CN109745008B - Adsorbable microscope detection device and laser scanning microscope - Google Patents
Adsorbable microscope detection device and laser scanning microscope Download PDFInfo
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- CN109745008B CN109745008B CN201910099558.2A CN201910099558A CN109745008B CN 109745008 B CN109745008 B CN 109745008B CN 201910099558 A CN201910099558 A CN 201910099558A CN 109745008 B CN109745008 B CN 109745008B
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Abstract
The embodiment of the invention provides an adsorbable microscope detection device and a laser scanning microscope. The adsorbable microscope detection device comprises an adsorption shell and a micro microscope probe, wherein a movement device for driving the micro microscope probe to move up and down is arranged on the inner side of the adsorption shell, and a first light path for receiving pulse laser signals and transmitting and outputting the pulse laser signals to autofluorescent substances in living cells and a second light path for collecting and transmitting fluorescent signals and second harmonic signals generated by exciting the autofluorescent substances are arranged in the micro microscope probe. The adsorbable microscope detection device and the laser scanning microscope provided by the embodiment of the invention realize three-dimensional detection imaging of the adsorbable microscope detection device on the cell structure in the skin tissue through the miniaturized adsorption shell, the built-in motion device and the micro microscope probe provided with the first light path and the second light path, and have the advantages of simple structure and convenience in use.
Description
Technical Field
The embodiment of the invention relates to the technical field of laser scanning microscopes, in particular to an adsorbable 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, related 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 an adsorbable microscope detection device and a laser scanning microscope.
In a first aspect, an embodiment of the present invention provides an adsorbable microscope detection device, including:
Adsorption shell and set up in micro-microscope probe in the adsorption shell, the adsorption shell inboard is provided with the motion, micro-microscope probe set up in on the motion, the motion is used for driving micro-microscope probe reciprocates, wherein:
the motion device with micro-microscope probe all set up in the absorption casing, be provided with first light path and second light path in the micro-microscope probe, wherein:
The first optical path is used for receiving the pulse laser signal and transmitting and outputting the pulse laser signal to an autofluorescent substance in a living body cell;
The second light path is used for collecting and conducting fluorescent signals and second harmonic signals generated by excitation of the autofluorescent substances.
In a second aspect, an embodiment of the present invention provides an adsorption three-dimensional nonlinear laser scanning microscope, including:
The embodiment of the invention provides an adsorbable microscope detection device, which comprises a fluorescence collection device, an air exhaust device, a scanning acquisition controller, a femtosecond pulse laser, an optical fiber coupling module and the first aspect of the invention, wherein the fluorescence collection device and the optical fiber coupling module are connected with the adsorbable microscope detection device in an optical fiber communication mode, the fluorescence collection device and the adsorbable microscope detection device are electrically connected with the scanning acquisition controller, and the air exhaust device is electrically connected with the adsorbable 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 adsorbable microscope detection device;
The adsorbable microscope detection device is used for outputting the pulse laser signal to an autofluorescent substance in a living body cell after receiving the pulse laser signal, acquiring a fluorescent signal and a second harmonic signal generated after excitation of the autofluorescent substance, and outputting the fluorescent signal and the second harmonic signal 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 a scanning galvanometer in 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 external adsorption space of the adsorbable microscope detecting device so as to form negative pressure in the external adsorption space.
