Single objective multi-mode microscopic imaging system

Abstract

The application provides a single-objective multi-mode microscopic imaging system, and relates to the technical field of microscopic imaging.The system comprises a laser light source, an excitation light path module, a modulation scanning module, a detection light path module, an imaging camera and a controller.The excitation light path module comprises an expansion-constriction lens group, a shaping unit, a first slit and a first relay lens group.The modulation scanning module comprises a dynamic light splitting unit in a non-fixed position, a first scanning galvanometer, a scanning lens, a barrel lens and an objective lens.The detection light path module comprises a first lens, a second relay lens group, a second slit in a non-fixed state, an electronically adjustable lens, a third relay lens group and a second scanning galvanometer.The modulation scanning module is used for transmitting a laser beam to the back focal plane of the objective lens and relaying sample fluorescence collected by the objective lens to the detection light path module.The controller is used for switching between oblique light sheet imaging mode and confocal imaging mode according to the position of the dynamic light splitting unit and the state of the second slit.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microscopic imaging, in particular to a single-objective multi-mode microscopic imaging system. BACKGROUND

[0002] With the development of research fields such as life science, neuroscience and developmental biology, the demand for three-dimensional high-resolution imaging of living samples is also increasing. Light sheet fluorescence microscopy (LSFM) and oblique plane microscopy (OPM) have been widely used in scientific research and clinical research.

[0003] In related technologies, LSFM adopts two objectives to undertake excitation and collection tasks respectively, and achieves lateral excitation and vertical detection by forming intersecting light paths. However, such a double-objective system has many limitations, such as large device size, limited sample fixation and rotation, fixed imaging angle, and the like, and is difficult to meet the needs of flexible observation of complex sample structures. OPM introduces an oblique angle light sheet after the back focal plane of a single objective, so that the excitation path and the collection path share one objective, thereby simplifying the system light path. However, such a single-objective system is difficult to meet the resolution requirement, and thus still needs to introduce complex light path adjustment, such as introducing multiple objectives or optical elements for large-scale adjustment, to perform multi-mode switching, thereby not only increasing the device complexity and the difficulty of mode switching, but also increasing the cost. Therefore, how to simplify the mode switching of the microscopic imaging system is crucial. SUMMARY

[0004] The present application provides a single-objective multi-mode microscopic imaging system.

[0005] According to a first aspect of the present application, a single-objective multi-mode microscopic imaging system is provided, which comprises:

[0006] a laser light source, an excitation light path module, a modulation scanning module, a detection light path module, an imaging camera and a controller;

[0007] The excitation light path module comprises an expansion and contraction lens group, a shaping unit, a first slit and a first relay lens group.

[0008] The modulation scanning module comprises a dynamic light splitting unit with non-fixed position, a first scanning galvanometer, a scanning lens, a barrel lens and an objective.

[0009] The detection light path module comprises a first lens, a second relay lens group, a second slit with non-fixed state, an electronically adjustable lens, a third relay lens group and a second scanning galvanometer.

[0010] The laser light source is configured to generate a laser beam;

[0011] The excitation light path module is configured to direct the laser beam to the modulation scanning module;

[0012] The modulation scanning module is configured to transmit the laser beam to a back focal plane of the objective lens and relay sample fluorescence collected by the objective lens to the detection light path module;

[0013] The detection light path module is configured to focus the sample fluorescence to the imaging camera;

[0014] The controller is configured to switch between an oblique light sheet imaging mode and a confocal imaging mode according to a position of the dynamic light splitting unit and a state of the second slit.

[0015] Optionally, the controller is configured to cause the microscopic imaging system to be in the oblique light sheet imaging mode when the dynamic light splitting unit is in the offset position and the second slit is in the removed state, and to be in the confocal imaging mode when the dynamic light splitting unit is in the reference position and the second slit is in the inserted state.

[0016] Optionally, the excitation light path module is configured to direct the laser beam to the modulation scanning module, including:

[0017] The expansion and contraction lens group is configured to adjust a spot size of the laser beam;

[0018] The shaping unit is configured to perform shaping processing on the laser beam;

[0019] The first slit is configured to perform optical filtering processing on the laser beam and cause the laser beam to reach the first relay lens group;

[0020] The first relay lens group is configured to transmit the laser beam to the dynamic light splitting unit.

