CN109745006B - Separation type adsorption device, microscope detection device and laser scanning microscope - Google Patents
Separation type adsorption device, microscope detection device and laser scanning microscope Download PDFInfo
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- CN109745006B CN109745006B CN201910099533.2A CN201910099533A CN109745006B CN 109745006 B CN109745006 B CN 109745006B CN 201910099533 A CN201910099533 A CN 201910099533A CN 109745006 B CN109745006 B CN 109745006B
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Abstract
The embodiment of the invention provides a separation type adsorption device, a microscope detection device and a laser scanning microscope. The separated adsorption device comprises an outer shell, a base with a sucker, a cover glass and a motion device, wherein the sucker is embedded into a sucker hole, and the cover glass is fixed at a sucker sealing port to form an inner space and an outer adsorption space; the fixed support of the moving device is fixed on the side wall of the outer shell, and the limiting block is in relative sliding connection with the fixed support to drive the micro microscope probe to move up and down. The separated adsorption device, the microscope detection device and the laser scanning microscope provided by the embodiment of the invention form an external adsorption space for adsorbing the adsorption device on the skin and a built-in space for arranging the probe through the shell, the sucker and the cover glass, so that after the device is adsorbed on the skin, the micro microscope probe outputs an internal signal and receives an external signal through the cover glass, the whole device is miniaturized, the vibration influence of life body activities on the probe is avoided, and the operation is simple and the use is convenient.
Description
Technical Field
The embodiment of the invention relates to the technical field of laser scanning microscopes, in particular to a separation type adsorption device, a microscope detection device and a laser scanning microscope.
Background
With the continuous development of medicine and biology, people have made remarkable progress on the research of cell morphology, tissue structure or fiber state in intestines and stomach in animal life bodies, and particularly, the related technology of obtaining biological cell morphology of living bodies by exciting by pulse laser radiation in a near infrared region and detecting by a suitable high-sensitivity receiver to obtain fluorescence signals and second harmonic signals has achieved remarkable results.
The related detection device for acquiring the morphology of the biological cells based on the fluorescence signal, the second harmonic signal and the CARS (Coherent anti-stokes raman scattering) signal is important in the application of the above technology. The existing imaging equipment for detecting human cells or tissues is mainly a three-dimensional nonlinear laser scanning microscope, wherein the laser scanning microscope is in a mechanical arm-based laser scanning microscope at present, namely a detection device of the laser scanning microscope is arranged on a mechanical arm, the detection device is moved through adjustment of the mechanical arm, and then different tissue structures of a human body are detected in an aligned mode.
However, in the detection device based on the mechanical arm in the three-dimensional nonlinear laser scanning microscope, due to the fact that the volume of the detection device is large, the probe corresponds to a large skin area of a human body, so that in a specific operation, the detection device is easily affected by human body shake, the resolution requirements of three-dimensional nonlinear laser scanning imaging are generally high, and the detection device is easily affected by vibration, so that the imaging quality is affected.
Disclosure of Invention
Aiming at the technical problems in the prior art, the embodiment of the invention provides a separation type adsorption device, a microscope detection device and a laser scanning microscope.
In a first aspect, an embodiment of the present invention provides a separation type adsorption apparatus for setting a micro-microscope probe, including:
the micro microscope probe comprises an outer shell, a base, a cover glass and a moving device for driving the micro microscope probe to move up and down, wherein a sucker is arranged on the base, the sucker is embedded into a sucker hole formed in the bottom of the outer shell, and the outer shell is detachably connected with the base through magnetic field force, wherein:
the cover glass is fixed at the sealing port of the sucker to form an inner space and an outer adsorption space of the adsorption device;
the motion device set up in the built-in space, the motion device includes fixed bolster and stopper, wherein:
The fixed support is fixed on the side wall of the outer shell, and the limiting block is in relative sliding connection with the fixed support and used for driving the micro-microscope probe to move up and down, wherein the micro-microscope probe is aligned with the cover glass in the forward direction.
In a second aspect, an embodiment of the present invention provides an adsorption microscope detection device, including:
the utility model provides a miniature microscope probe and a disconnect-type adsorption equipment for setting up miniature microscope probe that this invention embodiment provided in a first aspect, wherein, miniature microscope probe is detachable to be fixed on the stopper, miniature microscope probe includes probe casing and fixed plate, probe casing top is provided with first through-hole and second through-hole, probe casing bottom is provided with the third through-hole, wherein:
a first light path is arranged in a first channel formed between the first port and the third port, and a second light path is arranged in a second channel formed between the second port and the third port, wherein:
the first optical path sequentially comprises a collimating lens, a micro-electromechanical scanning galvanometer, a first lens, a second lens, a dichroic mirror and an objective lens which are positioned between the first port and the third port, wherein the first optical path is used for conducting laser signals received by the first port from the first port to the third port;
The second light path sequentially comprises the objective lens and the dichroic mirror, wherein the objective lens and the dichroic mirror are positioned between the third port and the second port, and the second light path is used for conducting optical signals collected by the objective lens from the third port to the second port.
In a third aspect, an embodiment of the present invention provides an adsorption three-dimensional nonlinear laser scanning microscope, including:
the fluorescence collection device, the air exhaust device, the scanning acquisition controller, the femtosecond pulse laser, the optical fiber coupling module and the adsorption type microscope detection device provided by the second aspect of the embodiment of the invention, wherein the fluorescence collection device and the optical fiber coupling module are connected with the adsorption type microscope detection device in an optical fiber communication mode, the fluorescence collection device and the adsorption type microscope detection device are electrically connected with the scanning acquisition controller, and the air exhaust device is electrically connected with the adsorption type microscope detection device, wherein:
the femtosecond pulse laser is used for outputting pulse laser signals to the optical fiber coupling module;
the optical fiber coupling module is used for coupling the pulse laser signals output by the femtosecond pulse laser and transmitting the pulse laser signals to the micro microscope probe in the adsorption microscope detection device;
The adsorption microscope detection device is used for receiving the pulse laser signals, outputting the pulse laser signals to autofluorescent substances in living cells, acquiring fluorescent signals and second harmonic signals generated after the autofluorescent substances are excited, and outputting the fluorescent signals and the second harmonic signals to the fluorescent collection device;
the fluorescence collection device is used for respectively converting the fluorescence signal and the second harmonic signal into corresponding electric signals after receiving the fluorescence signal and the second harmonic signal;
the scanning acquisition controller is used for controlling the micro microscope probe to scan the pulse laser signal and acquire the electric signal;
the air extracting device is used for extracting air from the outer adsorption space of the adsorption type microscope detecting device so as to form negative pressure in the outer adsorption space.
