Modularized fluorescence microscopic imaging device capable of imaging in real time

Abstract

The invention relates to a modularized fluorescence microscopic imaging device capable of imaging in real time, which can realize lossless long-term imaging by using a mild LED light source and intelligent exposure control, reduce the influence of phototoxicity on a biological sample and keep the activity of the sample. According to the invention, continuous fluorescence signal detection can be carried out on cells in the detection hole, the same group of cells or the same cluster of cells. All events occurring in the whole time period can be completely recorded and reflected, and the method is particularly suitable for observing the growth and development process of all cells, especially the growth and development of neuronal cells.

Description

Technical Field

[0001] This invention relates to the field of microscopic imaging technology, specifically to a non-destructive, real-time imaging fluorescence microscopic imaging device that is compact in appearance, modularly assembled, and simple and flexible to use. Background Technology

[0002] With the continuous advancement and development of microscopic imaging technology, it has been widely applied in fields such as biology and chemistry, capturing image signals through optical detection imaging. However, there are several problems with using existing commercially available microscopic imaging equipment in biological experimental research. First, prolonged exposure to light can lead to phototoxicity, reducing the sensitivity of biological samples and even causing them to lose their activity. Organic molecules absorb light and degrade when reacting with oxygen, releasing reactive oxygen species such as superoxide radicals and hydrogen peroxide, which can damage cells and limit the actual observation time. Second, while commercial microscopes are powerful, their operation is complex and cumbersome, and their prices are high, limiting their widespread use and promotion. Third, the large size of traditional microscopes limits their use in confined spaces. Finally, although high-throughput biological screening technologies based on optical detection have been widely applied, the development of high-throughput imaging detection has been slow. Therefore, there is an urgent need to develop more flexible, compact, and modular high-throughput imaging microscopes to address the above problems. Summary of the Invention

[0003] Addressing the problems existing in current imaging microscopy technology on the market, we propose a novel solution: 1. Non-destructive real-time imaging: By using a gentle LED light source and intelligent exposure control, we can achieve non-destructive long-term imaging, reducing the impact of phototoxicity on biological samples and preserving sample viability. 2. Simple and flexible design: Our device is compact, easy to use, modularly integrated, and easy to operate. This makes it suitable for various experimental studies and can be easily integrated into other instruments. 3. Low cost: Our simple design and low cost facilitate market promotion and widespread adoption. In summary, our invention, the imaging microscope, solves some of the bottleneck problems in existing technologies, providing a more flexible, efficient, and cost-effective tool for biological research.

[0004] The objective of this invention is achieved through the following technical solutions.

[0005] A modular, real-time imaging microscopic imaging device includes a light source 1, a condenser lens 2, a first filter 3, a dichroic mirror 4, a second filter 5, a lens 6, a closed optical path 7, a camera 8, an objective lens 9, a digital triggering device 10, and a stage 11.

[0006] The light source 1 emits excitation light under the control of the digital trigger device 10. The light is focused by the condenser lens 2 and then filtered through the first filter 3 to obtain a specific wavelength of excitation light. The specific wavelength of excitation light is reflected by the dichroic mirror 4 to the objective lens 9, which illuminates the observed sample and excites the sample fluorescence signal. The fluorescence signal is collected by the objective lens 9, passes through the dichroic mirror 4, and is then filtered through the second filter 5 to obtain a specific wavelength of fluorescence. The light then passes through the optical path to the lens 6 and converges to the camera 8 to complete the imaging process.

[0007] Camera 8 can be configured differently depending on the sampling frame rate and pixel requirements. Digital triggering device 10 is connected to both light source 1 and camera 8, outputting trigger signals to both. These signals control the switching on / off of the light source and the camera's exposure during shooting. The light source is turned on during the camera's exposure time and turned off after the exposure ends. A gentler LED light source is used, and filters and achromatic lenses enhance the purity of the excitation light, ensuring long-term non-phototoxicity to living organisms and achieving non-destructive imaging.

[0008] Lens 6 is connected to camera 8, and the signal is focused onto camera 8 to form an image based on the parameters of lens 6. Lens 6 is connected to a closed optical path 7 to ensure that the optical path is not interfered with by other light rays, thus ensuring signal fidelity and clarity.

