A laser scanning confocal microscope
By combining a beam splitter and a filter system, the problem of insufficient resolution of fluorescence signals at different wavelengths was solved, enabling high-resolution image acquisition and improving the imaging effect of the laser scanning confocal microscope.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Utility models(China)
- Current Assignee / Owner
- CHANG YI GUANG KE (SU ZHOU) JI SHU YOU XIAN GONG SI
- Filing Date
- 2025-08-15
- Publication Date
- 2026-05-29
AI Technical Summary
Existing laser scanning confocal microscopes struggle to achieve optimal resolution when processing fluorescence signals of different wavelengths, leading to difficulties in image analysis.
A beam splitting mechanism is used to separate fluorescence signals of different wavelengths into corresponding filtering systems. Combined with components such as pinhole structures, collimating lenses, gratings and slits, stray light is filtered out, improving optical resolution and signal-to-noise ratio.
High-resolution imaging of fluorescence signals at different wavelengths was achieved, stray light interference was reduced, and image resolution and signal-to-noise ratio were improved.
Smart Images
Figure CN224303935U_ABST
Abstract
Description
Technical Field
[0001] This utility model relates to the field of microscopes, and in particular to a laser scanning confocal microscope. Background Technology
[0002] Laser scanning confocal microscopy (LSCM) is a modern biomedical imaging instrument. It adds a laser scanning device to fluorescence microscopy, using ultraviolet or visible light to excite fluorescent probes, thereby obtaining fluorescent images of fine structures within cells or tissues, and observing structures such as calcium carbonate at the subcellular level. 2+ LSCM detects changes in physiological signals such as pH value and membrane potential, as well as cell morphology. LSCM exhibits high sensitivity, producing high-contrast, high-resolution images that are non-destructive to the sample, highly reliable, and reproducible, giving it significant advantages in the field of detection and analysis.
[0003] In existing technologies, sample detection typically requires multi-dimensional processing, often necessitating the application of various fluorescent labels to the sample, resulting in the emission of fluorescence signals at multiple wavelengths. However, the varying processing capabilities of imaging units for different wavelengths during the collection and analysis of these fluorescence signals hinder optimal image resolution and thus impede image analysis. Utility Model Content
[0004] The purpose of this invention is to provide a laser scanning confocal microscope that can form high-resolution images when acquiring fluorescence signals of different wavelengths.
[0005] To achieve the above objectives, this utility model provides the following technical solution:
[0006] A laser scanning confocal microscope includes a beam splitting mechanism, multiple filter systems, and multiple detection systems. The beam splitting mechanism is used to transmit different fluorescence beams with wavelengths in different ranges to different filter systems. Each filter system corresponds to a detection system, and each filter system is configured for a different wavelength range to filter out stray light doped in the fluorescence beam. The detection system is used to receive the fluorescence beams leaving the filter systems and convert them into electrical signals.
[0007] Optionally, each of the filtering systems includes a focusing lens and a pinhole structure. The pinhole structure includes a projection surface and a through-hole-shaped focusing aperture formed on the projection surface. The focusing lens is used to focus at least a portion of the fluorescence beam onto the projection surface and through the focusing aperture. The projection surface is used to block defocused light rays.
[0008] Optionally, each of the filtering systems further includes a collimating lens disposed on the side of the pinhole structure away from the focusing lens.
[0009] Optionally, the laser scanning confocal microscope further includes multiple filtering systems, which are disposed between the filtering system and the detection system for selectively projecting fluorescence beams of different wavelengths.
[0010] Optionally, the filtering system includes a grating and a slit, the grating dispersing the fluorescence beam arriving at the filtering system, the grating being used to transmit only a portion of the fluorescence beam with a wavelength matching the wavelength to the detection system.
[0011] Optionally, the filtering system includes a bandpass filter.
[0012] Optionally, the beam splitting mechanism includes a plurality of wavepass filters, each corresponding to a filter system, and the cutoff wavelength of the wavepass filters is matched with that of the filter system.
