Tomography imaging system integrating optical microscopic imaging and large sample tissue slicing

Abstract

A tomography imaging system integrating optical microscopic imaging and large sample biological tissue slicing. By means of integration and synchronization of physical slicing and the imaging system, massive three-dimensional image information of an isolated organ or biological tissue can be acquired in a short time. Moreover, the system uses a remote focusing technology to increase an axial scanning speed, greatly shortening the imaging time, and increasing the image data acquisition throughput. In addition, the system is equipped with a dual-excitation optical path for dual-color imaging, so that two different tissue component information can be acquired at the same time.

Description

A tomographic imaging system that combines optical microscopy with large tissue sections Technical Field

[0001] The present application relates to the field of optical microscopy technology, and in particular to a tomography imaging system that integrates optical microscopy and large-sample tissue sections. Background Art

[0002] Existing imaging methods for ex vivo organs or tissues primarily involve manual slicing of the tissue, placing the slices on glass slides, and imaging them using a wide-field fluorescence microscope (or slide scanner). This method is prone to introducing distortion during slicing, hindering 3D image stitching. Furthermore, the extensive manual labor involved in slicing and imaging consumes significant manpower and time when imaging large tissue samples.

[0003] Summary of the Invention

[0004] To solve the above problems, this application adopts the following technical solutions:

[0005] The present application provides a tomographic imaging system that integrates optical microscopy imaging and large sample tissue sectioning, comprising a first optical path unit, a second optical path unit, a third lens (8), a third reflector (9), a seventh lens (28), a first dichroic mirror (10), a fourth lens (11), a second dichroic mirror (12), a second objective lens (13), an automatic sectioning system (14) and a light detection module (15), wherein the first optical path unit comprises a first polarization beam splitter (1), a first lens (2), a second lens (3), a first quarter wave plate (4), a first objective lens (5), a first reflector (6), and a first group of two-axis galvanometer scanning mirrors (7) arranged in sequence along the propagation direction of the light beam, and the second optical path unit comprises a second polarization beam splitter (21), a fifth lens (22), a sixth lens (23), a second quarter wave plate (24), a third objective lens (25), a second reflector (26), and a second group of two-axis galvanometer scanning mirrors (27) arranged in sequence along the propagation direction of the light beam, wherein:

[0006] A first excitation light beam is vertically incident on the first polarization beam splitter (1), and is reflected by the first polarization beam splitter (1), and then sequentially transmits through the first lens (2), the second lens (3), and the first quarter-wave plate (4), and is focused on the first reflector (6) by the first objective lens (5); the first excitation light beam is reflected by the first reflector (6) and then reversely transmits through the first objective lens (5), the first quarter-wave plate (4), the second lens (3), the first lens (2), and the first polarization beam splitter (1); the excitation light beam The light is then scanned by the first group of dual-axis galvanometer scanning mirrors (7), transmitted through the third lens (8), reflected by the third reflector (9), transmitted through the first dichroic mirror (10) and the fourth lens (11), and beam expansion is completed; the expanded excitation light beam is transmitted through the second dichroic mirror (12), and is focused on the sample to be tested by the second objective lens (13); the emission light generated by the excitation is transmitted through the second objective lens (13), reflected by the second dichroic mirror (12), and finally detected by the light detection module (15), thereby completing the scanning imaging of the sample;

[0007] The second excitation light beam is vertically incident on the second polarization beam splitter (21), and is reflected by the second polarization beam splitter (21), and then sequentially transmits through the fifth lens (22), the sixth lens (23), and the second quarter wave plate (24), and is focused on the second reflector (26) by the third objective lens (25); the second excitation light beam is reflected by the second reflector (26), and then reversely transmits through the third objective lens (25), the second quarter wave plate (24), the sixth lens (23), the fifth lens (22), and the second polarization beam splitter (21). (21); the second excitation light beam is scanned by the second group of dual-axis galvanometer scanning mirrors (27), then transmitted through the seventh lens (28), reflected by the first dichroic mirror (10), transmitted through the fourth lens (11) and completed beam expansion; the expanded excitation light beam is transmitted through the second dichroic mirror (12), and is focused on the sample to be tested by the second objective lens (13); the emission light generated by the excitation is transmitted through the second objective lens (13), then reflected by the second dichroic mirror (12), and finally detected by the light detection module (15), completing the scanning imaging of the sample;