[0009] Dichroic mirror 4 has the characteristic of "long wavelengths pass through, short wavelengths reflect," meaning it reflects short-wavelength light and transmits long-wavelength light. Therefore, the short-wavelength excitation light from light source 1 is reflected to the sample by the dichroic mirror, and the long-wavelength fluorescence generated by the sample will pass through the dichroic mirror.

[0010] When the optical signal passes through the first filter 3, the wavelength range of the light passing through the first filter 3 can be limited.

[0011] The optical signal passes through the second filter 5, and the wavelength range of the light passing through the second filter 5 can be limited.

[0012] A multi-well plate is placed on the stage 11 for placing samples.

[0013] More specifically, light source 1 is an LED light;

[0014] More specifically, the wavelength range of the LED lights is 400-660nm; more specifically, they are red LEDs, blue LEDs, and green LEDs.

[0015] More specifically, the LED light is a blue LED, the first filter 3 has a wavelength transmission range of 450-500nm; the second filter 5 has a wavelength transmission range of 500-540nm;

[0016] More specifically, the LED light is a green LED, the first filter 3 has a wavelength transmission range of 540-575nm; the second filter 5 has a wavelength transmission range of 575-625nm.

[0017] More specifically, the LED light is a red LED, the first filter 3 has a wavelength transmission range of 625-650nm; the second filter 5 has a wavelength transmission range of 650-700nm;

[0018] More specifically, the digital trigger device 10 is a device that transmits TTL level signals, such as a microcontroller.

[0019] Specifically, lens 6 is an achromatic lens.

[0020] Specifically, the exposure time is 1-1000ms, and more specifically, it is 1-100ms. More specifically, the exposure time accounts for less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% of a single imaging cycle.

[0021] Specifically, the magnification of objective lens 9 is 4-20 times, and the numerical aperture of objective lens 9 is 0.17-0.8.

[0022] Specifically, camera 8 can be an industrial camera.

[0023] Specifically, the stage 11 has a multi-well plate on it, with the number of microwells in the multi-well plate being 24, 48, 96, or 384. Multiple sizes of multi-well plates can be replaced as needed. The stage 11 can be an X-shaped stage, allowing for the replacement of different culture dishes as needed, such as 3.5cm or 6cm diameter culture dishes.

[0024] More specifically, the imaging device also includes a support module comprising a column 12, a column clamping block 13, and an XYZ platform 14.

[0025] More specifically, the imaging device also includes a temperature and humidity control module for a cell culture incubator 15.

[0026] Compared with the prior art, the advantages of this invention are:

[0027] (1) Long-term non-destructive fluorescence imaging. This invention uses a gentler LED light source and enhances the purity of the excitation light through filters and achromatic lenses to avoid stray light pollution. On this basis, a digital triggering device controls the light source to be lit only at the moment of imaging, greatly reducing the illumination time. This allows the system to observe biological samples for a long time while significantly reducing phototoxicity, thus achieving long-term non-destructive imaging.

[0028] (2) Real-time imaging. This invention enables continuous fluorescence signal detection on cells in the detection well, the same group of cells, or the same cluster of cells. It can completely record and reflect all events that occur throughout the entire time period. It is particularly suitable for observing the growth and development process of all cells, especially the growth and development of neurons. Because neurons are more fragile and have more demanding environmental requirements compared to other cells, this technology is very useful for studying cell growth and development processes.

[0029] (3) Multi-resolution, multi-band imaging. This invention is equipped with objectives of different numerical apertures (0.17-0.8), which can flexibly adjust the magnification and resolution as needed. It is equipped with LED light sources and filter groups of different wavelengths, which can perform microscopic imaging of a variety of fluorescent groups (400-660nm). Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the structure of Embodiment 1 of the present invention.

[0031] Figure 2 This is a schematic diagram of the structure of Embodiment 2 of the present invention.

[0032] Figure 3 This is a schematic diagram of the structure of Embodiment 3 of the present invention.

[0033] Figure label:

[0034] 1-Light source, 2-Condenser lens, 3-First filter, 4-Dichromatic mirror, 5-Second filter, 6-Lens, 7-Closed optical path, 8-Camera, 9-Objective lens; 10-Digital trigger device, 11-Stage, 12-Column, 13-Column clamp, 14-XYZ platform, 15-Temperature and humidity control module for cell culture incubator. Detailed Implementation

[0035] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The following embodiments are only used to more clearly illustrate the technical solutions of the present invention, and are therefore merely examples and should not be used to limit the scope of protection of the present invention.