The adsorbable microscope detection device and the laser scanning microscope provided by the embodiment of the invention comprise an adsorption shell and a micro microscope probe arranged in the adsorption shell, wherein the adsorbable microscope detection device is adsorbed on the skin tissue of a living body to be detected through the miniaturized adsorption shell, so that the micro microscope probe detects the cell structure in the skin tissue, and performs three-dimensional detection through up-and-down movement adjustment of an operation device, and simultaneously, the micro microscope probe excites autofluorescent substances in cells through two built-in light paths and pulse laser signals output through the light paths, and acquires fluorescent signals and second harmonic signals generated by exciting the autofluorescent substances, so that the three-dimensional laser scanning microscope provided with the adsorbable microscope detection device performs three-dimensional imaging on the cell structure through the obtained fluorescent signals and the second harmonic signals, and the device has a simple structure and is convenient to use.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of an adsorbable microscope detection device according to an embodiment of the present invention;
FIG. 2 is a schematic cross-sectional view of an adsorbable microscope probe according to an embodiment of the present invention;
FIG. 3 is a schematic cross-sectional view of an adsorbable microscope detecting device according to another embodiment of the present invention;
FIG. 4 is a schematic cross-sectional view of an adsorbable microscope detecting device according to another embodiment of the present invention;
FIG. 5 is a schematic cross-sectional view of an adsorbable microscope detecting device according to another embodiment of the present invention;
FIG. 6 is a schematic diagram of an adsorption three-dimensional nonlinear laser scanning microscope according to an embodiment of the present invention;
FIG. 7 is a schematic diagram of a fluorescence collection device according to an embodiment of the present invention;
FIG. 8 is a schematic diagram of detecting human facial skin tissue by an adsorption three-dimensional nonlinear laser scanning microscope according to an embodiment of the present invention;
Fig. 9 is a schematic diagram of detecting skin tissue of a chest of a human body by using an adsorption three-dimensional nonlinear laser scanning microscope according to an embodiment of the present invention;
fig. 10 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. 11 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. 12 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 invention;
fig. 13 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 an adsorbable microscope detection device, and fig. 1 is a schematic structural diagram of the adsorbable microscope detection device provided by the embodiment of the present invention, as shown in fig. 1, where the adsorbable microscope detection device includes:
The absorption casing 11 and set up the miniature microscope probe 12 in absorption casing 11, absorption casing 11 inboard is provided with the motion device 13, and miniature microscope probe 12 sets up on motion device 13, and motion device 13 is used for driving miniature microscope probe 12 and reciprocates, wherein:
The motion device 13 and the micro microscope probe 12 are both arranged in the adsorption shell 11, and a first light path and a second light path are arranged in the micro microscope probe 12, wherein:
the first optical path is used for receiving the pulse laser signal and transmitting and outputting the pulse laser signal to an autofluorescent substance in a living body cell;
the second light path is used for collecting and conducting fluorescent signals and second harmonic signals generated by excitation of the autofluorescent substances.
Specifically, the adsorbable microscope detection device provided by the embodiment of the invention comprises an adsorption shell 11 and a micro microscope probe 12 in the adsorption shell 11, wherein the adsorption shell 11 is miniaturized and is used for being adsorbed on the surface of the skin of a living body, the micro microscope probe 12 in the adsorption shell 11 is used for detecting cell structures in the skin tissue of the living body, the micro microscope probe 12 moves up and down through a movement device 13 arranged in the adsorption shell 11 so as to realize three-dimensional detection of the cell structures in the skin tissue of the living body, a first optical path for receiving pulse laser signals and transmitting the pulse laser signals to an autofluorescent substance in the living body and a second optical path for collecting and transmitting fluorescent signals generated by exciting the autofluorescent substance and a second harmonic signal are arranged in the micro microscope probe 12, and the first optical path and the second optical path are arranged, so that the adsorbable microscope detection device can complete excitation of the autofluorescent substance in the skin cell of the living body and acquire the fluorescent signals generated by excitation and the fluorescent signals used for imaging the cell structures, and the movement device 13 can drive the micro microscope probe 12 to move up and down under the driving of a motor.
The adsorbable microscope detection device provided by the embodiment of the invention comprises an adsorption shell and a micro microscope probe arranged in the adsorption shell, wherein the adsorbable microscope detection device is adsorbed on skin tissues of a living body to be detected through the miniaturized adsorption shell, so that the micro microscope probe detects cell structures in the skin tissues and carries out three-dimensional detection through up-and-down movement adjustment of an operation device, and simultaneously, the micro microscope probe excites autofluorescent substances in cells through two built-in light paths and pulse laser signals output through the light paths, and obtains fluorescent signals and second harmonic signals generated by exciting the autofluorescent substances, so that a three-dimensional laser scanning microscope provided with the adsorbable microscope detection device carries out three-dimensional imaging on the cell structures through the obtained fluorescent signals and the second harmonic signals.