[0008] Three-dimensional imaging of large-sized tissue samples: the first group of dual-axis galvanometer scanning mirrors (7) and / or the second group of dual-axis galvanometer scanning mirrors (27) scan and image the sample within the imaging field; after completing the imaging of a single area, the sample is horizontally moved in the X / Y direction with micron accuracy through precise control of the displacement stage carrying the tissue sample to perform scanning imaging of the next area; this process is repeated to complete the scanning imaging of the tissue sample on the same focusing plane; then, the imaged portion of the sample is horizontally cut off by the automatic slicing system (14), and then the new sample surface is imaged after refocusing by the remote focusing module; the above slicing and imaging process is repeated until the imaging data acquisition of the entire sample is completed.

[0009] In some embodiments, the transmission optical element of the first optical path unit and the transmission optical element of the second optical path unit are the same or equivalent optical systems as the transmission optical element of the microscope optical path (mainly composed of the third lens or the seventh lens, the fourth lens and the second objective lens).

[0010] In some embodiments, the focal plane is quickly adjusted by changing the distance between the first reflector (6) and the first objective lens (5).

[0011] In some of the embodiments, the focal plane is quickly adjusted by changing the distance between the second reflector (26) and the third objective lens (25).

[0012] In some embodiments, the tomographic imaging system can simultaneously acquire two different biological tissue components.

[0013] This application adopts the above technical solution, and its beneficial effects are as follows:

[0014] This paper proposes a tomographic imaging system that integrates optical microscopy with large-sample tissue sectioning. This system integrates optical microscopy with automated serial sectioning of biological tissues, enabling automated sectioning and imaging of isolated organs or tissues in a short time, acquiring massive amounts of three-dimensional image information. Furthermore, the system utilizes remote focusing technology to increase axial scanning speed, significantly shortening imaging time and improving image data acquisition throughput. Furthermore, the system features dual excitation optical paths, enabling simultaneous acquisition of information on two distinct tissue components. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments of the present application or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0016] FIG1 is a schematic structural diagram of a tomographic imaging system for integrating optical microscopy imaging and large sample tissue sectioning, provided in an embodiment of the present application. DETAILED DESCRIPTION

[0017] The following describes in detail embodiments of the present application, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present application, and should not be construed as limiting the present application.

[0018] In the description of this application, it should be understood that the terms "upper", "lower", "horizontal", "inside", "outside", etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing this application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on this application.

[0019] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of the technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. Throughout the description of this application, "plurality" means two or more, unless otherwise specifically defined.

[0020] In order to make the purpose, technical solutions and advantages of this application more clear, this application is further described in detail below with reference to the accompanying drawings and embodiments.

[0021] Please refer to FIG1 , which is a schematic diagram of the structure of a tomographic imaging system for integrating multiphoton microscopy and large sample tissue sections provided in this embodiment, including:

[0022] A first optical path unit, a second optical path unit, a third lens (8), a third reflector (9), a seventh lens (28), a first dichroic mirror (10), a fourth lens (11), a second dichroic mirror (12), a second objective lens (13), an automatic slicing system (14) and an optical detection module (15), wherein the first optical path unit comprises a first polarization beam splitter (1), a first lens (2), a second lens (3), a first quarter wave plate (4), a first objective lens (5), a first reflector (6) and a first group of two-axis galvanometer scanning mirrors (7) arranged in sequence along the propagation direction of the light beam, and the second optical path unit comprises a second polarization beam splitter (21), a fifth lens (22), a sixth lens (23), a second quarter wave plate (24), a third objective lens (25), a second reflector (26) and a second group of two-axis galvanometer scanning mirrors (27) arranged in sequence along the propagation direction of the light beam, wherein:

[0023] A first excitation light beam is vertically incident on the first polarization beam splitter (1), and is reflected by the first polarization beam splitter (1), and then sequentially transmits through the first lens (2), the second lens (3), and the first quarter-wave plate (4), and is focused on the first reflector (6) by the first objective lens (5); the first excitation light beam is reflected by the first reflector (6) and then reversely transmits through the first objective lens (5), the first quarter-wave plate (4), the second lens (3), the first lens (2), and the first polarization beam splitter (1); the excitation light beam The light is then scanned by the first group of dual-axis galvanometer scanning mirrors (7), transmitted through the third lens (8), reflected by the third reflector (9), transmitted through the first dichroic mirror (10) and the fourth lens (11), and beam expansion is completed; the expanded excitation light beam is transmitted through the second dichroic mirror (12), and is focused on the sample to be tested by the second objective lens (13); the emission light generated by the excitation is transmitted through the second objective lens (13), reflected by the second dichroic mirror (12), and finally detected by the light detection module (15), thereby completing the scanning imaging of the sample;

[0024] The second excitation light beam is vertically incident on the second polarization beam splitter (21), and is reflected by the second polarization beam splitter (21), and then sequentially transmits through the fifth lens (22), the sixth lens (23), and the second quarter wave plate (24), and is focused on the second reflector (26) by the third objective lens (25); the second excitation light beam is reflected by the second reflector (26), and then reversely transmits through the third objective lens (25), the second quarter wave plate (24), the sixth lens (23), the fifth lens (22), and the second polarization beam splitter (21). (21); the second excitation light beam is scanned by the second group of dual-axis galvanometer scanning mirrors (27), then transmitted through the seventh lens (28), reflected by the first dichroic mirror (10), transmitted through the fourth lens (11) and completed beam expansion; the expanded excitation light beam is transmitted through the second dichroic mirror (12), and is focused on the sample to be tested by the second objective lens (13); the emission light generated by the excitation is transmitted through the second objective lens (13), then reflected by the second dichroic mirror (12), and finally detected by the light detection module (15), completing the scanning imaging of the sample;

[0025] Three-dimensional imaging of large-sized tissue samples: the first set of dual-axis galvanometer scanning mirrors (7) and / or the second set of dual-axis galvanometer scanning mirrors (27) scan and image the sample within the imaging field; after completing the imaging of a single area, the sample is horizontally moved in the X / Y direction with micron accuracy through precise control of the displacement stage carrying the tissue sample to perform scanning imaging of the next area; this process is repeated to complete the scanning imaging of the tissue sample on the same focusing plane; then, the imaged portion of the sample is horizontally cut off by the automatic slicing system (14), and the remote focusing module is refocused to perform the above-mentioned imaging operation on the new sample surface; the above slicing and imaging process is repeated until the imaging data acquisition of the entire sample is completed.

[0026] Taking imaging of a mouse brain as an example, the following is described: In the embodiment, wavelength 1 is 1280 nanometers, which is used to excite the fluorescent dye Alexa680 that marks blood vessels; wavelength 2 is 920 nanometers, which is used to image green fluorescent protein (GFP) that marks nerve cells.

[0027] In this embodiment, the first dichroic mirror is used to reflect light below 1000 nanometers and transmit light above 1040 nanometers.

[0028] In this embodiment, the second dichroic mirror is used to reflect light below 840 nanometers and transmit light above 900 nanometers.