[0021] Optionally, the modulation scanning module is configured to transmit the laser beam to a back focal plane of the objective lens and relay sample fluorescence collected by the objective lens to the detection light path module, including:

[0022] The dynamic light splitting unit is configured to reflect the laser beam to the first scanning galvanometer;

[0023] The first scanning galvanometer is configured to perform beam scanning on the laser beam and reflect the laser beam to the scanning lens;

[0024] The scanning lens and the barrel lens are configured to relay the laser beam to a back focal plane of the objective lens, so that the objective lens forms an excitation light sheet at a sample;

[0025] the objective lens is used for collecting the sample fluorescence;

[0026] the scanning lens and the cylindrical lens are used for relaying the sample fluorescence to the first scanning galvanometer;

[0027] the first scanning galvanometer is used for beam scanning of the sample fluorescence and reflecting the scanned sample fluorescence to the dynamic light splitting unit;

[0028] the dynamic light splitting unit is used for transmitting the sample fluorescence to the first lens.

[0029] Optionally, the probe light path module is used for focusing the sample fluorescence to the imaging camera, comprising:

[0030] the first lens and the second relay lens group are used for relaying the sample fluorescence after focusing by the electronically adjustable lens to the second slit;

[0031] the second slit is used for relaying the sample fluorescence to the second scanning galvanometer through the third relay lens group;

[0032] the second scanning galvanometer is used for beam scanning of the sample fluorescence and reflecting the scanned sample fluorescence to the imaging camera.

[0033] Optionally, the collimation unit comprises a cylindrical lens group.

[0034] Optionally, the dynamic light splitting unit comprises a non-fixed dichroic mirror, which is arranged on a non-fixed support.

[0035] Optionally, the first slit and the second slit are arranged at positions conjugate to the sample plane.

[0036] the first slit is used for adjusting the thickness and shape of the laser beam;

[0037] the second slit is used for inhibiting out-of-focus signals by spatial filtering in a confocal mode.

[0038] Optionally, the electronically adjustable lens is arranged at a sample imaging plane conjugate position of the probe light path, and is used for adjusting the focal length according to the driving voltage set by the controller.

[0039] Optionally, the controller is used for driving the first scanning galvanometer to scan in a gradient voltage and driving the electronically adjustable lens to adjust the focal length in a continuous voltage in an oblique light sheet imaging mode.

[0040] The controller is configured to drive the first scanning galvanometer with a gradient voltage, drive the second scanning galvanometer with an inverted gradient voltage, and drive the electronically adjustable lens with a gradient voltage for focal length adjustment in a confocal imaging mode.

[0041] In summary, the single objective multi-mode microscopic imaging system provided by the present application has at least the following beneficial effects: the single objective multi-mode microscopic imaging system includes a laser light source, an excitation light path module, a modulation scanning module, a detection light path module, an imaging camera, and a controller. The controller can switch between the oblique light sheet imaging mode and the confocal imaging mode according to the position of the dynamic light splitting unit and the state of the second slit, without introducing complex light path adjustment, so as to conveniently realize the switching of the microscopic imaging mode, simplify the switching process, and improve the efficiency of the switching of the microscopic imaging mode. BRIEF DESCRIPTION OF DRAWINGS

[0042] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the specific embodiments or prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0043] Figure 1 A schematic diagram of a single objective multi-mode microscopic imaging system provided by an embodiment of the present application;

[0044] Figure 2 A schematic diagram of an oblique light sheet microscopic imaging mode provided by an embodiment of the present application;

[0045] Figure 3 A schematic diagram of a confocal microscopic imaging mode provided by an embodiment of the present application;

[0046] Figure 4 A control logic schematic diagram of a single objective multi-mode microscopic imaging system provided by an embodiment of the present application. DETAILED DESCRIPTION

[0047] In order to make the above and other features and advantages of the present application clearer, the following further describes the present application with reference to the drawings. It should be understood that the specific embodiments given herein are for the purpose of explanation and are only exemplary, but are not limiting.

[0048] In the following description, many specific details are set forth in order to provide a thorough understanding of the present application. It will be apparent, however, to one skilled in the art that the present application can be practiced without specific details, which are not necessary to practice the present application. In other instances, well-known steps or procedures are not described in detail in order to avoid obscuring the present application.