The split type adsorption device for arranging the micro-microscope probe, the adsorption type microscope detection device and the adsorption type three-dimensional nonlinear laser scanning microscope provided by the embodiment of the invention are detachably connected together by adopting the magnetic attraction effect between the shell body and the base, and the suction cup and the cover glass form an external adsorption space capable of enabling the adsorption device to be adsorbed on the skin of a living body and an internal space for placing the micro-microscope probe, wherein the cover glass is fixed at a sealing port of the suction cup to realize sealing contact, and the external adsorption space is formed through the internal space of the suction cup; the motion device is arranged in the built-in space, and is integrally fixed on the side wall of the outer shell, the micro-microscope probe vertically moves through the relative vertical sliding of the limiting block and the fixed support, and the micro-microscope probe is positively aligned with the cover glass so as to output internal signals and receive external signals through the cover glass, the whole device is miniaturized, after the device is adsorbed on the skin of a human body, the vibration influence of the life body motion on the micro-microscope probe in the adsorption device can be avoided, and the outer shell and the base are detachably connected through magnetic field force, so that the cover glass can be replaced by conveniently operating personnel through detaching the base, and the operation is simple and the use is convenient.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of a separation type adsorption device for setting a micro-microscope probe according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of a suction cup on a base of a separation type suction device for setting a probe of a micro-microscope according to an embodiment of the present invention;
FIG. 3 is a schematic diagram of a combined structure of a separation type adsorption device for setting a micro-microscope probe according to an embodiment of the present invention;
fig. 4 is a schematic structural diagram of a probe fixing frame in a separation type adsorption device for setting a micro-microscope probe according to an embodiment of the present invention;
FIG. 5 is a schematic cross-sectional view of a separation type adsorption apparatus for setting a micro-microscope probe according to an embodiment of the present invention;
fig. 6 is a schematic structural diagram of a micro-microscope probe in an adsorption microscope probe device according to an embodiment of the present invention;
Fig. 7 is a schematic structural diagram of a micro-microscope probe in the adsorption microscope detection device according to the second embodiment of the present invention;
FIG. 8 is a schematic diagram of an adsorption three-dimensional nonlinear laser scanning microscope according to an embodiment of the present invention;
FIG. 9 is a schematic structural view of the fluorescence collecting apparatus according to the embodiment of the present invention;
fig. 10 is a schematic diagram of detecting skin tissue of a face of a human body by an adsorption three-dimensional nonlinear laser scanning microscope according to an embodiment of the present invention;
FIG. 11 is a schematic diagram of detecting human chest skin tissue by an adsorption three-dimensional nonlinear laser scanning microscope according to an embodiment of the present invention;
fig. 12 is a schematic diagram of simultaneous detection of skin tissues of a human body by a plurality of detection devices of an adsorption three-dimensional nonlinear laser scanning microscope provided by an embodiment of the present invention;
FIG. 13 is a schematic diagram of detecting animal skin tissue by an adsorption three-dimensional nonlinear laser scanning microscope according to an embodiment of the present invention;
FIG. 14 is a schematic diagram of a box-type combined structure of an adsorption three-dimensional nonlinear laser scanning microscope according to an embodiment of the present invention;
fig. 15 is a schematic diagram of a box-type combined structure of an adsorption three-dimensional nonlinear laser scanning microscope according to an embodiment of the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The existing relevant detection equipment for acquiring the biological cell morphology based on the fluorescence signal and the second harmonic signal is mainly a three-dimensional nonlinear laser scanning microscope, the morphology of the laser scanning microscope is mainly a laser scanning microscope based on a mechanical arm at present, namely a detection device of the laser scanning microscope is arranged on the mechanical arm, the detection device is moved through adjustment of the mechanical arm, and then different tissue structures of a human body are detected in an aligned mode. However, the laser scanning microscope has a large volume, and the probe corresponds to a large skin area of a human body, so that in a specific operation, the detection device is easily affected by human body shake, and the imaging quality is affected.
In order to more stably image the morphology of a biological cell and acquire structural information thereof, an embodiment of the present invention provides a separation type adsorption device for setting a micro-microscope probe, and fig. 1 is a schematic structural diagram of the separation type adsorption device for setting a micro-microscope probe according to the embodiment of the present invention, as shown in fig. 1, the separation type adsorption device includes:
the shell 11, base 13, coverslip 12 and the motion device 14 that drives micro-microscope probe 15 reciprocates are provided with sucking disc 131 on the base 13, and sucking disc 131 imbeds in the sucking disc hole 113 that shell 11 bottom was seted up, and shell 11 passes through magnetic field force with base 13 and can dismantle the connection, wherein:
the cover glass 12 is fixed at the sealing port 1311 of the sucker 131 to form an internal space and an external adsorption space of the adsorption device;
the movement device 14 is disposed in the built-in space, and the movement device 14 includes a fixing bracket 141 and a stopper 142, wherein:
the fixing bracket 141 is fixed on the side wall of the outer casing 11, and the limiting block 142 is connected with the fixing bracket 141 in a sliding manner, so as to drive the micro-microscope probe 15 to move up and down, wherein the micro-microscope probe 15 is aligned with the cover glass 12 in the forward direction.
