CN113310964B - Digital scanning optical sheet microscopic imaging system compatible with microfluidic chip - Google Patents
Digital scanning optical sheet microscopic imaging system compatible with microfluidic chip Download PDFInfo
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Abstract
The invention discloses a digital scanning optical sheet microimaging system compatible with a microfluidic chip, which comprises: an inverted microscope detection unit; the staggered layer supporting unit comprises a high-position supporting frame and a low-position supporting frame, wherein the high-position supporting frame is arranged across the two-dimensional translation table of the inverted microscope detection unit, and the low-position supporting frame is arranged on one side of the inverted microscope; the sample scanning unit passes through the high-position support frame of the staggered layer support unit and is fixed with the two-dimensional translation table of the inverted microscope detection unit, and is used for fixing the microfluidic chip sample and driving the microfluidic chip sample to perform scanning motion along the Z-axis direction; the digital scanning optical sheet lighting unit comprises a lighting objective lens and a digital scanning optical sheet forming element group. The invention overcomes the problem of space constraint by using the inverted microscope as a detection light path through the inverted microscope and the staggered layer supporting device, and realizes the long-time, large-visual-field, high-speed, high-resolution and high-flux three-dimensional imaging of free-moving living organisms.
Description
Technical Field
The invention belongs to the field of biomedical microscopic imaging, and particularly relates to a digital scanning optical sheet microscopic imaging system compatible with a microfluidic chip.
Background
The light sheet microscopic imaging system is an emerging biomedical imaging system, and an illumination light path and a detection light path of the light sheet microscopic imaging system are usually mutually independent and in an orthogonal state. The simplest light sheet can be generated by a cylindrical lens, which is a static Gaussian light sheet. The light level of such a light sheet is not adjustable; the stripe is easily generated when the tissue with uneven refractive index is encountered, so that the illumination is uneven; meanwhile, the laser beam is pressed into a sheet shape, and the fluorescence excitation efficiency is not high enough.
The common light sheet microscope is generally incompatible with a microfluidic chip, lacks a necessary control means for a biological sample, can obtain good effect when shooting a static sample, but is difficult to carry out high-speed high-resolution high-throughput three-dimensional imaging on living organisms. Some light sheet microscopes are also compatible with the microfluidic chip, but the sample in the microfluidic chip is imaged in an inclined illumination and detection mode, the imaging mode is influenced by the scanning stroke, and under the condition that the vertical height of imaging is kept unchanged, when the visual field is increased along the scanning dimension, the scanning stroke is increased, so that large-visual-field high-speed three-dimensional imaging cannot be realized.
Disclosure of Invention
Aiming at the defects or the improvement requirements of the prior art, the invention provides a digital scanning light sheet micro-imaging system compatible with a micro-fluidic chip, aiming at expanding the installation space of elements by combining an inverted microscope with a support frame designed in a staggered layer, successfully combining clamping, controlling and moving elements of the micro-fluidic chip and element groups formed by the digital scanning light sheet and enabling the lighting light path and the detection light path of the digital scanning light sheet to be orthogonal, thereby solving the technical problem that the lighting light path and the micro-fluidic sample of the digital scanning light sheet cannot be combined due to the local installation of the elements in the prior art.
To achieve the above object, according to one aspect of the present invention, there is provided a digital scanning optical sheet micro-imaging system compatible with a microfluidic chip, comprising: the device comprises a staggered layer supporting unit, an inverted microscope detection unit, a sample scanning unit with a microfluidic chip clamping piece and a digital scanning optical sheet lighting unit;
the inverted microscope detection unit comprises a two-dimensional translation table for carrying objects and an imaging light path for collecting fluorescence signals and imaging;
the staggered-layer supporting unit comprises a high-position supporting frame and a low-position supporting frame, wherein the high-position supporting frame spans the two-dimensional translation table of the inverted microscope detection unit, and the low-position supporting frame is arranged on one side of the inverted microscope;
the sample scanning unit passes through the high-position support frame of the staggered layer support unit and is fixed with the two-dimensional translation table of the inverted microscope detection unit, and is used for fixing a microfluidic chip sample and driving the microfluidic chip sample to perform scanning motion along the Z-axis direction;
the digital scanning light sheet lighting unit comprises a lighting objective lens and a digital scanning light sheet forming element group; the illumination objective lens is arranged on the high-position support frame and is positioned on the other side of the water vat; the digital scanning light sheet forming element group is arranged on the low-position support frame, the collimated laser forms a laser light sheet through the digital scanning light sheet forming element group shape and the illumination objective lens, and the light sheet beam waist is located in the water tank.