[0049] Reference Figure 1 The application provides a general optical path schematic diagram of a single-objective multi-mode microscopic imaging system, which comprises a laser light source, an excitation light path module, a modulation scanning module, a detection light path module, an imaging camera and a controller.

[0050] The excitation light path module comprises, in sequence along an optical axis, an expansion-condensation lens group, a shaping unit, a first slit SLIT1 and a first relay lens group; the modulation scanning module comprises a non-fixed-position dynamic light splitting unit, a first scanning galvanometer GALVO1, a scanning lens L6, a barrel lens L5 and an objective lens OBJ; and the detection light path module comprises, in sequence along an optical axis, a first lens L7, a second relay lens group, a non-fixed-state second slit SLIT2, an electronically tunable lens ETL, a third relay lens group and a second scanning galvanometer GALVO2.

[0051] In addition, the excitation light path module can be used for transmitting a laser beam to the modulation scanning module, the modulation scanning module can be used for transmitting the laser beam to a back focal plane of the objective lens and transmitting sample fluorescence collected by the objective lens to the detection light path module, and the detection light path module can be used for transmitting the sample fluorescence to the imaging camera.

[0052] The laser light source LASER can be used for generating a laser beam, and can be a fiber laser, or can also be a semiconductor laser, or can also be a solid-state laser, etc., and the application does not make any limitation in this regard.

[0053] In addition, the objective lens can be a high-numerical-aperture objective lens, which can be a water immersion objective lens, or can also be an oil immersion objective lens, etc., and the numerical aperture thereof can be greater than 0.8, or can also be greater than 0.9, etc., so as to maintain the light sheet uniformity of a larger effective field of view under high-resolution imaging conditions, or can also be adjusted according to actual needs, etc., and the application does not make any limitation in this regard.

[0054] Optionally, the shaping unit can comprise a cylindrical lens group CL1, which can be a single lens, or can also be a multi-lens combination, etc., and the application does not make any limitation in this regard.

[0055] In addition, the dynamic light splitting unit DM is a light splitting device arranged in a non-fixed position, that is, the position thereof can be moved and changed, so that when the dynamic light splitting unit is in a reference position, the subsequent excitation light sheet can be vertically incident on a sample, and in the oblique light sheet imaging mode, the subsequent excitation light sheet can be incident on the sample at an oblique angle, so that by moving the position of the dynamic light splitting unit, the incidence angle of the laser beam can be adjusted, the position compensation of the laser beam on the back focal plane of the objective lens is realized, and the good focusing position of the excitation light sheet on the sample plane can be maintained when different magnification objective lenses are replaced.

[0056] Optionally, the dynamic light splitting unit can be a non-fixed dichroic mirror, which can be arranged on a non-fixed support.

[0057] The non-fixed support can be a movable support, and the non-fixed dichroic mirror arranged on the movable support can be controlled to move. In addition, the non-fixed dichroic mirror can select a specific filter according to the excitation wavelength of the laser and the emission wavelength of the fluorescence, which is not limited in the present application.

[0058] Therefore, in the embodiments of the present application, the controller can control the non-fixed dichroic mirror by using the non-fixed support to adjust the position of the non-fixed dichroic mirror, and further adjust the tilt angle of the laser beam formed by the final objective lens.

[0059] In addition, the second slit can be a non-fixed slit, that is, it can be a slit that can be controlled by electric control or other means to realize state change, and it can be a pluggable slit, such as being controlled by electric control to be in an inserted state or a removed state, etc. In addition, the size of the second slit can be adapted to the objective lens and other optical elements, which is not limited in the present application.

[0060] The expansion and contraction lens group can include a second lens L1 and a third lens L2, the shaping unit can include a cylindrical lens CL1, the first relay lens group can include a fourth lens L3 and a fifth lens L4, the second relay lens group can include a sixth lens L8 and a seventh lens L9, and the third relay lens group can include an eighth lens L10 and a ninth lens L11.

[0061] Optionally, the excitation light path module can further include a mirror M1 for reflecting the laser beam. The detection light path module can further include a filter FILTER that can selectively transmit sample fluorescence, etc. The present application is not limited in this regard.