Specifically, the outer casing 11 and the base 13 in the split type adsorption device for setting the micro microscope probe 15 provided by the embodiment of the invention are made of materials capable of mutually and magnetically attracting, or are internally provided with mutually and magnetically attracting magnetic objects, so that the two magnetic objects can be detachably connected together through magnetic field force, the base 13 is provided with the suction cup 131, the bottom of the outer casing 11 is provided with the suction cup hole 113 for embedding the suction cup 131, the suction cup 131 is provided with the sealing port 1311 communicated with the space in the suction cup 131, and when the cover glass 12 covers the sealing port 1311, an internal space and an external adsorption space capable of enabling the adsorption device to adsorb on the skin of a living body are formed in the split type adsorption device; the moving device 14 for driving the micro-microscope probe 15 to move up and down is arranged in the built-in space, the moving device 14 is integrally fixed on the side wall of the outer shell 11, the limiting block 142 and the fixed support 141 thereof slide up and down relatively, the micro-microscope probe 15 is fixed on the limiting block 142 and moves up and down under the driving of the limiting block 142, and after the fixed support 141 and the micro-microscope probe 15 are fixed, the micro-microscope probe 15 is aligned with the cover glass 12 forward so as to output internal signals and receive external signals through the cover glass 12, thereby realizing zooming and three-dimensional imaging of a microscope.
The outer casing 11, the base 13, the cover glass 12 and the moving device 14 in fig. 1 are combined into a whole to form a separation type adsorption device for arranging the micro-microscope probe 15, and the micro-microscope probe 15 is fixed on the combined adsorption device to form the separation type adsorption device provided with the micro-microscope probe 15, wherein the micro-microscope probe 15 can be fixed on the limiting block 142 of the moving device 14 in an adhesive manner, can be detachably and fixedly connected on the limiting block 142 in a bolt connection manner, namely, the moving device 14 and the micro-microscope probe 15 are connected through bolts, through holes in the limiting block 142 and first bolt holes 151 in the micro-microscope probe 15, and other fixing devices can be added between the limiting block 142 and the micro-microscope probe 15 to connect the moving device 14 and the micro-microscope probe 15 through the through holes in the limiting block 142 and second bolt holes 152 in the micro-microscope probe 15.
The split type adsorption device for arranging the micro-microscope probe provided by the embodiment of the invention adopts the magnetic attraction effect between the shell body and the base to be detachably connected together, and the sucker and the cover glass form an external adsorption space capable of enabling the adsorption device to be adsorbed on the skin of a living body and an internal space for placing the micro-microscope probe, wherein the cover glass is fixed at a sealing port of the sucker to realize sealing contact, and the external adsorption space is formed through the internal space of the sucker; the motion device is arranged in the built-in space, and is integrally fixed on the side wall of the outer shell, the micro-microscope probe vertically moves through the relative vertical sliding of the limiting block and the fixed support, and the micro-microscope probe is positively aligned with the cover glass so as to output internal signals and receive external signals through the cover glass, the whole device is miniaturized, after the device is adsorbed on the skin of a human body, the vibration influence of the life body motion on the micro-microscope probe in the adsorption device can be avoided, and the outer shell and the base are detachably connected through magnetic field force, so that the cover glass can be replaced by conveniently operating personnel through detaching the base, and the operation is simple and the use is convenient.
On the basis of the above embodiments, the suction cup in the separation type suction apparatus for setting a micro-microscope probe according to the embodiment of the present invention further includes a suction port, and fig. 2 is a schematic structural diagram of the suction cup set on the base in the separation type suction apparatus for setting a micro-microscope probe according to the embodiment of the present invention, as shown in fig. 2, the suction port 1313 is connected with the sealing port 1311, and is used for being sucked on a living body to be tested through an external suction space. That is, in the separation type adsorption device provided by the embodiment of the invention, the suction cup 131 arranged on the base 13 further comprises an adsorption port 1313 in addition to the sealing port 1311, the sealing port 1311 is communicated with the adsorption port 1313 to form an internal space of the suction cup, and when the cover glass is sealed and fixed on the sealing port 1311, the internal space of the suction cup forms an external adsorption space through which the adsorption port 1313 is adsorbed on a living body to be detected.
On the basis of the above embodiments, the suction cup in the separation type adsorption device for setting a micro microscope probe according to the embodiment of the present invention further includes an air extraction port, as shown in fig. 2, where the air extraction port 1312 is connected with the external adsorption space and is used for extracting the gas in the external adsorption space. Namely, the suction cup in the separation type adsorption device provided by the embodiment of the invention further comprises an air suction port 1312, wherein the air suction port 1312 is communicated with the external adsorption space, and air in the external adsorption space can be sucked through the air suction port 1312, so that negative pressure in the external adsorption space is formed. Under the action of external pressure, the separated adsorption device is adsorbed on tissues such as skin of a living body to be detected.
On the basis of the above embodiments, the motion device in the separation type adsorption device for setting a micro microscope probe provided by the embodiment of the invention further comprises a probe fixing frame, the probe fixing frame is detachably and fixedly connected with the limiting block, and the micro microscope probe is detachably fixed on the limiting block through the probe fixing frame. That is, as shown in fig. 1, the moving device in the separation type adsorption device for setting a micro-microscope probe according to the embodiment of the present invention further includes a probe fixing frame 143, wherein the probe fixing frame 143 may be fixed on the stopper by means of screw connection, and the micro-microscope probe is fixed on the stopper by means of the probe fixing frame 143. Fig. 3 is a schematic structural diagram of a combined separation type adsorption device for setting a micro-microscope probe, as shown in fig. 3, a base 13 and an outer shell 11 are detachably connected together through magnetic force, a sucker on the base 13 is embedded into a sucker hole of the outer shell, a cover glass is fixed on the sucker, a micro-microscope probe 15 is fixed on a limiting block through a probe fixing frame, namely, one side of the probe fixing frame is fixed with the micro-microscope probe, the other side is fixed with the limiting block through a through hole on the limiting block and a second bolt hole 152 shown in fig. 1, and meanwhile, the micro-microscope probe is aligned with the cover glass in the forward direction. Fig. 4 is a schematic structural diagram of a probe fixing frame in a separation type adsorption device for setting a micro-microscope probe according to an embodiment of the present invention, as shown in fig. 4, the probe fixing frame is provided with a limiting block through hole 1431 and a probe through hole 1432, the limiting block through hole 1431 is used for connecting and fixing a limiting block, and the probe through hole 1432 is used for connecting the micro-microscope probe.