Preferably, when the digital scanning light sheet microimaging system compatible with the microfluidic chip works, the microfluidic chip is fixed on the microfluidic chip clamping piece and is arranged in the water tank, the illumination light sheet formed by the digital scanning light sheet illumination unit is projected on a sample through the transparent side surface of the water tank and the side surface with optical flatness of the microfluidic chip, and the excited fluorescence is collected and imaged by the inverted microscope detection unit through the lower bottom surface with optical flatness of the microfluidic chip and the transparent bottom surface of the water tank.
Preferably, the sample scanning unit of the digital scanning optical sheet microimaging system compatible with the microfluidic chip comprises a microfluidic chip clamping piece and a supporting piece; the microfluidic chip clamping piece penetrates through the staggered layer supporting unit through a supporting piece and is fixed on a two-dimensional translation table of the inverted microscope detection unit, and the two-dimensional translation table drives the microfluidic chip clamping piece to move in a horizontal plane.
Preferably, the digital scanning optical sheet microimaging system compatible with the microfluidic chip comprises a sample scanning unit, a sample scanning unit and a micro-fluidic chip, wherein the sample scanning unit comprises a Z-axis displacement table and a piezoelectric scanning table;
the Z-axis translation stage is used for driving the microfluidic chip clamping piece to move at a low speed along the Z axis so that the microfluidic chip is placed in the water tank; the piezoelectric scanning platform is connected with the microfluidic chip clamping piece and is used for driving the microfluidic chip clamping piece to move along the Z axis at a high speed; the micro-fluidic chip control part is connected with the micro-fluidic chip through a hydraulic pipeline, and the side surface and the lower bottom surface of the micro-fluidic chip are optical flat surfaces.
Preferably, the support member of the digital scanning light sheet microimaging system compatible with the microfluidic chip comprises a first L-shaped adapter member and a second L-shaped adapter member, wherein one side of the first L-shaped adapter member is parallel to one side of the second L-shaped adapter member in a staggered manner, and the other side of the first L-shaped adapter member is arranged in a parallel manner to form an Contraband-shaped support member, wherein the two sides in the staggered manner are connected through a Z-axis translation stage, the other side of the first L-shaped adapter member is fixed with the two-dimensional translation stage of the detection unit of the inverted microscope, the other side of the second L-shaped adapter member is connected with the piezoelectric scanning stage, and the piezoelectric scanning stage is connected with the microfluidic chip clamping member.
Preferably, in the digital scanning light sheet microimaging system compatible with the microfluidic chip, the microfluidic chip clamping piece comprises vertical clamping arms parallel to each other, a flat fixing piece fixed to the top ends of the clamping arms, and a pair of Contraband-type fixing pieces fixed to the bottom ends of the two arms of the clamping arms respectively, and the clamping arms are connected with the piezoelectric scanning platform through the flat fixing pieces and fix the microfluidic chip through Contraband-type fixing pieces.
Preferably, the sample scanning unit of the digital scanning optical sheet microimaging system compatible with the microfluidic chip comprises a microfluidic chip control part; the micro-fluidic chip control part is connected with the micro-fluidic chip through a hydraulic pipeline, and the side surface and the lower bottom surface of the micro-fluidic chip are optical flat surfaces;
preferably, in the digital scanning light sheet micro-imaging system compatible with the microfluidic chip, the digital scanning light sheet forming element group comprises an XY galvanometer, a Z galvanometer, a scanning lens, a sleeve lens and a plurality of reflectors for adjusting the trend of a light path, and the gaussian digital scanning light sheet forming element group is fixed on the low-position support frame.