[0036] It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning understood by those skilled in the art. The terms "first," "second," etc., in the specification, claims, and accompanying drawings of this disclosure are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate for the embodiments of this disclosure described herein.

[0037] Example 1

[0038] like Figure 1As shown, a non-destructive, real-time imaging microscopic device includes a light source 1, a condenser lens 2, a first filter 3, a dichroic mirror 4, a second filter 5, a lens 6, a closed optical path 7, a camera 8, an objective lens 9, a digital triggering device 10, and a stage 11.

[0039] Light source 1 emits excitation light, which is focused by condenser lens 2 and then filtered through a first filter 3 to obtain a specific wavelength of excitation light. The specific wavelength of excitation light is reflected by dichroic mirror 4 to objective lens 9, illuminating the observed sample and exciting a sample fluorescence signal. The fluorescence signal is collected by objective lens 9, passes through dichroic mirror 4, and is then filtered through a second filter 5 to obtain a specific wavelength of fluorescence. The light then passes through the optical path to lens 6 and converges to camera 8 to complete the imaging process.

[0040] Light source 1 is an LED lamp, controlled by a digital trigger device 10. Camera 8 can be configured differently depending on the sampling frame rate and pixel requirements. The digital trigger device 10 is connected to both light source 1 and camera 8, outputting trigger signals to both. These signals control the light source's on / off state and the camera's exposure. The light source is turned on during the camera's exposure time and turned off after the exposure. A gentler LED light source is used, and filters and achromatic lenses enhance the purity of the excitation light, ensuring long-term non-phototoxicity to living organisms and achieving non-destructive imaging.

[0041] Lens 6 is connected to camera 8, and the signal is focused onto camera 8 to form an image based on the parameters of lens 6. Lens 6 is connected to a closed optical path 7 to ensure that the optical path is not interfered with by other light rays, thus ensuring signal fidelity and clarity.

[0042] A multi-well plate is placed on the stage 11 for placing samples.

[0043] Dichroic mirror 4 has the characteristic of "long wavelengths pass through, short wavelengths reflect," meaning it reflects short-wavelength light and transmits long-wavelength light. Therefore, the short-wavelength excitation light from light source 1 is reflected to the sample by the dichroic mirror, and the long-wavelength fluorescence generated by the sample will pass through the dichroic mirror.

[0044] When the optical signal passes through the first filter 3, the wavelength range of the light passing through the first filter 3 can be limited.

[0045] The optical signal passes through the second filter 5, and the wavelength range of the light passing through the second filter 5 can be limited.

[0046] The lens of objective lens 9 can be changed according to the required magnification (4x, 10x, 20x, etc.).

[0047] In some embodiments, the LED is a blue LED, the first filter 3 has a wavelength transmission range of 450-500nm, and the second filter 5 has a wavelength transmission range of 500-540nm.

[0048] In some embodiments, the LED is a green LED, the first filter 3 has a wavelength transmission range of 540-575nm, and the second filter 5 has a wavelength transmission range of 575-625nm.

[0049] In some embodiments, the LED is a red LED, the first filter 3 has a wavelength transmission range of 625-650nm, and the second filter 5 has a wavelength transmission range of 650-700nm.

[0050] In some embodiments, the exposure time is set to 5ms and the imaging cycle is 100ms.

[0051] In some embodiments, the exposure time is set to 10ms and the imaging cycle is 100ms.

[0052] In some embodiments, the exposure time is set to 20ms and the imaging cycle is 100ms.

[0053] In some embodiments, the exposure time is set to 30ms and the imaging cycle is 100ms.

[0054] In some embodiments, the exposure time is set to 40ms and the imaging cycle is 100ms.

[0055] In some embodiments, the exposure time is set to 50ms and the imaging cycle is 100ms.

[0056] In some embodiments, the exposure time is set to 40ms and the imaging cycle is 200ms.

[0057] In some embodiments, the exposure time is set to 50ms and the imaging cycle is 300ms.

[0058] In some embodiments, camera 8 can be an industrial camera.

[0059] In some embodiments, the digital triggering device 10 may be a microcontroller.

[0060] In some embodiments, the stage 11 is topped with a multi-well plate, the multi-well plate having 24, 48, 96, or 384 wells. Multiple sizes of multi-well plates can be replaced as needed. The stage 11 can be an X-shaped stage, allowing for the replacement of different culture dishes as required, such as 3.5cm or 6cm diameter culture dishes.