[0029] The above embodiment provides a tomographic imaging system that integrates optical microscopy and large tissue sample sections and operates as follows:

[0030] Taking the excitation light path of wavelength 1 (1280 nanometers) as an example: the 1280-nanometer laser is vertically incident on the first polarization beam splitter and reflected by the first polarization beam splitter, and then transmits through the first lens, the second lens, the first quarter-wave plate, and is focused on the first reflector by the first objective lens; the excitation light beam is reflected by the first reflector and then transmits in reverse through the first objective lens, the first quarter-wave plate, the second lens, the first lens, and the first polarization beam splitter in sequence; the excitation light beam is scanned by the first group of dual-axis galvanometer scanning mirrors, and then transmits through the third lens, reflected by the third reflector, and then transmits through the first dichroic mirror and the fourth lens in sequence (that is, the third lens and the fourth lens perform beam expansion); the expanded excitation light beam transmits through the second dichroic mirror and is focused on the mouse brain to be tested by the second objective lens; the fluorescence generated by the excitation transmits through the second objective lens, then reflected by the second dichroic mirror, and finally detected by the light detection module, completing the scanning imaging of the mouse brain blood vessels in the imaging field.

[0031] The slicing / imaging process of the excitation light path of wavelength 2 (920 nanometers) is the same as that of the excitation light path of wavelength 1, wherein the 920-nanometer excitation light beam is vertically incident on the second polarization beam splitter and reflected by the second polarization beam splitter, and then transmits through the fifth lens, the sixth lens, and the second quarter-wave plate, and is focused on the second reflector by the third objective lens; after being reflected by the second reflector, the excitation light beam is reversely transmitted through the third objective lens, the second quarter-wave plate, the sixth lens, the fifth lens, and the second polarization beam splitter in sequence; the excitation light beam is scanned by the second group of dual-axis galvanometer scanning mirrors, and then transmits through the seventh lens, is reflected by the first dichroic mirror, and then transmits through the fourth lens (i.e., the seventh lens and the fourth lens perform beam expansion); after beam expansion, the excitation light beam transmits through the second dichroic mirror and is focused on the mouse brain to be tested by the second objective lens; the fluorescence generated by the excitation transmits through the second objective lens, is then reflected by the second dichroic mirror, and is finally detected by the light detection module, completing the scanning imaging of the mouse brain nerve cells in the imaging field.

[0032] Three-dimensional imaging of the mouse brain: The galvanometer scanning mirror scans and images the mouse brain sample within the imaging field of view. After completing imaging of a single area (i.e., the objective lens imaging field of view), the mouse brain sample is horizontally moved in the X / Y directions with micron accuracy through precise control of the translation stage to scan and image the next area. This process is repeated to complete scanning and imaging of the mouse brain on the same focal plane. Subsequently, the vibratome horizontally resects the imaged portion of the mouse brain sample, and the remote focusing module refocuses the sample and performs the above imaging operation on the new sample surface. The above slicing and imaging process is repeated until imaging data of the entire mouse brain's nerve cells and blood vessels are collected.

[0033] This paper proposes a tomographic imaging system that integrates optical microscopy with large-sample tissue sectioning. This system integrates optical microscopy with automated serial sectioning of biological tissues, enabling automated sectioning and imaging of isolated organs or tissues in a short time, acquiring massive amounts of three-dimensional image information. Furthermore, the system utilizes remote focusing technology to increase axial scanning speed, significantly shortening imaging time and improving image data acquisition throughput. Furthermore, the system features dual excitation optical paths, enabling simultaneous acquisition of information on two distinct tissue components.

[0034] The above are merely preferred embodiments of the present application and only specifically describe the technical principles of the present application. These descriptions are intended only to explain the principles of the present application and should not be construed in any way as limiting the scope of protection of the present application. Based on the explanations herein, any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present application, as well as other specific implementations of the present application that can be conceived by those skilled in the art without inventive effort, shall be included within the scope of protection of the present application.