[0062] It should be noted that, Figure 1 The optical elements in the above-mentioned embodiments are only illustrative, and can be adjusted as needed, such as being added or removed, or changing the position, etc., which is not limited in the present application.

[0063] It can be understood that in the confocal mode, the second slit can be used to suppress the out-of-focus signal by spatial filtering, so that when the oblique filter imaging mode and the confocal imaging mode are switched, the optical elements will not interfere.

[0064] The single-objective multi-mode microscopic imaging system provided by the embodiment of the present application comprises a laser light source, an excitation light path module, a modulation scanning module, a probe light path module, an imaging camera and a controller. The controller can switch between the oblique light sheet imaging mode and the confocal imaging mode according to the position of the dynamic light splitting unit and the state of the second slit. The switching of the microscopic imaging mode can be conveniently realized without introducing a complex light path adjustment, the switching process is simplified, and the efficiency of the switching of the microscopic imaging mode is improved.

[0065] Figure 2 The light path schematic diagram in the oblique light sheet microscopic imaging mode provided by the embodiment of the present application is shown.

[0066] As shown in the single-objective multi-mode microscopic imaging system, Figure 2 the fiber laser LASER can generate a laser beam, the expansion and contraction lens group can be used to adjust the spot size of the laser beam, the shaping unit CL1 can be used to perform shaping processing on the laser beam, the first slit SLIT1 can be used to perform filtering processing on the laser beam, and the laser beam reaches the first relay lens group, the first relay lens group can be used to transmit the laser beam to the dynamic light splitting unit DM, the dynamic light splitting unit DM can be used to reflect the laser beam to the first scanning galvanometer GALVO1, the first scanning galvanometer can be used to perform beam scanning on the laser beam, and reflect the laser beam to the scanning lens L6, the scanning lens L6 and the barrel lens L5 can be used to relay the laser beam to the back focal plane of the objective lens OBJ, so that the objective lens forms an excitation light sheet at the sample.

[0067] In addition, the objective lens can be used to collect sample fluorescence, the scanning lens and the barrel lens can be used to relay the sample fluorescence to the first scanning galvanometer, the first scanning galvanometer can be used to perform beam scanning on the sample fluorescence, and reflect the scanned sample fluorescence to the dynamic light splitting unit, and the dynamic light splitting unit is used to transmit the sample fluorescence to the first lens.

[0068] In addition, the first lens and the second relay lens group can be used to focus the sample fluorescence after being adjusted by the electronic adjustable lens, and relay the sample fluorescence to the second slit, and the second slit can be used to relay the sample fluorescence to the second scanning galvanometer through the third relay lens group, and the second scanning galvanometer can be used to perform beam scanning on the sample fluorescence, and reflect the scanned sample fluorescence to the imaging camera.

[0069] It can be understood that, according to Fourier optics, the fourth lens L3 and the fifth lens L4 in the first relay lens group need to constitute a 4f relay system. The expansion and contraction lens group can include the second lens L1 and the third lens L2, and the shaping unit can include the cylindrical lens CL1, the sixth lens L8 and the seventh lens L9 in the second relay lens group, which need to constitute a 4f relay system, so that the image plane does not drift when the second slit SLIT2 is inserted or removed, ensuring the confocal imaging capability. The eighth lens L10 and the ninth lens L11 of the third relay lens group need to constitute a 4f relay system to make the image plane after focusing by the electronic adjustable lens finally image to the imaging camera, thereby ensuring the stability and reliability of the image plane.

[0070] It can be understood that, as a laser light source, the fiber laser can generate a laser beam, which, after adjusting the beam diameter by the second lens L1 and the third lens L2 of the expansion and contraction lens group, enters the shaping unit composed of the cylindrical lens CL1 to generate a laser beam with uniform spatial distribution and long and flat shape. After the laser beam passes through the first slit SLIT1 to filter out stray light, it is relayed by the fourth lens L3 and the fifth lens L4 of the first relay lens group and transmitted to the non-fixed dichroic mirror DM. The non-fixed dichroic mirror DM can reflect the laser beam to the first scanning galvanometer GALVO1, and then the first scanning galvanometer GALVO1 performs beam scanning on the laser beam and reflects the scanned laser beam to the scanning lens L6 and the cylindrical lens L5. The scanning lens L6 and the cylindrical lens L5 can relay the laser beam to the back focal plane of the objective lens OBJ, so that the objective lens forms an excitation light sheet at the sample.