[0061] Example 2

[0062] like Figure 2As shown, a modular, real-time imaging fluorescence microscopy device includes an imaging module and a support module. The imaging module includes a light source 1, a condenser lens 2, a first filter 3, a dichroic mirror 4, a second filter 5, a lens 6, a closed optical path 7, a camera 8, an objective lens 9, a digital triggering device 10, and a stage 11; the support module includes a column 12, a column clamping block 13, and an XYZ platform 14.

[0063] Light source 1 emits excitation light, which is focused by condenser lens 2 and then filtered through a first filter 3 to obtain a specific wavelength of excitation light. The specific wavelength of excitation light is reflected by dichroic mirror 4 to objective lens 9, illuminating the observed sample and exciting a sample fluorescence signal. The fluorescence signal is collected by objective lens 9, passes through dichroic mirror 4, and is then filtered through a second filter 5 to obtain a specific wavelength of fluorescence. The light then passes through the optical path to lens 6 and converges to camera 8 to complete the imaging process.

[0064] Light source 1 is an LED light, controlled by a digital trigger device 10. Camera 8 can be configured differently depending on the sampling frame rate and pixel requirements. The digital trigger device 10 is connected to both light source 1 and camera 8, outputting trigger signals to both. These signals control the light source's on / off state and the camera's exposure. The light source is on during the camera's exposure time and off after exposure. A gentler LED light source is used, employing a lens to enhance signal intensity without increasing the light source frequency, enabling long-term, non-phototoxic imaging of biological subjects.

[0065] Lens 6 is connected to camera 8, and the signal is focused onto camera 8 to form an image based on the parameters of lens 6. Lens 6 is connected to a closed optical path 7 to ensure that the optical path is not interfered with by other light rays, thus ensuring signal fidelity and clarity.

[0066] A multi-well plate is placed on the stage 11 for placing samples.

[0067] Dichroic mirror 4 has the characteristic of "long wavelengths pass through, short wavelengths reflect," meaning it reflects short-wavelength light and transmits long-wavelength light. Therefore, the short-wavelength excitation light from light source 1 is reflected to the sample by the dichroic mirror, and the long-wavelength fluorescence generated by the sample will pass through the dichroic mirror.

[0068] When the optical signal passes through the first filter 3, the wavelength range of the light passing through the first filter 3 can be limited.

[0069] The optical signal passes through the second filter 5, and the wavelength range of the light passing through the second filter 5 can be limited.

[0070] The lens of objective lens 9 can be changed according to the required magnification (4x, 10x, 20x, etc.).

[0071] The light source, exposure time, color filter parameters, etc. can be set as in Example 1.

[0072] The imaging module is fixed to the column 12 via the column clamp 13, and the column 12 is fixed to the XYZ platform 14. The column 12 can move horizontally on the XYZ platform 14, and through program control, it can move to detect samples with different micropores in the multi-well plate on the stage 11.

[0073] In some embodiments, camera 8 can be an industrial camera.

[0074] In some embodiments, the digital triggering device 10 may be a microcontroller.

[0075] In some embodiments, the stage 11 has a perforated plate on top, and the number of micropores in the perforated plate is 24, 48, 96, or 384. Various perforated plates of different specifications can be replaced as needed. The stage 11 can be an X-shaped stage.

[0076] Example 3

[0077] A real-time imaging micro-fluorescence imaging device includes a temperature and humidity control module, an imaging module, a support module, and a base plate.

[0078] like Figure 2 , Figure 3 As shown, the imaging module includes a light source 1, a condenser lens 2, a first filter 3, a dichroic mirror 4, a second filter 5, a lens 6, a closed optical path 7, a camera 8, an objective lens 9, a digital triggering device 10, and a stage 11; the support module includes a column 12 and a column clamping block 13; the base plate is an XYZ platform 14; and the temperature and humidity control module is a cell culture incubator 15.

[0079] Light source 1 emits excitation light, which is focused by condenser lens 2 and then filtered through a first filter 3 to obtain a specific wavelength of excitation light. The specific wavelength of excitation light is reflected by dichroic mirror 4 to objective lens 9, illuminating the observed sample and exciting a sample fluorescence signal. The fluorescence signal is collected by objective lens 9, passes through dichroic mirror 4, and is then filtered through a second filter 5 to obtain a specific wavelength of fluorescence. The light then passes through the optical path to lens 6 and converges to camera 8 to complete the imaging process.