Fig. 5 is a schematic cross-sectional structure of a combined separation type adsorption device for setting a micro-microscope probe according to an embodiment of the present invention, as shown in fig. 5, a suction cup 131 is embedded in a suction cup hole of an outer housing 11, a cover glass 12 is fixed on the suction cup 131, a micro-microscope probe 15 is fixed on a limiting block, and the micro-microscope probe 15 is aligned to the cover glass in a forward direction, and is driven by a motion device 14 to move up and down. The micro microscope probe 15 is located in the built-in space 17, and the separation type adsorption device is adsorbed on the skin and other tissues of the living body to be detected by the negative pressure formed in the external adsorption space 18.
On the basis of the above embodiments, the outer casing of the separation type adsorption device for setting a micro microscope probe provided by the embodiment of the invention is further provided with a through hole for placing an air extraction pipeline, and the air extraction pipeline is connected with the air extraction port through the through hole in a ventilation way. Namely, the outer shell body of the separation type adsorption device for arranging the micro microscope probe is also provided with a through hole penetrating through the upper part and the lower part of the outer shell body, and an air exhaust pipeline for exhausting the external adsorption space penetrates through the through hole and is connected with an air exhaust hole on the sucker in an air-through manner and is in tight contact.
On the basis of the above embodiments, the separation type adsorption device for setting a micro microscope probe provided by the embodiment of the invention further comprises a motor, wherein the motor is arranged in the built-in space, and the separation type adsorption device comprises:
the motor is rotationally connected with the limiting block through the lead screw and used for driving the limiting block to move up and down. Namely, the separation type adsorption device for arranging the micro-microscope probe is further provided with a motor, and the motor is used for driving the limiting block provided with the micro-microscope probe to move up and down so as to adjust the external focal length of the micro-microscope probe, thereby realizing three-dimensional detection with different depths and different layers.
On the basis of the above embodiments, the outer housing in the separation type adsorption device for setting a micro microscope probe according to the embodiment of the present invention includes a first housing and a second housing, as shown in fig. 1, wherein:
the first shell 111 is internally provided with an accommodating space, the movement device 14 is arranged in the accommodating space, the first shell 111 and the second shell 112 are detachably and fixedly connected, the first shell is provided with a through hole 114 for placing an air extraction pipeline, and the air extraction pipeline is connected with an air extraction opening 1312 through the through hole 114. Namely, the outer casing of the separation type adsorption device for setting a micro-microscope probe provided by the embodiment of the invention comprises two parts, namely a first casing 111 and a second casing 112, wherein the first casing 111 is provided with an accommodating space for setting the movement device 14, namely, the sucker hole 113 in the embodiment is also arranged at the bottom of the first casing 111, the sucker is embedded into the sucker hole of the first casing 111, and the cover glass and the second casing 112 are combined to form an external adsorption space and an internal space in the embodiment, wherein the first casing 111 and the second casing 112 can be detachably and fixedly connected through screws, so that the whole device is convenient to assemble, disassemble and replace parts.
The embodiment of the invention also provides an adsorption type microscope detection device, which comprises a micro microscope probe and the separation type adsorption device provided by the embodiments, wherein the micro microscope probe is detachably fixed on a limiting block and comprises a probe shell and a fixing plate, the top end of the probe shell is provided with a first port and a second port, and the bottom end of the probe shell is provided with a third port, wherein:
a first light path is arranged in a first channel formed between the first port and the third port, and a second light path is arranged in a second channel formed between the second port and the third port, wherein:
the first optical path sequentially comprises a collimating lens, a micro-electromechanical scanning galvanometer, a lens, a dichroic mirror and an objective lens which are positioned between the first port and the third port, wherein the first optical path is used for conducting laser signals received by the first port from the first port to the third port;
the second light path sequentially comprises an objective lens and a dichroic mirror which are positioned between the third port and the second port, wherein the second light path is used for conducting optical signals collected by the objective lens from the third port to the second port.
Specifically, fig. 6 is a schematic structural diagram of a micro-microscope probe in the adsorption microscope detection device provided by the embodiment of the invention, as shown in fig. 6, three ports are formed on a probe housing 150 of the micro-microscope probe, two channels are formed between the three ports, two optical paths are arranged in the two channels, namely, a first optical path and a second optical path, a section of overlapped part is formed between the two channels formed between the three ports, a dichroic mirror 1544 and an objective lens 1545 which are shared by the two optical paths are arranged in the overlapped part of the channels, wherein the first optical path is an emission optical path, and sequentially comprises a collimating lens 157, a micro-electromechanical scanning galvanometer 1541, a lens 1542, a dichroic mirror 1543 and the objective lens 1544 which are positioned between the first port 154 and the third port 156, and the objective lens is fixed on the probe housing 150 through a fixing frame 159, namely, laser signals enter from the first port 154, are conducted through the first optical path, and parallel light incident to the first port exits through the dichroic mirror 1545 in a parallel light manner after passing through the two lenses; the second optical path is a collecting optical path, and is used for outputting the collected optical signals from the second port 155 to a collecting device connected with the micro microscope probe; and the laser signal transmitted by the first optical path can vertically and normally enter the objective lens 1544 under the reflection of the dichroic mirror 1543, the dichroic mirror reflects the laser signal of the first optical path, the laser signal collected by the second optical path is transmitted, and the pitching angle of the dichroic mirror 1543 can be adjusted.
