Miniaturized multichannel microscope for stimulated Raman scattering and fluorescence microscopic imaging

Abstract

The invention discloses a miniaturized multichannel microscope combining stimulated Raman scattering (SRS) and fluorescence microscopic imaging, which is suitable for the fields of biomedical detection, material analysis, living body imaging and the like. The multi-channel microscope comprises the following contents: a multi-channel microscope main body structure, a Kohler illumination structure, a sample displacement table structure, an optical filter switching card group, a PD reflector bracket and CCD reflector bracket structure, an objective lens and a PI console. A laser light source enters the CCD reflecting mirror frame and the PD reflecting mirror frame from a light through hole behind the microscope or a Kohler structure light through hole, laser is controlled by the pull rod to enter different reflecting mirror sets in the CCD reflecting mirror frame and the PD reflecting mirror frame, and the laser finally reaches the CCD imaging module and the PD imaging module along different reflecting mirror set channels. And finally collecting photon information for imaging.

Description

Technical Field

[0001] The present invention relates to the field of microscopic imaging technology, and particularly to a miniaturized multi-channel microscope combining stimulated Raman scattering (SRS) and fluorescence microscopy, which is applicable to fields such as biomedical detection, material analysis, and in vivo imaging. Background Art

[0002] As a core tool in modern scientific research, microscopic imaging technology plays an irreplaceable role in biomedicine, materials science, and clinical diagnosis. Traditional microscope imaging systems usually only support a single imaging modality (such as fluorescence or Raman), making it difficult to meet the multi-dimensional analysis needs of complex samples and users. Moreover, the equipment is large in volume and high in cost, making it difficult to meet the requirements of clinical or on-site rapid detection. Although stimulated Raman scattering (SRS) technology can achieve label-free chemical imaging, its signal is weak and needs to be complemented with fluorescence imaging to improve detection sensitivity. In addition, most multi-modal microscopic systems on the current market switch imaging modes in a time-division multiplexing manner. For example, fluorescence and SRS alternate imaging is achieved by mechanically switching filter plates or adjusting the laser path. Such solutions have significant drawbacks and their stability is severely limited. On the other hand, the progress of miniaturized microscopy technology makes it possible to make the equipment portable, but existing miniaturized systems usually only support a single imaging mode, so it is also difficult to perform multi-channel microscopic imaging.

[0003] Therefore, there is an urgent need for an integrated and miniaturized multi-channel microscopic imaging device that can synchronously or quickly switch between SRS and fluorescence imaging modes and achieve high-resolution and high-throughput sample analysis. Summary of the Invention

[0004] The purpose of the present invention is to design a miniaturized multi-channel microscope that can achieve synchronous or alternating detection of stimulated Raman and fluorescence imaging through a compact optical design, and has high sensitivity, fast imaging, and portability.

[0005] To achieve the object of the present invention, the present invention proposes the following technical solutions:

[0006] A miniaturized multi-channel microscope combining stimulated Raman scattering and fluorescence microscopy, comprising the following steps:

[0007] 1.1 Stimulated Raman Scattering Microscope Mode:

[0008] Step S1.1.1, the laser outputs two beams of excitation light, namely pump light and Stokes light. After the two beams of light are combined in space and time through a series of optical elements, they jointly reach the light passing hole of the main body structure of the microscope and enter the CCD mirror holder in the main body structure;

[0009] Step S1.1.2: By pulling the lever of the CCD mirror mount, the mirror group for the stimulated Raman microscopy imaging mode is matched. The light beam is reflected into the silver-coated mirror and then vertically reflected into the upper objective lens. The light beam is focused on the sample by the objective lens, and the light beam carrying photon information is collected by the condenser lens.

[0010] Step S1.1.3: Pull the lever of the PD mirror mount to switch to the mirror group for the stimulated Raman microscopy imaging mode. A mirror is placed obliquely in this mirror group, causing the light beam to be reflected to the left side of the microscope.