[0013] Optionally, the laser scanning confocal microscope further includes a laser, a dichroic mirror, a scanning system, an objective lens system, and a control system. The laser emits laser beams of various wavelengths. The laser beams are transmitted to the scanning system under the action of the dichroic mirror. The scanning system vibrates controllably under the control system. The objective lens system works in conjunction with the scanning system to focus the laser beams at any position on the focal plane of the sample and scan point by point, exciting the generation of the fluorescence beam. The fluorescence beam is transmitted to the dichroic mirror via the objective lens system and the scanning system. The dichroic mirror, with different delivery directions for the fluorescence beam and the laser beam, transmits the fluorescence beam to the beam splitter. The detection system converts the fluorescence beam into an electrical signal, and the control system analyzes several of the electrical signals to form a scanning image.
[0014] Optionally, another filtering system is provided between the laser and the dichroic mirror.
[0015] The beneficial effects of this invention are as follows: by using a beam splitting mechanism to transfer fluorescence of different wavelengths to different filter systems for processing, multiple channels are formed. At this time, the wavelength range that the filter system needs to match is relatively small, ensuring that the optical resolution of all bands reaches the optimal state.
[0016] Furthermore, the pinhole structure filters out light that remains out of focus after being focused by the focusing lens. When the laser scans the sample point by point, only the tiny area focused by the objective lens system is excited. The resulting fluorescence beam returns through the objective lens, and only the light focused at the focusing aperture can pass through. Scattered light or fluorescence from other locations cannot pass through the projection surface because it is not focused at the focusing aperture, thus being physically blocked. This blocks the defocus signal and helps improve the resolution of the scanned image.
[0017] Furthermore, by using a collimating lens to shape the fluorescence beam passing through the pinhole structure into a parallel beam with a smaller diameter, it helps to prevent the fluorescence beam from diffusing and becoming difficult for the detection system to receive.
[0018] Furthermore, by using a filtering system, the selectivity of the fluorescence beam wavelength is improved, thereby suppressing cross-color problems between different channels.
[0019] Furthermore, when fluorescent beams of multiple wavelengths are incident on the grating, due to the diffraction and interference of the grating, the light of different wavelengths will be deflected at different angles according to the grating equation, thus causing dispersion and being spatially separated into a series of colored beams. Wavelength filtering is achieved by selectively allowing light to pass through and blocking it through slits.
[0020] Furthermore, the spatial position of the slit can be controllably varied, which helps to adjust the wavelength of the fluorescence beam reaching the detector at any time, thereby adapting to different fluorescence signals and exhibiting high versatility.
[0021] Furthermore, filtering is achieved through bandpass filters, which have a simple structure and high versatility.
[0022] Furthermore, the combination of several wavepass filters can effectively transmit fluorescence with wavelengths in different ranges in different directions, thereby delivering different fluorescence signals to different filtering and detection systems for imaging processing.
[0023] Furthermore, a set of filtering systems is set up near the laser to help reduce laser beam scattering, ensuring that only the area near the focal plane on the sample is accurately illuminated and excited, avoiding stray light interference from the non-focal plane, and improving the signal-to-noise ratio.
[0024] The above description is only an overview of the technical solution of this utility model. In order to better understand the technical means of this utility model and to implement it in accordance with the contents of the specification, the preferred embodiments of this utility model are described in detail below with reference to the accompanying drawings. Attached Figure Description
[0025] Figure 1 This is a schematic diagram of the structure of the laser scanning confocal microscope shown in Embodiment 1 of this utility model;
[0026] Figure 2 This is a schematic diagram of the pinhole structure shown in Embodiment 1 of this utility model;
[0027] Figure 3 This is a schematic diagram of the structure of the laser scanning confocal microscope shown in Embodiment 2 of this utility model;
[0028] Figure 4 This is a schematic diagram of the structure of the laser scanning confocal microscope shown in Embodiment 3 of this utility model;
[0029] Figure 5 These are scanned images obtained from detecting the same sample in Embodiment 1 and Comparative Example 1 of this utility model.