Preferably, in the digital scanning optical sheet microimaging system compatible with the microfluidic chip, the high-position support frame comprises two arms which are parallel to each other and adjustable in distance.
Preferably, in the digital scanning light sheet microimaging system compatible with the microfluidic chip, the lower support frame is a bread board.
In general, compared with the prior art, the above technical solution contemplated by the present invention can achieve the following beneficial effects:
according to the invention, the inverted microscope and the staggered-layer supporting device are adopted, the inverted microscope is used as a detection light path, the problem of space constraint is solved, the digital scanning light sheet imaging technology and the microfluidic technology are combined, the advantages of no stripe, high controllability of light and energy concentration of the digital scanning light sheet are brought into play, the illumination uniformity is improved, and the signal-to-noise ratio, the contrast and the axial resolution capability of an image are improved; the micro-fluidic control capability is also exerted, the micro-fluidic improves the operability and controllability of living organisms, and the imaging flux is improved. The advantages of the two technologies are complementary, the problem that the traditional optical sheet imaging is difficult to be used for free motion living body imaging is solved, and the three-dimensional imaging of the free motion living body with large visual field, long time and high space-time resolution under a common inverted microscope is realized; particularly, a transverse illumination mode is adopted on the micro-fluidic chip, so that the acquisition speed of three-dimensional data is improved; the three points are combined to realize the long-time, large-visual-field, high-speed, high-resolution and high-flux three-dimensional imaging of the free-moving living organisms.
Drawings
FIG. 1 is a schematic diagram of an optical path structure of a digital scanning optical sheet micro-imaging system compatible with a microfluidic chip provided by the invention;
FIG. 2 is a schematic structural diagram of a digital scanning optical sheet micro-imaging system compatible with a microfluidic chip provided by the invention;
FIG. 3 is a three-dimensional structure diagram of a split-level supporting unit provided by the present invention;
FIG. 4 is a three-dimensional structure diagram of a high-position supporting frame of the split-level supporting unit provided by the present invention;
FIG. 5 is a three-dimensional block diagram of a sample scanning unit provided by the present invention;
FIG. 6 is a three-dimensional block diagram of a microfluidic chip holder;
FIG. 7 is a three-dimensional structural view of a water vat;
FIG. 8 is a schematic structural diagram of a microfluidic chip provided in an embodiment of the present invention;
FIG. 9 is a refractive index design diagram of a microfluidic chip provided in an embodiment of the present invention;
fig. 10 is a three-dimensional structural view of a microfluidic chip control element and an illumination detection mode provided in an embodiment of the present invention;
fig. 11 is a schematic view of the installation of the microfluidic chip provided by the embodiment of the invention.
The same reference numbers will be used throughout the drawings to refer to the same or like elements or structures, wherein:
the device comprises a laser 1, an optical fiber 2, a collimating head 3, a reflector 4, an XY galvanometer 5, a Z galvanometer 6, a scanning lens 7, a sleeve lens 8, an illumination objective lens 9, a water tank 10, a piezoelectric scanning table 11, a microfluidic chip holder 12, a microfluidic chip 13, a Z axis displacement table 14, a detection objective lens 15, a color filter block 16, a reflector 17, a sleeve lens 18, a camera 19, an inverted microscope 20, a second L-shaped adapter 21, a first L-shaped adapter 22, a flat plate 23, a strip-shaped connector 24, a support column 25, a digital scanning optical sheet 26, a reflector 27, a panel plate 28, a constant flow pump 29, an electromagnetic valve 30 and an air pump 31.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The common static Gaussian light sheet microscope has stripes and uneven illumination; the existing digital scanning light sheet is not combined with a microfluidic chip or adopts a transverse light sheet illumination mode, so that a biological sample can not be controlled, high-speed three-dimensional imaging can not be carried out, and high-flux imaging can not be carried out. However, the loading and control device of the microfluidic chip is complicated, the optical path system for forming the digital scanning light sheet also has a complicated structure, and the illumination optical path and the detection optical path need to be orthogonal, so that the loading and control device of the microfluidic chip and the optical path for forming the digital scanning light sheet are difficult to erect in the imaging field of the microscope.