On the basis of the above embodiments, the adsorption microscope detection device provided by the embodiment of the present invention further includes:
the liquid lens is positioned between the collimating lens and the micro-electromechanical scanning galvanometer to form a new first optical path, and the new first optical path sequentially comprises the collimating lens, the liquid lens, the micro-electromechanical scanning galvanometer, the lens, the dichroic mirror and the objective lens which are positioned between the first through hole and the third through hole. That is, the first optical path in the micro-microscope probe in the adsorption type microscope detecting device provided by the embodiment of the invention can be added with the liquid lens 158, fig. 7 is a schematic diagram of a second structure of the micro-microscope probe in the adsorption type microscope detecting device provided by the embodiment of the invention, and as shown in fig. 7, the zoom function is realized by adding the liquid lens 158 on the micro-microscope probe structure shown in fig. 7. The liquid lens 158 is located between the collimator lens 157 and the mems scanning galvanometer 1541, and the laser signal is collimated after passing through the collimator lens 157 in the original optical path structure shown in fig. 7. After the liquid lens 158 is added, a voltage or a current is applied to the liquid lens 158 to cause the surface of the liquid lens 158 to bend correspondingly, and thus, different optical powers are generated for the parallel light. The specific light path is: the laser signal is emitted from the optical fiber, is parallel incident to the liquid lens 158 after passing through the collimating lens 157, generates corresponding optical power according to the loaded voltage or current signal from the liquid lens 158, and the emitted converging or diverging light is converged on the sample after passing through the micro-electromechanical scanning galvanometer 1541, the lens 1542, the dichroic mirror 1543 and the objective lens 1544. The focal power change introduced by the liquid lens 158 can enable the focal point of the laser signal emitted from the opening of the objective lens 1544 to move back and forth in the depth direction, the response speed of the liquid lens 158 is very fast, and the scanning frequency of the liquid lens 158 is in the order of KHz, so that rapid scanning imaging in the depth direction can be realized. The liquid lens 158 is equivalent to a parallel plate glass when no voltage or current signal is applied, and does not have optical power to the laser signal and does not shift the focal point after the objective lens. When the device is specifically used, the liquid lens 158 is complementary to the moving device, the position of the objective lens 1544 is adjusted through the moving device, after coarse adjustment to the corresponding depth position, the system is switched to a liquid lens 158 zooming scanning mode, and rapid three-dimensional imaging is performed on a sample, wherein when the adsorption type microscope detecting device is not provided with the moving device, zooming adjustment can be performed only through the liquid lens.
The embodiment of the invention also provides an adsorption type three-dimensional nonlinear laser scanning microscope, fig. 8 is a schematic structural diagram of the adsorption type three-dimensional nonlinear laser scanning microscope provided by the embodiment of the invention, as shown in fig. 8, the laser scanning microscope comprises:
fluorescence collection device 56, air exhaust device 52, scan collection controller 531, femtosecond pulse laser 541, fiber coupling module 542 and adsorption microscope detection device 51 that the embodiment provided above, fluorescence collection device 56 and fiber coupling module 542 all are connected with adsorption microscope detection device 51 fiber communication, fluorescence collection device 56 and adsorption microscope detection device 51 all are connected with scan collection controller 531 electricity, air exhaust device 52 is connected with adsorption microscope detection device 51 electricity, wherein:
a femtosecond pulse laser 541 for outputting a pulse laser signal to the optical fiber coupling module 542;
the optical fiber coupling module 542 is used for coupling the pulse laser signal output by the femtosecond pulse laser 541 and transmitting the pulse laser signal to a micro microscope probe in the adsorption microscope detection device;
the adsorption microscope detecting device is used for receiving the pulse laser signals, outputting the pulse laser signals to autofluorescent substances in living cells, acquiring fluorescent signals and second harmonic signals generated after the autofluorescent substances are excited, and outputting the fluorescent signals and the second harmonic signals to the fluorescent collecting device 56;
The fluorescence collection device 56 is configured to convert the fluorescence signal and the second harmonic signal into corresponding electrical signals after receiving the fluorescence signal and the second harmonic signal, respectively;
the scanning acquisition controller 531 is used for controlling the micro microscope probe to scan the pulse laser signal and synchronously acquire the electric signal;
and the air extracting device 52 is used for extracting air from the external adsorption space of the adsorption type microscope detecting device so as to form negative pressure in the external adsorption space.
Specifically, the adsorption type three-dimensional nonlinear laser scanning microscope provided by the embodiment of the invention comprises a fluorescence collection device 56, an air extraction device 52, a scanning acquisition controller 531, a femtosecond pulse laser 541, an optical fiber coupling module 542 and an adsorption type microscope detection device 51, so that the three-dimensional nonlinear laser scanning microscope which can be adsorbed on human skin or deep into human intestines and stomach for detection is formed, wherein the femtosecond pulse laser 541 can emit pulse laser signals for exciting autofluorescent substances in human skin cells to generate multiphoton fluorescence signals and second harmonic signals, and the fluorescence signals and the second harmonic signals of 500-600nm are excited by using the femtosecond pulse laser 541 of 920nm, and the autofluorescent substances such as FAD or NADH in cells are excited by using the femtosecond pulse laser 541 of 780nm to generate corresponding fluorescence signals and second harmonic signals;
The fluorescence collection device 56 integrates two signal collection light paths, namely a fluorescence signal collection light path and a second harmonic signal collection light path, so as to realize the collection of fluorescence signals and second harmonic signals respectively; the scanning acquisition controller 531 controls a scanning galvanometer in the micro microscope probe to scan the pulse laser signal and excite the autofluorescent substance to generate a fluorescent signal and a second harmonic signal, and acquires a first electric signal and a second electric signal obtained by converting the fluorescent signal and the second harmonic signal by the fluorescent collection device 56; the air extraction device 52 mainly comprises an air extraction pump connected with an air extraction pipeline, wherein the air extraction pipeline is connected with an air extraction opening in the embodiment, an air extraction valve is arranged in the air extraction pipeline and is electrically connected with the air extraction device 52, the air extraction device 52 controls the air extraction flow of the air extraction pipeline by adjusting the opening and closing of the air extraction valve, so that the air extraction control of an external adsorption space is realized, the negative pressure in the external adsorption space is further adjusted, the adsorption device is adsorbed on the skin, intestines and stomach and other tissues of a living body under the action of atmospheric pressure, and the adsorption three-dimensional nonlinear laser scanning microscope can comprise a two-photon scanning microscope, a multiphoton scanning microscope and the like according to classification, wherein a small aperture diaphragm is additionally arranged when a femtosecond pulse laser can be replaced by a common continuous laser, and the adsorption three-dimensional nonlinear laser scanning microscope can also be adjusted to be a confocal microscope. The resolution of the adsorption three-dimensional nonlinear laser scanning microscope can be set to 800nm, the imaging field of view can be 200 micrometers by 200 micrometers, and the imaging speed can be 26 frames (256×256 pixels) or 13 frames (512×512 pixels).