[0030] Legend: 1-Laser, 2-Dichroic mirror, 3-Scanning system, 4-Objective lens system, 5-Beam splitting mechanism, 51-First wavepass filter, 52-Second wavepass filter, 53-Reflector, 6-Filtering system, 61-Focusing lens, 62-Pinhole structure, 621-Projection surface, 622-Focusing aperture, 63-Collimating lens, 7-Detection system, 8-Filtering system, 81-Grating, 82-Slit, 83-Bandpass filter. Detailed Implementation
[0031] The technical solution of this utility model will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this utility model. Based on the embodiments of this utility model, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this utility model.
[0032] In the description of this utility model, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the accompanying drawings and are only for the convenience of describing this utility model and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this utility model. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.
[0033] In the description of this utility model, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "joining" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this utility model based on the specific circumstances.
[0034] Furthermore, the technical features involved in the different embodiments of this utility model described below can be combined with each other as long as they do not conflict with each other.
[0035] This utility model application protects a laser scanning confocal microscope, including a beam splitting mechanism 5, multiple filter systems 6, and multiple detection systems 7. The beam splitting mechanism 5 is used to transmit different fluorescence beams with wavelengths in different ranges to different filter systems 6. The filter systems 6 correspond one-to-one with the detection systems 7. Each filter system 6 is used to filter out stray light doped in the fluorescence beam in different wavelength ranges. The detection system 7 is used to receive the fluorescence beam leaving the filter system 6 and convert it into an electrical signal.
[0036] The fluorescence of different wavelengths is transferred to different filter systems 6 for processing by the beam splitter 5. At this time, the wavelength range that the filter system 6 needs to cooperate with is relatively small, so as to ensure that the optical resolution of all bands reaches the best state.
[0037] Please refer to the following examples for details.
[0038] Example 1:
[0039] Please see Figure 1 The laser scanning confocal microscope shown in a preferred embodiment of this application includes a laser 1, a dichroic mirror 2, a scanning system 3, an objective lens system 4, a beam splitting mechanism 5, multiple filter systems 6, multiple detection systems 7, and a control system.
[0040] Laser 1 emits laser beams of various wavelengths, which directly reach dichroic mirror 2. Dichroic mirror 2 faces the laser beam at a 45° angle. Due to the wavelength difference, dichroic mirror 2 reflects the laser beam while transmitting the fluorescence beam. The laser beam, after being reflected by dichroic mirror 2, reaches scanning system 3. Scanning system 3 includes galvanometers arranged along two mutually perpendicular axes, which change the direction of light propagation through reflection. The control system drives the galvanometers to swing rapidly, thereby controlling the laser beam to deflect in different directions. With the cooperation of objective lens system 4, the laser beam is focused on various points on the focal plane of the sample. As the galvanometers swing, the laser beam sweeps across various points on the focal plane of the sample, thereby exciting the fluorescent markers carried on the sample to generate fluorescence beams. The fluorescence beam travels along a path opposite to that of the laser beam, passing sequentially through objective lens system 4 and scanning system 3, reaching dichroic mirror 2, and then directly passing through dichroic mirror 2 to reach beam splitting mechanism 5.
[0041] In this embodiment, the beam splitting mechanism 5 includes a first wavepass filter 51 and a second wavepass filter 52, and multiple filtering systems 6 are respectively the first filtering system and the second filtering system. Fluorescent beams passing through the dichroic mirror 2 directly reach the first wavepass filter 51. Fluorescent beams with wavelengths greater than the cutoff wavelength of the first wavepass filter 51 directly pass through the first wavepass filter 51 and reach the first filtering system. Fluorescent beams with wavelengths less than the cutoff wavelength of the first wavepass filter 51 are reflected by the first wavepass filter 51 and reach the second wavepass filter 52. Fluorescent beams with wavelengths less than the cutoff wavelength of the second wavepass filter 52 directly pass through the second wavepass filter 52, and fluorescent beams with wavelengths greater than the cutoff wavelength of the second wavepass filter 52 are reflected by the second wavepass filter 52 and reach the second filtering system. In some embodiments, the second wavepass filter 52 may be a reflector 53.