The invention provides a digital scanning light sheet microscopic imaging system compatible with a microfluidic chip, which has a schematic diagram shown in figure 1 and a structural diagram shown in figure 2 and comprises a staggered layer supporting unit, an inverted microscope detection unit, a sample scanning unit with a microfluidic chip clamping piece and a digital scanning light sheet lighting unit;
the laser passes through the microfluidic chip 13 from the side surface and forms a light sheet by scanning through the XY galvanometer 5. The sample can be driven by the Z-axis displacement table 14 to perform low-speed three-dimensional imaging, and can also be driven by the piezoelectric scanning table 11 to perform high-speed three-dimensional imaging. The light sheet enters from the side surface of the microfluidic chip, the scanning stroke is only influenced by the height of the chamber of the microfluidic chip and is irrelevant to the imaging visual field, and the chamber is low in height, so that large-visual-field high-speed dynamic imaging can be performed;
the inverted microscope detection unit comprises a two-dimensional translation table for carrying objects and an imaging light path for collecting fluorescence signals and imaging; the inverted microscope is used for detection, a detection light path does not need to be additionally built, the detection performance is excellent, and the stability is high. The two-dimensional translation stage of the inverted microscope is used for installing the sample scanning unit to realize position adjustment on the horizontal plane.
The staggered-layer supporting unit, as shown in fig. 3, includes a high-position supporting frame arranged across the two-dimensional translation stage of the inverted microscope detection unit, and a low-position supporting frame arranged at one side of the inverted microscope; preferably, the high-position support frame comprises two arms which are parallel to each other and have adjustable distance, as shown in fig. 4; the low-position support frame is preferably a bread board;
as shown in fig. 5, the sample scanning unit is fixed to the two-dimensional translation stage of the inverted microscope detection unit through the high-position support frame of the staggered support unit, and is configured to fix a microfluidic chip sample and drive the microfluidic sample to perform scanning motion along the Z-axis direction; the device comprises a micro-fluidic chip clamping piece, a micro-fluidic chip control piece, a Z-axis displacement table, a piezoelectric scanning table and a supporting piece; the microfluidic chip clamping piece penetrates through the staggered layer supporting unit through a supporting piece and is fixed on a two-dimensional translation table of the inverted microscope detection unit, and the two-dimensional translation table drives the microfluidic chip clamping piece to move in a horizontal plane; the Z-axis translation stage is used for driving the microfluidic chip clamping piece to move at a low speed along the Z axis so that the microfluidic chip is placed in the water tank; the piezoelectric scanning platform is connected with the microfluidic chip clamping piece and is used for driving the microfluidic chip clamping piece to move along the Z axis at a high speed; the micro-fluidic chip control part is connected with the micro-fluidic chip through a hydraulic pipeline, and the side surface and the lower bottom surface of the micro-fluidic chip are optical flat surfaces; preferably, the support member comprises a first L-shaped adapter member and a second L-shaped adapter member, wherein one side of the first L-shaped adapter member is staggered and parallel to one side of the second L-shaped adapter member, the other side of the first L-shaped adapter member is arranged in parallel to the other side of the second L-shaped adapter member, so that an Contraband-shaped support member is formed, the two sides which are staggered and parallel are connected through a Z-axis translation table, the other side of the first L-shaped adapter member is fixed with the two-dimensional translation table of the inverted microscope detection unit, the other side of the second L-shaped adapter member is connected with the piezoelectric scanning table, and the piezoelectric scanning table is connected with the microfluidic chip clamping member. The microfluidic chip clamping piece comprises vertical clamping arms parallel to each other, a flat fixing piece fixed to the top ends of the clamping arms, and a pair of Contraband-type fixing pieces fixed to the bottom ends of the two arms of the clamping arms respectively, as shown in fig. 6, wherein the clamping arms are connected with the piezoelectric scanning platform through the flat fixing pieces, and the microfluidic chip is fixed through Contraband-type fixing pieces.
The digital scanning light sheet lighting unit comprises a water tank, a lighting objective lens and a digital scanning light sheet forming element group.