[0011] Step S1.1.4: The two light beams are collected by the photodiode PD with a short-pass filter. Limited by the filtering effect of the short-pass filter, the Stokes light is filtered out, and the pump light is collected by the photodiode PD.

[0012] Step S1.1.5: Use a fast lock-in amplifier to achieve heterodyne detection and realize stimulated Raman scattering microscopy imaging.

[0013] 1.2 Fluorescence microscope mode:

[0014] Step S1.2.1: The laser outputs an excitation light source, which enters the Kohler illumination module through the light passing hole at the Kohler structure device.

[0015] Step S1.2.2: After the light beam enters the Kohler illumination system, the filter wheel assembly is assembled into the Kohler illumination structure by embedding. Different filters are switched by pushing and pulling the lever of the filter wheel assembly. The light beam enters the PD mirror mount after passing through the filter wheel assembly.

[0016] Step S1.2.3: Two sets of mirror groups with different placement methods are assembled in the PD mirror mount. The mode is switched by pushing and pulling the lever to reflect the light beam into the condenser lens. The light beam then passes through the sample, the objective lens, and reaches the silver-coated mirror.

[0017] Step S1.2.4: The silver-coated mirror reflects the light beam into the CCD mirror mount, which also has two sets of mirror groups with different placement methods. The mirror group is switched by pulling the lever to select the mirror group corresponding to the fluorescence imaging mode, and the light beam is reflected to the light output hole of the main structure. The corresponding filter is assembled at the light output hole and introduced into the CCD imaging module and the PMT.

[0018] Step S1.2.5: The imaging module and the PMT collect photon information to realize fluorescence microscopy imaging.

[0019] Preferably,

[0020] In the said step S1.1.1, the main structure of the microscope is processed with stainless steel material to ensure the stability of the microscope and provide a stable guarantee for imaging and signal reception.

[0021] In the above-mentioned steps S1.1.1 and S1.2.4, the overall CCD mirror holder is machined from hard aluminum material, fixed to the microscope main structure through a slide rail, and connected to a pull rod to facilitate the switching of different lens group modes; there are two types of lens groups with different placement methods in the CCD mirror holder, corresponding to different imaging mode selections. The pull rod is used to slide the CCD mirror holder for selection.

[0022] Preferably,

[0023] The silver-plated mirrors in steps S1.1.2 and S1.2.3 adopt an integral structure. The mirror holder is designed as a solid triangular body, and grooves are designed on the inclined surface to place the silver-plated mirrors, which can reflect the light beam vertically upward or horizontally into the CCD mirror holder.

[0024] The objective lenses described in steps S1.1.2 and S1.2.3 are installed in the PI console. The PI console is assembled in the microscope main structure, and its function is to move the objective lens in the Z-axis direction to meet the 3D imaging requirements in stimulated Raman scattering microscopy imaging; the sample stage is composed of three one-dimensional stainless steel displacement stages and connectors, with sufficient stability.

[0025] Preferably,

[0026] The overall PD mirror holder described in steps S1.1.3 and S1.2.2 is machined from hard aluminum material, fixed to the microscope main structure through a slide rail, and connected to a pull rod to facilitate the switching of different lens group modes.

[0027] Preferably,

[0028] In step S1.1.4, a short-pass filter is assembled on the photodiode PD to collect the pump light signal.

[0029] Preferably,

[0030] In step S1.1.5, a lock-in amplifier performs heterodyne amplification detection to achieve stimulated Raman scattering microscopy imaging.

[0031] Preferably,

[0032] The filter card group described in step S1.2.2 has three different types of filters, which are adapted to fluorescence microscopy imaging, and the filters are switched by connecting a pull rod.

[0033] Preferably,

[0034] In the CCD imaging module described in step S1.2.4, for easy observation, a CCD camera is used to replace the eyepiece structure in a traditional microscope, facilitating real-time observation of imaging information.

[0035] Advantages and beneficial effects of the present invention:

[0036] 1. In the selection of materials for the main structure of the microscope, related connectors, and the displacement stage, the present invention uses 304 stainless steel to maximize the stability of the mechanical structure, improve the overall stability of this microscope system, and provide guarantee for the stability required for fluorescence microscopy imaging and stimulated Raman scattering microscopy imaging.

