

4. The near-infrared multi-channel synchronous microscopic imaging system according to claim 1, characterized in that: The operating object translation stage includes three-axis combined motion, including three dimensions: X, Y and Z.

5. The near-infrared multi-channel synchronous microscopic imaging system according to claim 1, characterized in that: The switchable objective lens unit is a 10, 25 or 50 times near-infrared objective lens.

6. The near-infrared multi-channel synchronous microscopic imaging system according to claim 1, characterized in that: The switchable dichroic mirror filter unit includes a third dichroic mirror and a long-pass filter.

7. The near-infrared multi-channel synchronous microscopic imaging system according to claim 6, characterized in that: The cut-off wavelength of the third dichroic mirror is 1000nm to 1500nm, and the cut-off wavelength of the filter is 1000nm to 1500nm.

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