The water vat is fixed on the high-position support frame of the staggered support unit and is positioned right above the field of view of the detection unit of the inverted microscope as shown in fig. 7; the illumination objective lens is arranged on the high-position support frame and is positioned on one side of the low-position support frame of the water vat; the illumination objective lens needs to be placed above a two-dimensional translation stage of the inverted microscope and cannot be influenced by the two-dimensional translation stage, and the water vat is the same. The stage unit thus facilitates the mounting of the illumination objective and the water vat, as shown in fig. 2, 3.
The digital scanning optical sheet forming element group is arranged on the low-position support frame; the collimated laser forms laser sheet light through a digital scanning sheet forming element group, and the beam waist of the sheet light is positioned in the water tank through the illumination objective lens; the digital scanning optical sheet forming element group comprises an XY galvanometer, a Z galvanometer, a scanning lens, a sleeve lens and a plurality of reflectors for adjusting the trend of a light path, and the Gaussian digital scanning optical sheet forming element group is fixed on the low-position support frame. The digital scanning light sheet forming element group has the advantages of no stripe, high controllability of light and high energy concentration fluorescence excitation efficiency, greatly improves the image signal to noise ratio and the axial resolution capability of a common inverted microscope, but has more components and larger volume, and is difficult to integrate with an illumination objective lens on the same support frame. The invention preferably arranges the low-position support frame at the position of half the height of the microscope, which can match the height of the detection objective lens of the inverted microscope. Under the swing of the vibrating mirror, the light beam moves back and forth along one direction to form a light sheet, so that stripes can be eliminated, and the illumination uniformity is improved. When in imaging, the inverted microscope is used for detecting, the performance of a detection light path is excellent, and meanwhile, the optical sheet imaging improves the signal-to-noise ratio and the axial resolution of an image. The digital scanning light sheet lighting unit has the advantages of no stripe, high controllability of light and high energy concentration fluorescence excitation efficiency, and greatly improves the image signal-to-noise ratio and the axial resolution capability of a common inverted microscope.
When the device works, the microfluidic chip is fixed and detachably fixed on the microfluidic chip clamping piece, and is arranged in the water tank, the illuminating light sheet formed by the digital scanning light sheet illuminating unit is projected on a sample through the transparent side surface of the water tank and the side surface with optical flatness of the microfluidic chip, and excited fluorescence is collected and imaged by the inverted microscope detection unit through the lower bottom surface with optical flatness of the microfluidic chip and the transparent bottom surface of the water tank.
The microfluidic chip generally needs to be matched with a control device such as an injector or a constant flow pump, a three-dimensional moving device and the like; the light sheet microscopic imaging system is adopted, so that the illumination light path and the detection light path are required to be in an orthogonal state; therefore, the complex optical element, the micro-fluidic chip and the control device thereof, which are arranged on the light sheet illumination light path for improving the imaging effect, and the detection light path are very narrow in spatial position, the whole volume is large, and the illumination and imaging effects are poor. According to the invention, the space above the visual field of the detection light path is expanded by the inverted microscope, and the Z-axis motion and the imaging light path of the sample are integrated on the Z axis by combining the three-dimensional movement decomposition of the microfluidic chip, so that more space is vacated for the arrangement of the illumination light path, the illumination light path does not need to adopt oblique incidence, and the illumination light path is orthogonally incident on a horizontal plane, so that the illumination light path and the detection light path can be in an orthogonal state, and the space division discrimination capability is improved; however, such an arrangement presents a mounting conflict between the illumination objective, which needs to be placed above the two-dimensional translation stage of the inverted microscope and cannot be influenced by the two-dimensional translation stage, the water vat, which works similarly, and the XY plane motion system of the sample above the detection optical path. In order to solve the contradiction, the invention adopts the high-position support frame of the staggered layer support unit to install the illumination objective lens and the water cylinder which can not be influenced by the two-dimensional translation stage, and expands two layers of installation spaces, thereby realizing the integration of the inverted microscope detection unit and the sample scanning unit on the Z axis, simultaneously realizing the staggered layer design of high and low positions, fully utilizing the space on one side of the inverted microscope detection unit, improving the integration level and reducing the volume of the digital scanning light sheet illumination unit.