[0071] Then the objective lens OBJ can be used to collect sample fluorescence, the scanning lens L6 and the cylindrical lens L5 can be used to relay the sample fluorescence to the first scanning galvanometer GALVO1, and the first scanning galvanometer GALVO1 can be used to perform beam scanning on the sample fluorescence and reflect the scanned sample fluorescence to the non-fixed dichroic mirror DM. The non-fixed dichroic mirror DM can transmit the sample fluorescence to the first lens L7. The first lens L7 and the sixth lens L8 in the second relay lens group can relay the sample fluorescence after focusing by the electronic adjustable lens ETL to the seventh lens L9 in the second relay lens group, and then pass through the second slit SLIT2. The sample fluorescence can be relayed to the eighth lens L10 in the third relay lens group, and then the second scanning galvanometer GALVO2 can reflect the sample fluorescence to the ninth lens L11 in the third relay lens group. After filtering by the optical filter FILTER, the sample fluorescence can reach the imaging camera CAMERA.

[0072] Optionally, the first slit and the second slit can be arranged at positions conjugated to the sample plane, the first slit can be used to adjust the thickness and shape of the laser beam, i.e., to limit the thickness and shape of the laser beam. The second slit can be used to suppress out-of-focus signals by spatial filtering in the confocal mode, i.e., the second slit removes the detection light path in the oblique light sheet mode, which can avoid aberration or energy loss, and inserts the detection light path in the confocal mode and suppresses the out-of-focus signals by spatial filtering, so that when the imaging mode is switched, the optical elements can be ensured not to interfere, and the imaging quality can be optimized.

[0073] It can be understood that the shaping unit can be used to shape the laser beam to form a laser sheet, and accordingly, the first slit can filter the laser sheet, and the thickness of the laser sheet can be adjusted. Alternatively, the laser sheet is the laser beam after shaping, and therefore, in order to unify the description, the laser beam and the laser sheet can be collectively referred to as the laser beam.

[0074] Optionally, the electronic tunable lens can be arranged at a position conjugated to the sample imaging plane of the detection light path, and can be used to adjust the focal length according to the driving voltage set by the controller, so that rapid imaging of different depths can be achieved without moving the sample.

[0075] For example, the controller can apply a driving voltage corresponding to a target focal length to the electronic tunable lens ETL according to a preset voltage-focal length characteristic curve, so that the imaging focal plane can be quickly switched without moving the sample, and the image acquisition of different depth layers can be completed in cooperation with the exposure trigger of the camera, which is not limited in the present application.

[0076] Optionally, in the oblique light sheet imaging mode, the driving voltage of the electronic tunable lens can be a continuous voltage, so that the electronic tunable lens can continuously change the focal length, and the imaging camera and the electronic tunable lens remain synchronized, and the image is continuously triggered and acquired at a high speed. Therefore, in one complete scanning cycle of the electronic tunable lens, a large number of images can be acquired by the imaging camera, and since each frame of image corresponds to a specific focal point range, i.e., one oblique light sheet corresponds to one camera imaging, and the oblique light sheet is driven to scan horizontally by the first scanning galvanometer GALVO1, a three-dimensional reconstruction image can be constructed. Since the acquisition speed is extremely fast, the three-dimensional volume imaging of the living sample can be realized in the oblique light sheet imaging mode.

[0077] Optionally, in confocal imaging mode, the driving voltage of the electronically adjustable lens can be a stepped voltage, allowing the objective lens's focus to move layer by layer. During the stable holding period of a voltage step of the electronically adjustable lens, the first and second scanning mirrors can complete a full line scan cycle, and the imaging camera acquires the image of that layer synchronously. Thus, after acquisition, discrete multi-layer images of different depths can be obtained. Because the focal plane of each image layer is relatively stable and the signal-to-noise ratio is higher, better optical slicing effects can be achieved in conjunction with the second slit, making it widely applicable to scenarios with extremely high resolution requirements.