[0037] 2. The present invention uses an inverted structure. The inverted microscope adopts an inverted configuration, with the objective lens placed below the stage, while the condenser and Köhler illumination system are located above the stage. This design makes the inverted microscope more convenient and flexible for observing cells and tissues.

[0038] 3. The present invention adopts a self-designed Köhler illumination system to ensure that the pump light photons during each stimulated Raman scattering microscopy imaging can be collected by the condenser with the highest efficiency. At the same time, a display screen is used instead of a traditional eyepiece in the microscope, making the operation more convenient and suitable for wide-field imaging.

[0039] 4. The miniaturized multi-channel microscope designed by the present invention for stimulated Raman and fluorescence microscopy imaging can achieve high-sensitivity multimodal imaging through a compact coaxial optical path design, dynamic beam splitting technology, and multi-channel mode selection. Moreover, the device is small in volume and low in cost, and is suitable for clinical diagnosis and on-site rapid detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0041] Figure 1 It is the overall structure diagram of the miniaturized multi-channel microscope provided by the present invention;

[0042] Figure 2 It is the schematic diagram of the microscopy imaging system of the microscope of the present invention;

[0043] Figure 3 It is the diagram of the CCD mirror holder and PD mirror holder of the present invention; DETAILED DESCRIPTION OF THE EMBODIMENTS

[0044] The following will clearly and completely describe the design solutions in the embodiments of the present application in conjunction with the drawings.

[0045] In conjunction with Figure 1 、 Figure 2The design and microscopic imaging process of a miniaturized multi-channel microscope for stimulated Raman scattering (SRS) and fluorescence microscopy of the present invention are described as follows. The main structure of the microscope and the sample stage, and the displacement stage are made of stainless steel materials to improve the stability of the mechanical structure of the microscope. The overall structure is selected as an inverted structure. Compared with the upright microscope, in this design, the positions of the objective lens and the condenser, and the Köhler illumination system are inverted, making it more convenient and flexible to observe samples such as cells and tissues. At the same time, a PI control console is equipped in the objective lens to move the objective lens in the Z-axis direction to meet the requirements of 3D imaging in stimulated Raman scattering microscopy. The main structure of the microscope includes a CCD mirror holder and a PD mirror holder, both of which are equipped with slide rails to facilitate the switching of different lens groups by pulling the rods. The present invention also independently designs a Köhler illumination system, so that the pump light photons during stimulated Raman scattering microscopy can be collected by the condenser with the highest efficiency. When performing stimulated Raman scattering microscopy, the light beam enters the CCD mirror holder through the light passing hole after passing through the main structure of the microscope. By pulling the rod to match the lens group of the corresponding mode, the light beam is reflected to the silver-coated mirror, and then the light beam is vertically reflected into the objective lens. After the light beam is focused on the sample, the light beam carrying photon information is collected by the condenser and enters the PD mirror holder. Pull the rod to select the lens group of the corresponding mode, reflect the light beam to the photodiode PD, and a high-quality short-pass filter is added in front of the photodiode to filter out the Stokes light. After collecting the pump light information, it is transmitted to the lock-in amplifier for phase amplification to achieve stimulated Raman scattering microscopy. When performing fluorescence microscopy, the light beam filters out some stray light through the filter set, enters the PD mirror holder, is reflected into the condenser and then reaches the sample and the objective lens, and then is horizontally reflected into the CCD mirror holder by the silver-coated mirror. Select the lens group in the fluorescence mode, and at the same time, the PMT and the CCD imaging module collect the photon information to achieve fluorescence microscopy.