[0042] Please see Figure 2 The filtering system 6 includes a pinhole structure 62 and focusing lenses 61 and collimating lenses 63 disposed on both sides of the pinhole structure 62. The pinhole structure 62 includes a flat projection surface 621 perpendicular to the fluorescence beam and a through-hole-shaped focusing aperture 622 disposed in the middle of the projection surface 621. The fluorescence beam is focused on the position on the projection surface 621 corresponding to the focusing aperture 622 under the action of the focusing lens 61, passes through the focusing aperture 622, and reaches the collimating lens 63, where it is converted into a parallel beam. Since the light transmitted from the sample surface to the objective lens system 4 also includes light reflected from the laser beam and fluorescence excited by the fluorescence signal carried by the tissue outside the focal plane of the sample, the light entering the objective lens system 4 and transferred to the filtering system 6 includes stray light in addition to the fluorescence beam. After the laser is filtered out by the beam splitter 5, the stray light that cannot be focused to the position corresponding to the focusing aperture 622 is blocked by the projection surface 621, which helps to minimize interference and improve the resolution of the final scanned image.
[0043] Because different filter systems 6 process fluorescence beams with wavelengths in different wavelength ranges, the parameters of each filter system 6 differ. These differences include the distance between the pinhole structure 62 and the focusing lens 61, the distance between the pinhole structure 62 and the collimating lens 63, the curvature of the focusing lens 61 and the collimating lens 63, and the diameter of the focusing aperture 622. By setting multiple filter systems 6 to process fluorescence beams in different wavelength bands, the compatibility between the filter system 6 and the fluorescence beam is improved, thereby enhancing the focusing and filtering effects of the filter system 6 and ultimately improving the quality of the scanned image.
[0044] After leaving the filter system 6, the fluorescence beam shines directly into the corresponding detection system 7. The detection system 7 receives the optical signal and converts it into an electrical signal. The control system processes the electrical signal to obtain the scanned image. Because the filter system 6 filters out stray light and avoids signal noise caused by stray light, the resolution of the scanned image is improved.
[0045] In this embodiment, light propagates directly between adjacent structures, which is not conducive to the spatial layout of the internal structure of the device. In some embodiments, the direction of the light path can be adjusted by setting multiple reflective sheets to meet the spatial requirements of the mechanical structure. Since these methods are common techniques in the art, they will not be described in detail here.
[0046] This embodiment also includes two filtering systems 8, both positioned between the filtering system 6 and the detection system 7, to further filter out noise mixed in with the optical signal to be converted and improve the contrast of the scanned image. In some embodiments, the filtering system 8 is positioned on the side of the filtering system 6 away from the detection system 7.
[0047] The filtering system 8 includes a grating 81 and a slit 82. The fluorescence beam leaving the filtering system 6 enters the grating 81 and undergoes dispersion, causing light of different wavelengths to be distributed regularly in space. The slit 82 can be moved controllably, thereby selecting light beams of different wavelengths to pass through the slit 82 by spatial changes and blocking other light rays, achieving a filtering effect. It can also be adjusted in real time according to the signal to be detected, and has high selectivity.
[0048] In this embodiment, another filtering system 6 is provided between the laser 1 and the dichroic mirror 2. The laser beam passes through the focusing lens 61, the pinhole structure 62 and the collimating lens 63 in sequence before reaching the dichroic mirror 2. This helps to reduce the scattering of the laser beam, ensure that only the area near the focal plane on the sample is accurately irradiated and excited, avoid stray light interference from the non-focal plane, and improve the signal-to-noise ratio.
[0049] The pinhole structure 62 set near the laser 1 and the pinhole structure 62 set near the detection system 7 are conjugate points on the optical path, thereby ensuring that only the signal of the focal plane is efficiently detected.
[0050] Example 2:
[0051] Please see Figure 3 The only difference between this embodiment and the first embodiment is that the beam splitting mechanism 5 in this embodiment also includes a reflector 53. The fluorescence beam passing through the second wavepass filter 52 directly reaches the reflector 53 and reaches the third filter system under the action of the reflector 53.
[0052] Example 3:
[0053] Please see Figure 4 The only difference between this embodiment and Embodiment 1 is that the filtering system 8 in this embodiment only includes a bandpass filter 83.