[0078] Understandably, in oblique light plate microscopy, the tilt angle of the excitation plate formed by the final objective lens can be controlled by adjusting the incident angle of the laser beam through translation of the non-fixed dichroic mirror. When the DM is in the offset position, the excitation plate is tilted within the sample, such as... Figure 2 As shown, oblique light plate imaging can be achieved. In this case, there is no second slit at the image position, and the second scanning galvanometer does not deflect, acting as a reflector. When DM is reset to the reference position, the excitation light plate is perpendicular to the back focal plane of the objective lens. At this point, combined with the insertion of SLIT2 and the coordinated scanning of the scanning galvanometer, a line scanning confocal mode can be formed, as shown... Figure 3 As shown, confocal imaging is achieved, thereby improving image sharpness. The reference position is the location where the excitation plate is perpendicular to the rear focal plane of the objective lens.

[0079] Understandably, in confocal imaging mode, the controller can synchronously control the first and second scanning mirrors, enabling them to work in conjunction with the electronically adjustable lens. This allows the electronically adjustable lens to quickly adjust its focal length in the probe optical path and synchronize with the scanning of the excitation / probe optical path, achieving rapid Z-axis scanning and real-time scanning function correction during 3D imaging, thereby obtaining a distortion-free 3D image.

[0080] It should be noted that, Figure 3 The various optical components and Figure 1 , Figure 2 The optical elements in all of them are the same, and their functions and roles can be referred to the descriptions of the various embodiments of this application. To avoid repetition, they will not be repeated here.

[0081] Therefore, in the embodiment of the present application, the controller can make the microscopic imaging system in the oblique light sheet imaging mode when the dynamic light splitting unit is in the offset position and the second slit is in the removed state, and make the microscopic imaging system in the confocal imaging mode when the dynamic light splitting unit is in the reference position and the second slit is in the inserted state. Therefore, by controlling the position of the dynamic light splitting unit and the state of the second slit, the switching between the oblique light sheet imaging mode and the confocal imaging mode can be realized, which is relatively simple and convenient to operate, and does not need to introduce a complex optical path, thereby further improving the efficiency of the imaging mode switching.

[0082] Figure 4 A control logic diagram of the single-objective multi-mode microscopic imaging system provided in the embodiment of the present application is provided. The control logic of the single-objective multi-mode microscopic imaging system provided in the embodiment of the present application is described below in combination with Figure 4 The control logic of the single-objective multi-mode microscopic imaging system provided in the embodiment of the present application is described below in combination with

[0083] It can be understood that in the oblique light sheet imaging mode, the controller can move the position of the dynamic light splitting unit, so that the original vertical laser beam formed at the original reference position can be deflected by a certain angle along the axial direction to form an oblique incident excitation light sheet passing through the sample. Therefore, without moving the sample, the excitation and imaging of different depth surfaces of the sample can be realized by changing the angle of the laser beam.

[0084] For example, in the case where the DM is horizontally pulled to the L7 direction to deviate from the reference position, that is, at the position 1, and the SLIT2 is in the removed state. Since the GALVO1 is used to realize the fast excitation line scanning task, the controller can periodically drive the GALVO1 in a sine wave or sawtooth wave manner, so that the GALVO1 performs beam scanning under the driving of the step voltage. At this time, since the GALVO2 only acts as a mirror, it does not need to be driven by applying a driving voltage. Then, the controller can apply a continuous voltage to the ETL to make the ETL adjust the focus according to the driving of the driving voltage, thereby realizing the layer-by-layer image acquisition in the Z-axis direction. Since the SLIT2 is in the removed state in this mode, that is, no spatial filtering is performed in the detection light path, so as to retain all the fluorescence signals as much as possible, the imaging speed and the large field of view are ensured, and the inclined light sheet is completely imaged. The imaging camera automatically triggers image acquisition after each Z-axis voltage is stable, which can be used to construct a three-dimensional reconstruction image. During the entire imaging period, the driving voltage of the ETL is gradually increased, the excitation light sheet slides along the sample volume direction, the GALVO1 performs high-speed line scanning to form the lateral resolution, and the imaging camera synchronously acquires images at each stable light sheet layer, and finally the high-resolution volume image is synthesized.