Claims

1. A tomographic imaging system integrating optical microscopy imaging and large sample tissue sections, characterized in that: The invention comprises a first optical path unit, a second optical path unit, a third lens (8), a third reflector (9), a seventh lens (28), a first dichroic mirror (10), a fourth lens (11), a second dichroic mirror (12), a second objective lens (13), an automatic slicing system (14) and an optical detection module (15), wherein the first optical path unit comprises a first polarization beam splitter (1), a first lens (2), a second lens (3), a first quarter wave plate (4), a first objective lens (5), a first reflector (6) and a first group of dual-axis galvanometer scanning mirrors (7) which are sequentially arranged along the propagation direction of the light beam, and the second optical path unit comprises a second polarization beam splitter (21), a fifth lens (22), a sixth lens (23), a second quarter wave plate (24), a third objective lens (25), a second reflector (26) and a second group of dual-axis galvanometer scanning mirrors (27) which are sequentially arranged along the propagation direction of the light beam, wherein: A first excitation light beam is vertically incident on the first polarization beam splitter (1), and is reflected by the first polarization beam splitter (1), and then sequentially transmits through a first lens (2), a second lens (3), and a first quarter-wave plate (4), and is focused by the first objective lens (5) on the first reflector (6); the first excitation light beam is reflected by the first reflector (6), and then reversely transmits through the first objective lens (5), the first quarter-wave plate (4), the second lens (3), the first lens (2), and the first polarization beam splitter (1); the excitation light beam The light is then scanned by the first group of dual-axis galvanometer scanning mirrors (7), transmitted through the third lens (8), reflected by the third reflector (9), transmitted through the first dichroic mirror (10) and the fourth lens (11), and beam expansion is completed; the beam-expanded excitation light beam is transmitted through the second dichroic mirror (12), and is focused on the sample to be tested by the second objective lens (13); the emission light generated by the excitation is transmitted through the second objective lens (13), reflected by the second dichroic mirror (12), and finally detected by the light detection module (15), thereby completing the scanning imaging of the sample; The second excitation light beam is vertically incident on the second polarization beam splitter (21), and is reflected by the second polarization beam splitter (21), and then sequentially transmits through the fifth lens (22), the sixth lens (23), and the second quarter wave plate (24), and is focused on the second reflector (26) by the third objective lens (25); the second excitation light beam is reflected by the second reflector (26), and then reversely transmits through the third objective lens (25), the second quarter wave plate (24), the sixth lens (23), and the second quarter wave plate (24), and is focused on the second reflector (26) by the third objective lens (25). The fifth lens (22) and the second polarization beam splitter (21) are connected to the optical system; the second excitation light beam is scanned by the second group of dual-axis galvanometer scanning mirrors (27), then transmitted through the seventh lens (28), reflected by the first dichroic mirror (10), transmitted through the fourth lens (11) and beam expansion is completed; the beam expanded excitation light beam is transmitted through the second dichroic mirror (12), and then focused on the sample to be tested by the second objective lens (13); the emission light generated by the excitation is transmitted through the second objective lens (13), then reflected by the second dichroic mirror (12), and finally detected by the light detection module (15), thereby completing the scanning imaging of the sample; Three-dimensional imaging of large-sized tissue samples: the first group of dual-axis galvanometer scanning mirrors (7) and / or the second group of dual-axis galvanometer scanning mirrors (27) scan and image the sample within the imaging field of view; after completing the imaging of a single area, the sample is horizontally moved in the X / Y direction with micron accuracy through precise control of the displacement stage carrying the tissue sample to perform scanning imaging of the next area; this process is repeated to complete the scanning imaging of the tissue sample on the same focusing plane; then, the automatic slicing system (14) horizontally cuts off the imaged portion of the sample, and then the remote focusing module refocuses and performs imaging operations on the new sample surface; the above slicing and imaging processes are repeated until the imaging data acquisition of the entire sample is completed.

2. The tomography imaging system according to claim 1, characterized in that: The transmission optical element of the first optical path unit and the transmission optical element of the second optical path unit are the same or equivalent optical systems as the transmission optical element of the microscope optical path.

3. The tomography imaging system according to claim 1, characterized in that: The focal plane is quickly adjusted by changing the distance between the first reflector (6) and the first objective lens (5).

4. The tomography imaging system according to claim 1, characterized in that: The focal plane is quickly adjusted by changing the distance between the second reflecting mirror (26) and the third objective lens (25).

5. The tomography imaging system according to claim 1, characterized in that: The tomography imaging system can simultaneously acquire two different biological tissue components.

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