[0046] Combined with Figure 3 , the independently designed CCD mirror holder and PD mirror holder in the present invention are described as follows. Both mirror holders are made of hard aluminum materials and are equipped with slide rails and rods to facilitate the sliding of the two mirror holders in the main structure of the microscope and realize the propagation of the light beam in the two mirror holders in different modes. The slide rails assembled on the CCD mirror holder and the PD mirror holder are connected to the main structure of the microscope by screws, so that the whole mirror holder moves on the slide rail without affecting the slide rail, further improving the stability during the movement. At the same time, 2.5 mm diameter round bead grooves are designed on the surface of the mirror holder. When switching different microscopic imaging modes, the corresponding stagnation feeling between the spring screws embedded in the main structure of the microscope and the grooves makes it more convenient to judge whether the mirror holder reaches the optimal position, so that the light beam can propagate in the microscope structure as much as possible.

[0047] By designing different microscopic structures, the present invention changes the traditional upright structure to an inverted structure more suitable for observing cells and tissues, which better meets the requirements of fluorescence microscopy imaging and stimulated Raman scattering microscopy imaging. By designing the PD mirror mounts and CCD mirror mounts of different lens groups, the light beams in different modes can better enter the microscope, improving the photon information acquisition ability of the detector and thus enhancing the resolution of the imaging system. The self-designed Köhler illumination system enhances the efficiency of the condenser lens in collecting pump light photons in stimulated Raman scattering microscopy imaging, improving the resolution of stimulated Raman scattering microscopy imaging. The main structure of the microscope and the sample displacement stage are both made of stainless steel materials, enhancing the mechanical configuration of the overall structure and fully improving the stability. Since the photon collection part is the least sensitive to stability, the photodiode PD in the photon collection part is placed on the left side of the high PD mirror mount and connected to the main structure of the microscope through steel components.

[0048] A method for using a miniaturized multi-channel microscope combining stimulated Raman scattering and fluorescence microscopy imaging according to the present invention includes the following steps:

[0049] 1.1 Stimulated Raman scattering microscope mode:

[0050] In step S1.1.1, the laser outputs two excitation lights, namely pump light and Stokes light. After the two lights are combined in space and time through a series of optical elements, they jointly reach the light passing hole of the main structure of the microscope and enter the CCD mirror mount in the main structure;

[0051] In the above step S1.1.1, the main structure of the microscope is processed with stainless steel materials to ensure the stability of the microscope, providing a stable guarantee for imaging and signal reception;

[0052] In the above step S1.1.1 and step S1.2.4, the entire CCD mirror mount is processed with hard aluminum materials. It is fixed in the main structure of the microscope through a slide rail and is connected to a pull rod to facilitate the switching of different lens group modes; there are two different placement methods of lens groups in the CCD mirror mount. To adapt to different imaging mode selections, the pull rod is pulled to slide the CCD mirror mount for selection.

[0053] In step S1.1.2, the pull rod of the CCD mirror mount is pulled to match the lens group of the stimulated Raman microscopy imaging mode. The light beam is reflected into the silver-coated mirror and vertically reflected into the upper objective lens. The light beam is focused on the sample by the objective lens, and the light beam carrying photon information is collected by the condenser lens;

[0054] The silver-coated mirror in step S1.1.2 and step S1.2.3 adopts an integral structure. The mirror mount is designed as a solid triangular body, and a groove is designed on the inclined surface to place the silver-coated mirror, which can reflect the light beam vertically upward or horizontally into the CCD mirror mount;

[0055] The objective lenses described in step S1.1.2 and step S1.2.3 are installed in the PI console, which is assembled in the microscope main structure. Its function is to move the objective lens in the Z-axis direction to meet the 3D imaging requirements in stimulated Raman scattering microscopy imaging. The sample stage is composed of three one-dimensional stainless steel displacement stages and connectors, with sufficient stability.

[0056] In step S1.1.3, pull the rod of the PD mirror holder to switch to the stimulated Raman microscopy imaging lens group. A mirror is placed obliquely in this lens group, so that the light beam is reflected to the left side of the microscope.

[0057] The whole PD mirror holder described in step S1.1.3 and step S1.2.2 is machined from hard aluminum material, fixed to the microscope main structure through a slide rail, and connected to the rod for convenient switching between different lens group modes.