[0080] Light source 1 is an LED light, controlled by a digital trigger device 10. Camera 8 can be configured differently depending on the sampling frame rate and pixel requirements. The digital trigger device 10 is connected to both light source 1 and camera 8, outputting trigger signals to both. These signals control the light source's on / off state and the camera's exposure. The light source is on during the camera's exposure time and off after exposure. The use of a gentler LED light source, employing a lens, enhances signal intensity without increasing the light source frequency, enabling long-term, non-phototoxic imaging of biological subjects.

[0081] Lens 6 is connected to camera 8, and the signal is focused onto camera 8 to form an image based on the parameters of lens 6. Lens 6 is connected to a closed optical path 7 to ensure that the optical path is not interfered with by other light rays, thus ensuring signal fidelity and clarity.

[0082] A multi-well plate is placed on the stage 11 for placing samples.

[0083] Dichroic mirror 4 has the characteristic of "long wavelengths pass through, short wavelengths reflect," meaning it reflects short-wavelength light and transmits long-wavelength light. Therefore, the short-wavelength excitation light from light source 1 is reflected to the sample by the dichroic mirror, and the long-wavelength fluorescence generated by the sample will pass through the dichroic mirror.

[0084] When the optical signal passes through the first filter 3, the wavelength range of the light passing through the first filter 3 can be limited.

[0085] The optical signal passes through the second filter 5, and the wavelength range of the light passing through the second filter 5 can be limited.

[0086] The lens of objective lens 9 can be changed according to the required magnification (4x, 10x, 20x, etc.).

[0087] The imaging module is fixed to the column 12 via the column clamp 13, and the column 12 is fixed to the XYZ platform 14. The column 12 can move horizontally on the XYZ platform 14, and through program control, it can move to detect samples with different micropores in the multi-well plate on the stage 11.

[0088] The light source, exposure time, color filter parameters, etc. can be set as in Example 1.

[0089] In some embodiments, camera 8 can be an industrial camera.

[0090] In some embodiments, the digital triggering device 10 may be a microcontroller.

[0091] In some embodiments, the stage 11 has a perforated plate on top, and the number of micropores in the perforated plate is 24, 48, 96, or 384. Various perforated plates of different specifications can be replaced as needed. The stage 11 can be an X-shaped stage.

Claims

1. A modular, real-time imaging microscopic imaging device, characterized in that, The system includes a light source (1), a condenser lens (2), a first filter (3), a dichroic mirror (4), a second filter (5), a lens (6), a closed optical path (7), a camera (8), an objective lens (9), a digital trigger device (10), and a stage (11). The digital trigger device (10) is connected to the light source (1) and the camera (8) respectively, and is used to output trigger signals to the light source and the camera. The trigger signals are used to control the switching of the light source and the exposure of the camera. The light source is turned on during the camera's exposure time and turned off after the exposure ends.

2. The imaging device according to claim 1, characterized in that, The exposure time accounts for less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% of a single imaging cycle.

3. The imaging device according to claim 1, characterized in that, The light source is an LED lamp (1), with a wavelength range of 400-660nm.

4. The imaging device according to claim 1, characterized in that, The light source (1) is a blue LED, the first filter 3 has a wavelength transmission range of 450-500nm, and the second filter 5 has a wavelength transmission range of 500-540nm.

5. The imaging device according to claim 1, characterized in that, The light source (1) is a green LED, the first filter 3 has a wavelength transmission range of 540-575nm, and the second filter 5 has a wavelength transmission range of 575-625nm.

6. The imaging device according to claim 1, characterized in that, The light source (1) is a red LED, the first filter 3 has a wavelength transmission range of 625-650nm, and the second filter 5 has a wavelength transmission range of 650-700nm.

7. The imaging device according to claim 1, characterized in that, The digital triggering device (10) is a microcontroller.

8. The imaging device according to claim 1, characterized in that, The magnification of the objective lens (9) is 4-20 times, and the numerical aperture of the objective lens (9) is 0.17-0.

8.

9. The imaging apparatus according to any one of claims 1-8, characterized in that, The imaging device also includes a support module comprising a column (12), a column clamp (13), and an XYZ platform (14).

10. The imaging apparatus according to any one of claims 1-8, characterized in that, The imaging device also includes a temperature and humidity control module for a cell culture incubator (15).

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