The adsorption type three-dimensional nonlinear laser scanning microscope provided by the embodiment of the invention adopts a fluorescence collection device, an air exhaust device, a scanning acquisition controller, a femtosecond pulse laser, an optical fiber coupling module and an adsorption type microscope detection device, so that the three-dimensional nonlinear laser scanning microscope which can be adsorbed on human skin or deep into human intestines and stomach for detection is formed, the focal length is adjusted by adjusting the distance between the micro microscope probe and a cover glass, the three-dimensional scanning of the laser scanning microscope is realized, the fluorescence signal and a second harmonic signal of multiple photons are obtained by exciting an intracellular autofluorescent substance by the femtosecond pulse laser, the nonlinearity of the laser scanning microscope is realized, the fluorescence signal and the second harmonic signal are collected by the fluorescence collection device and are converted into corresponding electric signals, then the fluorescence image reflecting the cell tissue structure is obtained by the electric signals, and the like.
On the basis of the above embodiments, fig. 9 is a schematic structural diagram of a fluorescence collection device according to an embodiment of the present invention, as shown in fig. 9, where the fluorescence collection device according to an embodiment of the present invention includes an optical fiber universal interface 781, a first photomultiplier 782, a second photomultiplier 783, a first collection optical path located between the optical fiber universal interface 781 and the first photomultiplier 782, and a second collection optical path located between the optical fiber universal interface 781 and the second photomultiplier 783, where:
the first collecting light path sequentially comprises a coupling collecting lens 71, an infrared filter 72, a first dichroic mirror 73, a first filter 74 and a first collecting lens 75, wherein the first collecting light path is used for collecting fluorescent signals received by the fluorescent collecting device, and the first photomultiplier 782 is used for converting the fluorescent signals into first electric signals;
the second collecting optical path sequentially includes a coupling collecting lens 71, an infrared filter 72, a first dichroic mirror 73, a second dichroic mirror 76, a second filter 77, and a second collecting lens 78, where the second collecting optical path is used to collect the second harmonic signal received by the fluorescent collecting device, and the second photomultiplier 783 is used to convert the second harmonic signal into a second electric signal. Namely, the fluorescence collection device provided by the embodiment of the invention has a double-path signal collection function, and integrates two paths of light paths, wherein the first dichroic mirror 73 in the first collection light path is a dichroic mirror for transmitting fluorescence signals, reflecting second harmonic waves, the second dichroic mirror 76 and the first dichroic mirror 73 are the same dichroic mirror for reflecting the second harmonic waves, the first filter 74 is used for transmitting the fluorescence signals and filtering out other interference signals, the second filter 77 is used for transmitting corresponding second harmonic signals and filtering out other interference signals, for example, when 780nm femtosecond fiber lasers are used for exciting autofluorescence substances in skin cells on the surface of a human body, 390nm second harmonic signals and 450-600nm two-photon autofluorescence signals can be obtained, two paths of fluorescence can be separated through the dichroic mirror 73 reflecting the wavelengths above 420nm, and the first dichroic mirror 74 with the wavelengths below 420nm and the second filter 77 with the wavelengths of 450-600nm can be used for obtaining clean second harmonic signals and fluorescence signals respectively.
On the basis of the above embodiments, the adsorption three-dimensional nonlinear laser scanning microscope provided by the embodiment of the present invention further includes an industrial personal computer, as shown in fig. 8, wherein the industrial personal computer 532 is electrically connected with the scanning acquisition controller 531, where:
the industrial personal computer 532 is configured to acquire the first electrical signal and the second electrical signal acquired by the scan acquisition controller 531, generate a first fluorescent image based on the first electrical signal, and generate a second fluorescent image based on the second electrical signal. That is, the adsorption three-dimensional nonlinear laser scanning microscope provided by the embodiment of the invention further comprises an industrial personal computer 532 electrically connected with the scanning acquisition controller 531, wherein the industrial personal computer 532 generates a first fluorescent image based on a first electric signal and generates a second fluorescent image based on a second electric signal, and the first fluorescent image and the second fluorescent image can be respectively used for displaying cell structure and fiber structure information, wherein control software is installed on the industrial personal computer, and a control instruction is sent to the scanner through the control software so as to control the scanning acquisition controller to acquire the first electric signal and the second electric signal.
On the basis of the above embodiments, the adsorption three-dimensional nonlinear laser scanning microscope provided by the embodiment of the present invention further includes a display, as shown in fig. 8, where the display 55 is electrically connected to the industrial personal computer 532, and is used for displaying the first fluorescent image and the second fluorescent image. That is, the adsorption three-dimensional nonlinear laser scanning microscope provided by the embodiment of the invention further comprises a display 55 for displaying the first fluorescent image and the second fluorescent image, and the staff can directly acquire the related information of the first fluorescent image and the second fluorescent image through the display 55.
Based on the above embodiments, the adsorption type three-dimensional nonlinear laser scanning microscope provided by the embodiment of the invention has a plurality of adsorption type microscope detection devices. The fluorescence collection device and the optical fiber coupling module provided by the embodiment of the invention can be simultaneously connected with a plurality of adsorption type microscope detection devices in an optical fiber communication way, namely, a plurality of detection devices are integrated in one adsorption type three-dimensional nonlinear laser scanning microscope system, so that the simultaneous detection of different tissue parts of a living body is realized, and the comparison analysis is performed.
On the basis of the above embodiments, the adsorption three-dimensional nonlinear laser scanning microscope provided by the embodiment of the invention further comprises an adjusting optical fiber, which is used for optical fiber transmission connection between the fluorescence collection device and the optical fiber coupling module and the adsorption microscope detection device, wherein:
the length of the adjusting optical fiber is adjustable. The fluorescence collection device and the optical fiber coupling module in the adsorption three-dimensional nonlinear laser scanning microscope provided by the embodiment of the invention are respectively connected with the adsorption microscope detection device through the optical fiber with adjustable length in an optical fiber transmission way, so that the detection device is flexibly moved according to different experimental scene requirements, the limitation of the limited optical fiber length is avoided, the length of the adjusting optical fiber is adjustable, the application in various occasions is realized by changing the optical fibers with different lengths, and the optical fiber with different lengths can be changed at any time according to the requirements.