[0058] In step S1.1.4, the two light beams are collected by the photodiode PD with a short-pass filter. Limited by the filtering effect of the short-pass filter, the Stokes light is filtered out, and the pump light is collected by the photodiode PD.

[0059] In step S1.1.4, the photodiode PD is equipped with a short-pass filter to collect the pump light signal.

[0060] In step S1.1.5, a fast lock-in amplifier is used to achieve heterodyne detection and realize stimulated Raman scattering microscopy imaging.

[0061] In step S1.1.5, the lock-in amplifier performs heterodyne amplification detection to realize stimulated Raman scattering microscopy imaging.

[0062] 1.2 Fluorescence microscope mode:

[0063] In step S1.2.1, the laser outputs an excitation light source, which enters the Kohler illumination module through the light passing hole at the Kohler structure device.

[0064] After the light beam enters the Kohler illumination system, the filter card group is assembled in the Kohler illumination structure by embedding. Different filters are switched by pushing and pulling the rod of the filter card group. The light beam enters the PD mirror holder after passing through the filter card group.

[0065] The filter card group described in step S1.2.2 has three different types of filters, which are adapted to fluorescence microscopy imaging and are switched by a rod connection.

[0066] Step S1.2.3: Assemble two sets of mirror groups with different placement methods in the PD mirror holder. Control the mode switching by pushing and pulling the rod, reflect the light beam into the condenser lens, and then the light beam passes through the sample and the objective lens to reach the silver-coated mirror.

[0067] Step S1.2.4: The silver-coated mirror reflects the light beam into the CCD mirror holder, which also assembles two sets of mirror groups with different placement methods. Control the switching of the mirror groups by the rod, select the mirror group corresponding to the fluorescence imaging mode, reflect the light beam to the light outlet hole of the main structure, and assemble the corresponding filter on the light outlet hole, and introduce it into the CCD imaging module and the PMT.

[0068] In the CCD imaging module described in Step S1.2.4, for easy observation, a CCD camera is used to replace the eyepiece structure in the traditional microscope, which is convenient for real-time observation of imaging information.

[0069] Step S1.2.5: The imaging module and the PMT collect photon information to achieve fluorescence microscopy imaging.

[0070] The miniaturized multi-channel microscope of the present invention that combines stimulated Raman scattering (SRS) and fluorescence microscopy can achieve high-sensitivity multimodal imaging compared with the traditional microscope through a compact coaxial optical path design, dynamic spectroscopic technology, and multi-channel mode selection. The selection of the main body of the steel structure and the inverted structure enables better imaging efficiency and adaptability for both fluorescence microscopy imaging and stimulated Raman scattering microscopy imaging modes. Moreover, the device has a small volume and low cost, and is suitable for clinical diagnosis and on-site rapid detection.

Claims

1. A miniaturized multi-channel microscope combining stimulated Raman scattering and fluorescence microscopy, characterized in that: The following steps are involved: 1.1 Stimulated Raman Scattering Microscope Mode: Step S1.1.1, the laser outputs two beams of excitation light, namely pump light and Stokes light. The two beams are combined in space and time by a series of optical elements and arrive at the light hole of the microscope main structure together, and enter the CCD reflector frame in the main structure; Step S1.1.2, by pulling the rod of the CCD reflector frame to match the mirror group of the stimulated Raman microscopy imaging mode, the light beam is reflected to the silver-coated reflector, and vertically reflected into the objective lens above. The light beam is focused on the sample through the objective lens, and the light beam carrying the photon information is collected by the condenser; Step S1.1.3, pull the lever of the PD reflector frame to switch to the stimulated Raman microscopy imaging mode mirror group, in which a reflector is placed at an angle so that the light beam is reflected to the left side of the microscope; Step S1.1.4, the two light beams are collected by a photodiode PD with a short-pass filter. Due to the filtering effect of the short-pass filter, the Stokes light is filtered out and the pump light is collected by the photodiode PD; Step S1.1.5, using a fast lock-in amplifier to implement heterodyne detection and stimulated Raman scattering microscopy imaging; 1.2 Fluorescence Microscope Mode: Step S1.2.1, the laser outputs an excitation light source, which enters the Kohler illumination module through the light hole at the Kohler structure device; Step S1.2.2, after the light beam enters the Kohler illumination system, the filter card set is assembled in the Kohler illumination structure by embedding, and different filters are switched by pushing and pulling the filter card set pull rod. After the light beam passes through the filter card set, it enters the PD reflector frame; Step S1.2.3, assembling two sets of reflectors with different placement modes in the PD reflector frame, and controlling the switching of the modes by pushing and pulling the pull rod to reflect the light beam into the condenser, and then the light beam passes through the sample and the objective lens to reach the silver-coated reflector; Step S1.2.4, the silver-coated reflector reflects the light beam into the CCD reflector frame, which is also equipped with two sets of reflector groups with different placement methods. The mirror group is switched by pulling the lever to select the mirror group corresponding to the fluorescence imaging mode, and the light beam is reflected to the light exit hole of the main structure. The light exit hole is equipped with a corresponding filter and introduced into the CCD imaging module and PMT; Step S1.2.5, the imaging module and PMT collect photon information to achieve fluorescence microscopy imaging.