[0054] Comparative Example 1:
[0055] The only difference between this embodiment and Embodiment 1 is that this comparative example does not include a beam splitting mechanism 5 and a filtering system 8, and only includes a set of filtering systems 6 and a detection system 7.
[0056] The same group of samples labeled with two different fluorescent markers were observed using laser scanning confocal microscopy as described in Example 1 and Comparative Example 1, respectively. The fluorescence beams excited by the two fluorescent markers were blue and green, respectively. Please refer to [link to relevant documentation]. Figure 5 As can be seen from the comparison, the scanned image obtained in Example 1 has a higher resolution and effectively avoids color bleeding.
[0057] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
[0058] The embodiments described above are merely illustrative of several implementations of this utility model, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the utility model patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this utility model, and these all fall within the protection scope of this utility model. Therefore, the protection scope of this utility model patent should be determined by the appended claims.
Claims
1. A laser scanning confocal microscope, characterized in that, It includes a beam splitter (5), multiple filter systems (6) and multiple detection systems (7). The beam splitter (5) is used to transmit different fluorescence beams with wavelengths in different ranges to different filter systems (6). The filter systems (6) correspond one-to-one with the detection systems (7). Each filter system (6) is used to filter out stray light doped in the fluorescence beam. The detection system (7) is used to receive the fluorescence beam leaving the filter system (6) and convert it into an electrical signal.
2. The laser scanning confocal microscope as described in claim 1, characterized in that, Each of the filtering systems (6) includes a focusing lens (61) and a pinhole structure (62). The pinhole structure (62) includes a projection surface (621) and a through-hole-shaped focusing aperture (622) formed on the projection surface (621). The focusing lens (61) is used to focus at least a portion of the fluorescence beam onto the projection surface (621) and through the focusing aperture (622). The projection surface (621) is used to block defocused light.
3. The laser scanning confocal microscope as described in claim 2, characterized in that, Each of the filtering systems (6) further includes a collimating lens (63), which is disposed on the side of the pinhole structure (62) away from the focusing lens (61).
4. The laser scanning confocal microscope as described in claim 1, characterized in that, It also includes multiple filtering systems (8), which are disposed between the filtering system (6) and the detection system (7) for selectively projecting fluorescent beams of different wavelengths.
5. The laser scanning confocal microscope as described in claim 4, characterized in that, The filtering system (8) includes a grating (81) and a slit (82). The grating (81) disperses the fluorescence beam arriving at the filtering system (8) and is used to transmit only a portion of the fluorescence beam with a wavelength matching the wavelength to the detection system (7).
6. The laser scanning confocal microscope as described in claim 5, characterized in that, The width and position of the slit (82) can be controlled to adjust the matching wavelength.
7. The laser scanning confocal microscope as described in claim 4, characterized in that, The filtering system (8) includes a bandpass filter (83).
8. The laser scanning confocal microscope as described in claim 1, characterized in that, The beam splitting mechanism (5) includes a plurality of wavepass filters, each of which corresponds to one of the filtering systems (6), and its cutoff wavelength is matched with that of the filtering system (6).
9. The laser scanning confocal microscope as described in any one of claims 1 to 8, characterized in that, It also includes a laser (1), a dichroic mirror (2), a scanning system (3), an objective lens system (4), and a control system. The laser (1) is used to emit laser beams of various wavelengths. The laser beams are transmitted to the scanning system (3) under the action of the dichroic mirror (2). The scanning system (3) vibrates controllably under the drive of the control system. The objective lens system (4) cooperates with the scanning system (3) to focus the laser beams on any position of the focal plane of the sample and scan point by point to generate the fluorescence beam. The fluorescence beams are transmitted to the dichroic mirror (2) through the objective lens system (4) and the scanning system (3). The dichroic mirror (2) transmits the fluorescence beams to the beam splitter (5) because the delivery directions of the fluorescence beams and the laser beams are different. The fluorescence beams are converted into electrical signals by the detection system (7). The control system analyzes several of the electrical signals to form a scanning image.
10. The laser scanning confocal microscope as described in claim 9, characterized in that, Another filtering system (6) is provided between the laser (1) and the dichroic mirror (2).