The following are examples:
a digital scanning light sheet microscopic imaging system compatible with a microfluidic chip is shown in a schematic diagram of fig. 1 and a structural diagram of fig. 2, and comprises a staggered layer supporting unit, an inverted microscope detection unit, a sample scanning unit with a microfluidic chip clamping piece and a digital scanning light sheet lighting unit;
the inverted microscope detection unit comprises a two-dimensional translation table for carrying objects and an imaging light path for collecting fluorescence signals and imaging; fluorescence detection can be performed by selecting an appropriate detection objective lens 15, color filter block 16, and camera 19, and mounting these components on an inverted microscope. An IX70 inverted microscope, a bilayer IX73 inverted microscope, and a monolayer IX73 inverted microscope may be used.
The staggered-layer supporting unit, as shown in fig. 3, includes a high-position supporting frame arranged across the two-dimensional translation stage of the inverted microscope detection unit, and a low-position supporting frame arranged at one side of the inverted microscope; the high-position support frame comprises two arms which are parallel to each other and the distance of which can be adjusted; the low-position supporting frame is a bread board. In this embodiment, a split processing manner is adopted, two aluminum alloy flat plates 23 are manufactured as two arms, and then are stably connected by a water cylinder 10 and a strip-shaped connecting piece 24, and finally, the high position is supported right above the inverted microscope 20 by four connecting rods 25. Fig. 4 is a three-dimensional structural view of a stage unit above a microscope.
The sample scanning unit with the microfluidic chip holder is shown in fig. 5. Which is secured to the inverted microscope 20 by an L-shaped adapter 22. The Z-axis displacement table 14 is installed on the vertical surface of the L-shaped adaptor 22, the table top of the Z-axis displacement table 14 is connected with the L-shaped adaptor 21, the piezoelectric scanning table 11 is installed above the L-shaped adaptor 21, and the two L-shaped adaptors and the Z-axis displacement table form a support and penetrate through the high-position support frame. The micro-fluidic chip clamping piece 12 is arranged on the table top of the piezoelectric scanning table 11 and extends downwards from the middle through hole, and the micro-fluidic chip 13 is arranged at the tail end of the micro-fluidic chip clamping piece 12. The structure of the microfluidic chip clamping piece 12 is shown in fig. 6, and the unit is used for clamping the microfluidic chip and driving the microfluidic chip to perform three-dimensional scanning imaging. The sample clamping piece is designed to be matched with the microfluidic chip in size, the loading is stable, the clamping piece is provided with the transverse strip-shaped fence, the microfluidic chip can be prevented from being damaged by the stress of the guide pipe, the stability of three-dimensional scanning is improved, the microfluidic chip does not need to be disassembled, and the microfluidic chip can be used for a long time.
The side surface of the microfluidic chip 13 is made flat, as shown in fig. 8, where fig. 8a is a perspective view and fig. 8b is a side view, and the optical sheet generated by the digital scanning optical sheet lighting unit is incident into the chamber through the side surface of the microfluidic chip 13. The side surface of the chip is flattened, so that the effect of a glass surface is shown. The material proportion of the upper layer and the lower layer of the microfluidic chip can select the refractive index proportion, and the artifact of the transverse light sheet can be inhibited by the refractive index proportion. The positive refractive index ratio indicates that the refractive index of the upper layer material is greater than that of the lower layer material, and the negative refractive index ratio indicates that the refractive index of the upper layer material is less than that of the lower layer material. For the optical system of inverted detection, shallow artifacts can be eliminated by using the inverse index matching, as shown in fig. 9. Preferably, the refractive index matching fluid is introduced into the chip.
The microfluidic chip control part is shown in fig. 10, wherein the fluid layer has two ports including p.1 and p.2, and can be injected by an injector or a constant flow pump 29; the control layer in the figure has two ports, including v.1 and v.2, which can be controlled by a solenoid valve 30 and an air pump 31.