[0085] In the online scanning confocal mode, the tilted light sheet is no longer used, and the excitation light sheet in the vertical direction is output through the objective lens, and the scattered light is suppressed by the second slit to realize high-resolution layer imaging in the Z-axis. At this time, the controller can control the DM to return to the reference position, that is, the no displacement state as shown in Figure 3 At this time, after transmission through the modulation scanning module, the vertical excitation light sheet can be output. At the same time, the controller can control the second slit SLIT2 to be inserted between the L9 and L10 lenses to block the scattered light deviating from the focal point as much as possible, thereby improving the optical sectioning capability. The controller can apply a gradient voltage to the GALVO1 to perform lateral scanning of the light beam, and at the same time, apply a reverse gradient voltage to the GALVO2 to compensate by reverse scanning, offset the image drift caused by the GALVO1 scanning, so as to ensure the stability of the sample image as much as possible during the scanning process. The controller can apply a gradient voltage to the ETL to make the ETL realize single-layer fast scanning by focusing layer by layer or keeping the focal point constant, and the imaging camera can collect synchronous images according to the deflection period of the GALVO2 to construct two-dimensional or volumetric data.

[0086] Therefore, in the embodiment of the application, by adjusting the position of the dynamic light splitting unit and the state of the second slit, the switching between the tilted light sheet imaging mode and the confocal imaging mode can be quickly realized, which is simple to operate and efficient.

[0087] It can be understood that the scanning mode parameters can be preset in the controller, so that the controller can automatically switch modes during the acquisition period. For example, when the controller is controlled, it can first initialize the current mode, then adjust the position of the dynamic light splitting unit to change the angle of the excitation light sheet, and at the same time, control the state of the second slit to be inserted or removed according to the position of the dynamic light splitting unit, so that it corresponds to the position of the dynamic light splitting unit, then load the driving voltage to drive the ETL, and start the GALVO1 and GALVO2 scanning, then trigger the imaging camera exposure at the end of each frame, and reconstruct the image after the acquisition is completed. Therefore, the single-objective multi-mode microscopic imaging system provided in the embodiment of the application has a compact structure design, and can realize flexible imaging mode switching by combining the above control strategy. Therefore, not only can the biological sample be subjected to high-resolution three-dimensional fluorescence imaging, but also high-contrast two-dimensional structural images can be obtained, thereby realizing multi-functional integration on a single system.

[0088] Therefore, in the embodiments of the present application, by dynamically adjusting the excitation angle of the oblique light sheet and synchronously controlling the optical path, the excitation path and the detection path are shared, and the system structure is simplified. At the same time, through synchronous control of the dynamic light splitting unit, the first scanning galvanometer, the second scanning galvanometer, the electronically tunable lens and the second slit, not only the rapid switching between different imaging modes is realized, but also the high-resolution and rapid imaging of three-dimensional samples at different angles and depths is ensured. The microscopic system has compact structure, high degree of integration, low light toxicity, high throughput and high resolution, and can be widely used for tissue imaging, live cell tracking and high-throughput scene.

[0089] It should be noted that the optical elements in the single-objective multi-mode microscopic imaging system provided in the embodiments of the present application, such as the lenses in the relay lens group, need to meet the conjugate and optical constraints, so as to ensure that the imaging mode can be switched flexibly under the conditions of focusing and no distortion.

[0090] The single-objective multi-mode microscopic imaging system provided in the embodiments of the present application includes a laser light source, an excitation light path module, a modulation scanning module, a detection light path module, an imaging camera and a controller. The controller can make the microscopic imaging system in an oblique light sheet imaging mode when the dynamic light splitting unit is in an offset position and the second slit is in a removed state, and make the microscopic imaging system in a confocal imaging mode when the dynamic light splitting unit is in a reference position and the second slit is in an inserted state. In this way, by controlling the position of the dynamic light splitting unit and the state of the second slit, the switching between the oblique light sheet imaging mode and the confocal imaging mode can be realized without additional complex optical paths. This switching mode is more flexible and convenient to operate, and further improves the efficiency of the imaging mode switching.

[0091] The above-described technical features can be combined in any way. Although not all possible combinations of the technical features are described, any combination of the technical features should be considered to be covered by the present specification, as long as such a combination does not contradict.