In order to more clearly illustrate the application scenario of the adsorption type three-dimensional nonlinear laser scanning microscope provided by the embodiment of the present invention, a schematic diagram of detecting skin tissue of a human face by using the adsorption type three-dimensional nonlinear laser scanning microscope provided by the embodiment of the present invention will be further illustrated by a legend, as shown in fig. 10, the adsorption type microscope detecting device 51 is adsorbed on the human face by using the air pumping function of the air pumping device 52, wherein the first device 53 is integrated with a scanning acquisition controller and an industrial personal computer, the industrial personal computer is electrically connected with the display 55, the second device 54 is integrated with a femtosecond pulse laser, an optical fiber coupling module and a fluorescence collecting device, and the optical fiber coupling module and the fluorescence collecting device are all connected with the optical fiber transmission of the adsorption type microscope detecting device 51, wherein the working principle of the adsorption type three-dimensional nonlinear laser scanning microscope is the same as that of the above embodiments, and will not be repeated herein.
Fig. 11 is a schematic diagram of detecting skin tissue of a human chest by an adsorption three-dimensional nonlinear laser scanning microscope according to an embodiment of the present invention, as shown in fig. 11, the adsorption three-dimensional nonlinear laser scanning microscope detecting device 51 is adsorbed on the human chest by an air extracting function of the air extracting device 52, wherein a scanning acquisition controller and an industrial personal computer are integrated in the first device 53, the industrial personal computer is electrically connected with the display 55, a femtosecond pulse laser, an optical fiber coupling module and a fluorescence collecting device are integrated in the second device 54, and the optical fiber coupling module and the fluorescence collecting device are all connected with the optical fiber transmission of the adsorption three-dimensional nonlinear laser scanning microscope detecting device 51, wherein the operation principle of the adsorption three-dimensional nonlinear laser scanning microscope is the same as that of the above embodiments, and the description is omitted herein.
Fig. 12 is a schematic diagram of simultaneous detection of skin tissues of a human body by a plurality of detection devices of the adsorption three-dimensional nonlinear laser scanning microscope provided by the embodiment of the invention, as shown in fig. 12, by the air extraction function of the air extraction device 52, the plurality of detection devices of the adsorption three-dimensional nonlinear laser scanning microscope 51 are respectively and simultaneously adsorbed on the face, the chest and the legs of the human body, wherein a scanning acquisition controller and an industrial personal computer are integrated in a first device 53, the industrial personal computer is electrically connected with a display 55, a femtosecond pulse laser, an optical fiber coupling module and a fluorescence collection device are integrated in a second device 54, and the optical fiber coupling module and the fluorescence collection device are all in optical fiber transmission connection with the detection devices of the adsorption three-dimensional nonlinear laser scanning microscope 51, so that skin tissue structures of different parts of the human body can be detected simultaneously under the action of the detection devices of the adsorption three-dimensional nonlinear laser scanning microscope, the operation is simple and the use is convenient, and the length of the optical fiber can be adjusted due to the adoption of the optical fiber transmission connection between the detection devices of the adsorption three-dimensional nonlinear laser scanning microscope. The working principle of the adsorption three-dimensional nonlinear laser scanning microscope is the same as that of the above embodiments, and the description thereof is omitted here. Fig. 13 is a schematic diagram of detecting animal skin tissue by using an adsorption three-dimensional nonlinear laser scanning microscope according to an embodiment of the present invention, as shown in fig. 13, the adsorption three-dimensional nonlinear laser scanning microscope may also be used to adsorb the adsorption three-dimensional nonlinear laser scanning microscope detecting device 51 onto the animal skin tissue by using the air suction function of the air suction device 52, where the first device 53 integrates a scanning acquisition controller and an industrial personal computer, the industrial personal computer is electrically connected with the display 55, the second device 54 integrates a femtosecond pulse laser, an optical fiber coupling module and a fluorescence collecting device, and the optical fiber coupling module and the fluorescence collecting device are all connected with the optical fiber transmission of the adsorption three-dimensional nonlinear laser scanning microscope detecting device 51, and the working principle is the same as that of the above embodiments.
For the adsorption three-dimensional nonlinear laser scanning microscope provided by the above embodiments, another specific implementation manner is provided in the embodiment of the present invention, fig. 14 is a schematic diagram of a box-type combined structure of the adsorption three-dimensional nonlinear laser scanning microscope provided in the embodiment of the present invention, as shown in fig. 14, a scan collection controller 531, an industrial personal computer 532, an air extractor 52, a fluorescence collection device 56, and an integrated module 540 of integrating a femtosecond pulse laser and optical fiber coupling of the adsorption three-dimensional nonlinear laser scanning microscope are integrated together in a portable suitcase, the suitcase is internally provided with the industrial personal computer with a display screen, and a display 55 is integrated on a cover of the suitcase; the adsorption microscope detecting device 51 is adsorbed on skin tissue of a human body to be detected, is in optical fiber coupling and optical fiber communication connection with the fluorescence collecting device 56 in the box body, is connected with the air pump through an air extraction pipeline, and is electrically connected with the scanning acquisition controller 531 and the industrial personal computer 532 through a power plug. Fig. 15 is a schematic diagram of a box-type combined structure of an adsorption three-dimensional nonlinear laser scanning microscope according to an embodiment of the present invention, as shown in fig. 15, a display 55 integrated on a box cover is integrated with a box body provided with each module, so that the whole equipment can be moved conveniently, and a workplace can be replaced, and the display 55 can be placed on the box body externally when in use, so that a worker can obtain information on the display conveniently. After the adsorption type three-dimensional nonlinear laser scanning microscope is used, a worker can carry the equipment box, and the equipment can be conveniently replaced in a workplace, especially in a hospital, a laboratory or an outdoor place.
It should be further noted that, in the adsorption three-dimensional nonlinear laser scanning microscope provided in each embodiment, after the wavelength of the femto-second pulse laser is changed and the filtering range of each optical filter is adjusted, CARS signals can be collected on a part of fluorescent and non-SHG (Second Harmonic Generation ) signal active tissue, so that the adsorption three-dimensional nonlinear laser scanning microscope is adjusted to be an adsorption micro CARS microscope, and specific adjustment parameters can be set according to specific needs.
While the present invention has been described in connection with the embodiments illustrated in the drawings, it will be apparent to those skilled in the art that the present invention is not limited to the preferred embodiments of the present invention, and various modifications and variations can be made thereto by those skilled in the art on the basis of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.