As shown in fig. 11, the microfluidic chip 13 is inserted into a catheter and then mounted on the microfluidic chip holder 12, and then the microfluidic chip holder is mounted on the piezoelectric scanning stage. The step is only needed to be executed once, the microfluidic chip 13 can be installed on the microfluidic chip clamping piece 12 for a long time, and the sample loading and sample unloading can be realized only by operating the injector/constant flow pump 29 and the electromagnetic valve 30 for each imaging, so that the step is very convenient and has high flux. The controllability of the biological sample can be improved, and the microfluidic high-throughput performance can be exerted.
A digital scanning light sheet illumination unit as shown in fig. 3. Comprises a water vat 10, an illumination objective 9 and a digital scanning light sheet forming element group; the scanning device comprises an XY galvanometer, a Z galvanometer, a scanning lens, a sleeve lens and a plurality of reflectors for adjusting the trend of a light path, wherein a Gaussian digital scanning optical sheet forming element group is fixed on a low-position support frame. In the embodiment, laser is emitted by a laser 1, reaches a quasi-finger head 3 through an optical fiber 2, and is emitted as parallel light with the spot diameter of 1.5 mm. After being reflected by the mirror 4, the beam reaches the XY galvanometer 5 and is reflected to the Z galvanometer 6. The beam after passing through the galvanometer is incident on the scanning lens 7 to be subjected to flat field scanning, with a beam waist of 29 um. Finally, the beam passes through a reducing system consisting of a sleeve lens 8 and an objective lens 9, and the beam waist size is 7.25 um. The corset is located inside the water tub 10. The objective lens 9 and the water vat 10 are arranged on a high-position support frame above the inverted microscope, and other components are arranged on a bread board 28 of a low-position support frame.
Three-dimensional imaging is carried out on the drosophila larva abdominal neurons:
the fruit fly larvae are led into the microfluidic chip 13 through the injector/constant flow pump 29, the electromagnetic valve 31 is opened, and at the moment, the fruit fly larvae can only do two-dimensional free motion in the imaging visual field and can not be separated from the imaging visual field.
When shooting drosophila larva abdominal neurons, the frequency of the piezoelectric scanning platform 11 is 10Hz, and 2 three-dimensional images are acquired in each period, so that 20vps is achieved. The chamber design of micro-fluidic chip is 3000um 500um 90um, and the fruit fly larva can freely move in this space. The time for introducing the drosophila larvae into the microfluidic chip is less than 1 minute, the imaging time is random, and the neurons of the drosophila larvae are clear. The stroke of the piezoelectric scanning platform is 60um, the exposure time of the camera 19 is 4.5ms, the camera 19 selects an external edge trigger mode, the camera frame rate is 160fps, and the scanning step length is 7.5 um.
The embodiment is a compact imaging system built on the basis of a common inverted microscope, combines the advantages of no stripe of a digital scanning polished section, high controllability of light and energy concentration and the advantages of the manipulation performance of a microfluidic biological sample, and improves the acquisition data of three-dimensional imaging by a mode of transverse polished section illumination on a microfluidic chip, so that the invention realizes the long-time, large-field, high-speed, high-resolution and high-flux three-dimensional imaging of free-moving living organisms.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. A digital scanning optical sheet microimaging system compatible with a microfluidic chip is characterized by comprising: the device comprises a staggered layer supporting unit, an inverted microscope detection unit, a sample scanning unit with a microfluidic chip clamping piece and a digital scanning optical sheet lighting unit;
the inverted microscope detection unit comprises a two-dimensional translation table for carrying objects and an imaging light path for collecting fluorescence signals and imaging;
the staggered-layer supporting unit comprises a high-position supporting frame and a low-position supporting frame, wherein the high-position supporting frame spans the two-dimensional translation table of the inverted microscope detection unit, and the low-position supporting frame is arranged on one side of the inverted microscope;
the sample scanning unit passes through the high-position support frame of the staggered layer support unit and is fixed with the two-dimensional translation table of the inverted microscope detection unit, and is used for fixing a microfluidic chip sample and driving the microfluidic chip sample to perform scanning motion along the Z-axis direction;
the digital scanning light sheet lighting unit comprises a lighting objective lens and a digital scanning light sheet forming element group; the illumination objective lens is arranged on the high-position support frame and is positioned on the other side of the water vat; the digital scanning light sheet forming element group is arranged on the low-position support frame, and the collimated laser forms a laser light sheet through the digital scanning light sheet forming element group and the illumination objective lens, so that the beam waist of the light sheet is positioned in the water tank.