2. A miniaturized multi-channel microscope combining stimulated Raman scattering and fluorescence microscopy according to claim 1, characterized in that: In the step S1.1.1, the main structure of the microscope is processed with stainless steel to ensure the stability of the microscope and provide stable guarantee for imaging and signal reception; In the steps S1.1.1 and S1.2.4, the CCD reflector frame is made of hard aluminum material as a whole, is fixed in the main structure of the microscope by a slide rail, and is connected to a pull rod for convenient switching of different lens group modes; the CCD reflector frame contains two lens groups with different placement methods, which can be used to select different imaging modes, and the pull rod pulls the CCD reflector frame for sliding selection.

3. A miniaturized multi-channel microscope combining stimulated Raman scattering and fluorescence microscopy according to claim 1, characterized in that: The silver-coated reflector in step S1.1.2 and step S1.2.3 adopts an integral structure, and the frame is designed as a solid triangle. The silver-coated reflector is placed in the groove designed on the inclined surface, which can reflect the light beam vertically upward or horizontally into the CCD frame; The objective lens described in step S1.1.2 and step S1.2.3 is installed in the PI console, which is assembled in the main structure of the microscope. Its function is to move the objective lens in the Z-axis direction to meet the 3D imaging requirements in stimulated Raman scattering microscopy. The sample stage is composed of three one-dimensional stainless steel translation stages and connectors, and has sufficient stability.

4. A miniaturized multi-channel microscope combining stimulated Raman scattering and fluorescence microscopy according to claim 1, characterized in that: The PD reflective mirror frame described in step S1.1.3 and step S1.2.2 is made entirely of hard aluminum material, fixed to the main structure of the microscope via a slide rail, and connected to a pull rod for easy switching of different mirror group modes.

5. The miniaturized multi-channel microscope combining stimulated Raman scattering and fluorescence microscopy according to claim 1, characterized in that: In step S1.1.4, the photodiode PD is equipped with a short-pass filter to collect the pump light signal.

6. The miniaturized multi-channel microscope combining stimulated Raman scattering and fluorescence microscopy according to claim 1, characterized in that: In step S1.1.5, the phase-locked amplifier performs heterodyne amplification detection to achieve stimulated Raman scattering microscopy imaging.

7. The miniaturized multi-channel microscope combining stimulated Raman scattering and fluorescence microscopy according to claim 1, characterized in that: The filter card set described in step S1.2.2 has three sets of filters of different models, which are suitable for fluorescence microscopy imaging and are connected by a pull rod to switch the filters.

8. The miniaturized multi-channel microscope combining stimulated Raman scattering and fluorescence microscopy according to claim 1, characterized in that: The CCD imaging module described in step S1.2.4 uses a CCD camera to replace the eyepiece structure in a traditional microscope for easy observation, thereby facilitating real-time observation of imaging information.

Images