[0092] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and such modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A single objective multi-modal microscopic imaging system, characterized in that, The system comprises a laser light source, an excitation light path module, a modulation scanning module, a detection light path module, an imaging camera and a controller. The excitation light path module comprises an expansion and contraction beam lens group, a shaping unit, a first slit and a first relay lens group. The modulation scanning module comprises a non-fixed position dynamic light splitting unit, a first scanning galvanometer, a scanning lens, a barrel lens and an objective lens. The detection light path module comprises a first lens, a second relay lens group, a non-fixed state second slit, an electronically adjustable lens, a third relay lens group and a second scanning galvanometer. The laser light source is configured to generate a laser beam. The excitation light path module is configured to guide the laser beam to the modulation scanning module. The modulation scanning module is configured to transmit the laser beam to the back focal plane of the objective lens and relay sample fluorescence collected by the objective lens to the detection light path module. The detection light path module is configured to focus the sample fluorescence to the imaging camera. The controller is configured to switch between oblique light sheet imaging mode and confocal imaging mode according to the position of the dynamic light splitting unit and the state of the second slit.

2. The microscopic imaging system of claim 1, wherein The controller is configured to make the microscopic imaging system in oblique light sheet imaging mode when the dynamic light splitting unit is in an offset position and the second slit is in a removed state, and make the microscopic imaging system in confocal imaging mode when the dynamic light splitting unit is in a reference position and the second slit is in an inserted state. The excitation light path module is configured to guide the laser beam to the modulation scanning module, comprising 3. The microimaging system of claim 1, wherein, The expansion and contraction beam lens group is configured to adjust the spot size of the laser beam. The shaping unit is configured to perform shaping processing on the laser beam. The first slit is configured to perform optical filtering processing on the laser beam and make the laser beam reach the first relay lens group. The first relay lens group is configured to transmit the laser beam to the dynamic light splitting unit. The modulation scanning module is configured to transmit the laser beam to the back focal plane of the objective lens and relay sample fluorescence collected by the objective lens to the detection light path module, comprising 4. The microimaging system of claim 3, wherein, The dynamic light splitting unit is configured to reflect the laser beam to the first scanning galvanometer. The first scanning galvanometer is configured to perform beam scanning on the laser beam and reflect the laser beam to the scanning lens. The scanning lens and the barrel lens are configured to relay the laser beam to the back focal plane of the objective lens to make the objective lens form an excitation light sheet at a sample. The objective lens is configured to collect the sample fluorescence. The scanning lens and the barrel lens are configured to relay the sample fluorescence to the first scanning galvanometer. The first scanning galvanometer is configured to perform beam scanning on the sample fluorescence and reflect the scanned sample fluorescence to the dynamic light splitting unit. The dynamic light splitting unit is configured to transmit the sample fluorescence to the first lens. The detection light path module is configured to focus the sample fluorescence to the imaging camera, comprising 5. The microimaging system of claim 4, wherein, ​ The first lens and the second relay lens group are used to relay the sample fluorescence focused by the electronically tunable lens to the second slit; The second slit is used to relay the sample fluorescence to the second scanning galvanometer through the third relay lens group; The second scanning galvanometer is used to perform beam scanning on the sample fluorescence and reflect the scanned sample fluorescence to the imaging camera.

6. The microimaging system of claim 1, wherein, The collimating unit includes a cylindrical lens group.

7. The microscopy imaging system of claim 1, wherein, The dynamic light splitting unit includes a non-fixed dichroic mirror arranged on a non-fixed support.

8. The microscopic imaging system of claim 1, wherein, The first slit and the second slit are arranged at positions conjugate to the sample plane; The first slit is used to adjust the thickness and shape of the laser beam; The second slit is used to suppress out-of-focus signals by spatial filtering in a confocal mode.

9. The microscopic imaging system of claim 1, wherein, The electronically tunable lens is arranged at a position conjugate to the sample imaging plane of the probe light path and is used to adjust the focal length according to the driving voltage set by the controller.

10. The microscopic imaging system of claim 1, wherein, The controller is used to drive the first scanning galvanometer to scan with a gradient voltage and drive the electronically tunable lens to adjust the focal length with a continuous voltage in an oblique wedge imaging mode; The controller is used to drive the first scanning galvanometer to scan with a gradient voltage, drive the second scanning galvanometer to scan with an inverted gradient voltage, and drive the electronically tunable lens to adjust the focal length with a gradient voltage in a confocal imaging mode.

Images