2. The microfluidic chip-compatible digital scanning light sheet microimaging system of claim 1, wherein in operation, the microfluidic chip is fixed on the microfluidic chip holder and placed in a water tank, the illumination light sheet formed by the digital scanning light sheet illumination unit is projected on the sample through the transparent side surface of the water tank and the optically flat side surface of the microfluidic chip, and the excited fluorescence is collected and imaged by the inverted microscope detection unit through the optically flat lower bottom surface of the microfluidic chip and the transparent bottom surface of the water tank.
3. The microfluidic chip compatible digital scanning light sheet microimaging system of claim 1, wherein the sample scanning unit comprises a microfluidic chip holder and a support; the microfluidic chip clamping piece penetrates through the staggered layer supporting unit through a supporting piece and is fixed on a two-dimensional translation table of the inverted microscope detection unit, and the two-dimensional translation table drives the microfluidic chip clamping piece to move in a horizontal plane.
4. The microfluidic chip compatible digital scanning light sheet microimaging system of claim 3, wherein the sample scanning unit comprises a Z-axis displacement stage, a piezoelectric scanning stage;
the Z-axis displacement table is used for driving the microfluidic chip clamping piece to move at a low speed along the Z axis so that the microfluidic chip is placed in the water cylinder; the piezoelectric scanning platform is connected with the microfluidic chip clamping piece and is used for driving the microfluidic chip clamping piece to move along the Z axis at a high speed; the micro-fluidic chip control part is connected with the micro-fluidic chip through a hydraulic pipeline, and the side surface and the lower bottom surface of the micro-fluidic chip are optical flat surfaces.
5. The microfluidic chip compatible digital scanning light sheet microimaging system of claim 4, wherein the support member comprises a first and a second L-shaped adaptor, wherein one side of the first and second L-shaped adaptor is parallel and staggered, and the other side is parallel to each other, forming an Contraband-shaped support member, wherein the parallel and staggered sides are connected by a Z-axis translation stage, the other side of the first L-shaped adaptor is fixed to the two-dimensional translation stage of the inverted microscope detection unit, the other side of the second L-shaped adaptor is connected to the piezoelectric scanning stage, and the piezoelectric scanning stage is connected to the microfluidic chip holder.
6. The microfluidic chip-compatible digital scanning light sheet micro-imaging system according to claim 4, wherein the microfluidic chip holder comprises vertical and parallel holding arms, a flat plate holder fixed to the top ends of the holding arms, and a pair of Contraband type holders fixed to the bottom ends of the two arms of the holding arms, respectively, the holding arms are connected to the piezoelectric scanning platform through the flat plate holder, and the microfluidic chip is fixed by Contraband type holder.
7. The microfluidic chip compatible digital scanning light sheet microimaging system of claim 1, wherein the sample scanning unit comprises a microfluidic chip control; the micro-fluidic chip control part is connected with the micro-fluidic chip through a hydraulic pipeline, and the side surface and the lower bottom surface of the micro-fluidic chip are optical flat surfaces.
8. The microfluidic chip compatible digital scanning optical sheet micro-imaging system of claim 1, wherein the digital scanning optical sheet forming element group comprises an XY galvanometer, a Z galvanometer, a scanning lens, a sleeve lens and a plurality of reflectors for adjusting the direction of an optical path, and the digital scanning optical sheet forming element group is fixed on the lower support.
9. The microfluidic chip-compatible digital scanning light sheet microimaging system of claim 1, wherein the elevated holder comprises two arms parallel to each other and adjustable in distance.
10. The microfluidic chip compatible digital scanning light sheet microimaging system of claim 1, wherein the low support is a bread board.
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