The apparatus embodiments described above are merely illustrative, wherein elements illustrated as separate elements may or may not be physically separate, and elements shown as elements may or may not be physical elements, may be located in one place, or may be distributed over a plurality of network elements. Some or all of the modules may be selected according to actual needs to achieve the purpose of the solution of this embodiment. Those of ordinary skill in the art will understand and implement the present invention without undue burden.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (7)
1. A split adsorption device for setting up a micro-microscope probe, comprising:
the micro microscope probe comprises an outer shell, a base, a cover glass and a moving device for driving the micro microscope probe to move up and down, wherein a sucker is arranged on the base, the sucker is embedded into a sucker hole formed in the bottom of the outer shell, and the outer shell is detachably connected with the base through magnetic field force, wherein:
the cover glass is fixed at the sealing port of the sucker to form an inner space and an outer adsorption space of the adsorption device;
the motion device set up in the built-in space, the motion device includes fixed bolster and stopper, wherein:
The fixing support is fixed on the side wall of the outer shell, and the limiting block is in relative sliding connection with the fixing support and is used for driving the micro-microscope probe to move up and down, wherein the micro-microscope probe is aligned to the cover glass in the forward direction;
the sucking disc further comprises an adsorption port, wherein the adsorption port is communicated with the sealing port and is used for being adsorbed on a life body to be detected through the external adsorption space;
the sucking disc further comprises an air extraction opening, wherein the air extraction opening is communicated with the external adsorption space and is used for extracting air in the external adsorption space;
the motion device further comprises a probe fixing frame, the probe fixing frame is detachably and fixedly connected with the limiting block, and the micro microscope probe is detachably fixed on the limiting block through the probe fixing frame;
the outer shell is also provided with a through hole for placing an air extraction pipeline, and the air extraction pipeline is connected with the air extraction opening through the through hole in a ventilation way.
2. The utility model provides an absorption formula microscope detection device, its characterized in that, including miniature microscope probe and the disconnect-type adsorption equipment for setting up miniature microscope probe of claim 1, wherein, miniature microscope probe is detachable to be fixed on the stopper, miniature microscope probe includes probe casing and fixed plate, probe casing top is provided with first opening and second opening, probe casing bottom is provided with the third opening, wherein:
A first light path is arranged in a first channel formed between the first port and the third port, and a second light path is arranged in a second channel formed between the second port and the third port, wherein:
the first optical path sequentially comprises a collimating lens, a micro-electromechanical scanning galvanometer, a lens, a dichroic mirror and an objective lens which are positioned between the first through hole and the third through hole, wherein the first optical path is used for conducting laser signals received by the first through hole from the first through hole to the third through hole;
the second light path sequentially comprises the objective lens and the dichroic mirror, wherein the objective lens and the dichroic mirror are positioned between the third port and the second port, and the second light path is used for conducting optical signals collected by the objective lens from the third port to the second port.
3. The adsorption microscope probe of claim 2, wherein the micro microscope probe further comprises:
the liquid lens is positioned between the collimating lens and the micro-electromechanical scanning galvanometer to form a new first optical path, and the new first optical path sequentially comprises the collimating lens, the liquid lens, the micro-electromechanical scanning galvanometer, the lens, the dichroic mirror and the objective lens which are positioned between the first through hole and the third through hole.
4. An adsorption type three-dimensional nonlinear laser scanning microscope, comprising:
fluorescence collection device, air exhaust device, scanning collection controller, femtosecond pulse laser, fiber coupling module and claim 2 or 3 adsorption microscope detection device, fluorescence collection device with fiber coupling module all with adsorption microscope detection device fiber communication connection, fluorescence collection device with adsorption microscope detection device all with scanning collection controller electricity is connected, air exhaust device with adsorption microscope detection device electricity is connected, wherein:
the femtosecond pulse laser is used for outputting pulse laser signals to the optical fiber coupling module;
the optical fiber coupling module is used for coupling the pulse laser signals output by the femtosecond pulse laser and transmitting the pulse laser signals to the micro microscope probe in the adsorption microscope detection device;
the adsorption microscope detection device is used for receiving the pulse laser signals, outputting the pulse laser signals to autofluorescent substances in living cells, acquiring fluorescent signals and second harmonic signals generated after the autofluorescent substances are excited, and outputting the fluorescent signals and the second harmonic signals to the fluorescent collection device;
The fluorescence collection device is used for respectively converting the fluorescence signal and the second harmonic signal into corresponding electric signals after receiving the fluorescence signal and the second harmonic signal;
the scanning acquisition controller is used for controlling the micro microscope probe to scan the pulse laser signal and synchronously acquiring the electric signal;
the air extracting device is used for extracting air from the outer adsorption space of the adsorption type microscope detecting device so as to form negative pressure in the outer adsorption space.
5. The adsorptive three-dimensional nonlinear laser scanning microscope of claim 4, wherein said fluorescence collection device comprises a fiber optic universal interface, a first photomultiplier tube, a second photomultiplier tube, and a first collection optical path between said fiber optic universal interface and said first photomultiplier tube, a second collection optical path between said fiber optic universal interface and said second photomultiplier tube, wherein:
the first collecting light path sequentially comprises a coupling collecting lens, an infrared filter, a first dichroic mirror, a first filter and a first collecting lens, wherein the first collecting light path is used for collecting the fluorescent signals received by the fluorescent collecting device, and the first photomultiplier is used for converting the fluorescent signals into first electric signals;
The second collecting light path sequentially comprises the coupling collecting lens, the infrared filter, the first dichroic mirror, the second filter and the second collecting lens, wherein the second collecting light path is used for collecting the second harmonic signals received by the fluorescent collecting device, and the second photomultiplier is used for converting the second harmonic signals into second electric signals.
6. The adsorption three-dimensional nonlinear laser scanning microscope in accordance with claim 4, wherein the adsorption microscope detection device is a plurality of.
7. The adsorptive three-dimensional nonlinear laser scanning microscope of claim 6, further comprising a conditioning fiber for fiber optic transmission connection between said fluorescence collection device and said fiber optic coupling module, respectively, and said adsorptive microscope detection device, wherein:
the length of the conditioning fiber is adjustable.
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