On the basis of the above embodiments, the suction housing in the suction-type microscope detecting device provided by the embodiment of the present invention includes an outer housing, a base, and a cover glass, as shown in fig. 1, a suction cup 1121 is disposed on the base 112, the suction cup 1121 is embedded into a suction cup hole 1113 formed at the bottom of the outer housing 111, and the outer housing 111 is detachably connected with the base 112 through magnetic field force, wherein:
The cover glass 113 is fixed at the sealing port of the suction cup 1121 to form an inner space and an outer suction space of the suction type device, the moving device and the micro microscope probe are arranged in the inner space, and the micro microscope probe 12 is aligned with the cover glass 113 in the forward direction. Namely, the outer shell 111 and the base 112 of the adsorption shell in the adsorbable microscope detection device 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 112 is provided with a sucker 1121, the bottom of the outer shell 111 is provided with a sucker hole 1113 for embedding the sucker 1121, the sucker 1121 is provided with a sealing port communicated with the space in the sucker 1121, and when the cover glass 113 covers the sealing port, 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 adsorbable microscope detection device; and the motion device and the micro microscope probe are arranged in the built-in space, after the micro microscope probe is fixed, the micro microscope probe 12 is aligned to the cover glass 113 positively, so that an internal signal is output and an external signal is received through the cover glass 113, and thus the zooming and three-dimensional imaging of the microscope are realized, and the outer shell 111 and the base 112 are detachably connected through magnetic field force, so that a worker can replace the cover glass 113 by detaching the base 112 conveniently, and the operation is simple and the use is convenient.
On the basis of the above embodiments, the first light path in the adsorbable microscope detection device provided by the embodiment of the present invention includes a first light ray, a second light ray and a third light ray that are sequentially connected, where:
the pulse laser signal transmits a collimating lens, an analyzer, a polarization spectroscope and a first quarter wave plate in a first light path to a scanning galvanometer to form first light;
After forming a first light ray, the pulse laser signal is reflected by the scanning galvanometer and then transmitted to the polarization spectroscope again to form a second light ray;
After forming the second light, the pulse laser signal is reflected by the polarization spectroscope and then transmitted to the scanning mirror and the dichroic mirror to form a third light;
The second light path sequentially comprises an objective lens and a dichroic mirror for collecting and transmitting fluorescent signals and second harmonic signals. Fig. 2 is a schematic cross-sectional structure of an adsorbable microscope detecting device according to an embodiment of the present invention, wherein the schematic cross-sectional structure is related to an optical path in a micro microscope probe, as shown in fig. 2, when a first optical path in the adsorbable microscope detecting device according to an embodiment of the present invention processes a pulse laser signal, the pulse laser signal forms three light rays, namely, a first light ray, a second light ray and a third light ray, when the optical path is folded back, a collimating lens 121, a polarization analyzer 122, a polarizing beam splitter 123, a first quarter wave plate 124 to a scanning beam splitter 125 are disposed in the optical path forming the first light ray, a scanning beam splitter 125, a first quarter wave plate 124 and a polarizing beam splitter 123 are disposed in the optical path forming the second light ray, and a polarizing beam splitter 123, a transmissive scanning mirror 126, a dichroic mirror 127 and an objective lens 128 are disposed in the optical path forming the third light ray;
The pulse laser signal is emitted from the optical fiber, and then enters the polarization analyzer 122 after being collimated by the collimating lens 121, so that the collimated light is changed into linear polarized light, and the polarization direction of the linear polarized light is consistent with the transmission polarization direction of the polarization spectroscope 123, so that the linear polarized light directly transmits through the polarization spectroscope 123 after entering the polarization spectroscope 123, and enters the first quarter wave plate 124 positioned at the left side of the polarization spectroscope 123, wherein the fast axis direction of the first quarter wave plate forms an angle of +/-45 degrees with the polarization direction of the linear polarized light, and after passing through the first quarter wave plate 124, the linear polarized light is changed into circular polarized light to be incident on the scanning galvanometer 125, so that the first light is formed; after the first light is formed, the circularly polarized light reflected by the scanning galvanometer 125 enters the first quarter wave plate 124 again to become linearly polarized light, wherein the polarization direction of the linearly polarized light is perpendicular to the transmission polarization direction of the polarization spectroscope 123, so that the linearly polarized light is reflected on the light splitting surface of the polarization spectroscope 123 and exits from the lower part of the polarization spectroscope 123 to form a second light; after forming the second light, the emergent collimated light enters the scanning mirror 126 for converging, enters the objective lens 128, and forms a third light, wherein the focus of the collimated light is positioned at the relay image plane; scanning the scanning galvanometer 125 over the XY axis causes the pulsed laser signal focal point to scan over the entire relay image plane. The pulse laser signal is transmitted through a parallel plate as the dichroic mirror 127, and the dichroic mirror 127 discriminates the pulse laser signal from a fluorescence signal and a second harmonic signal generated by excitation of the autofluorescent substance according to wavelength, transmits the pulse laser signal, and reflects the fluorescence signal and the second harmonic signal. The relay image plane generated by converging the pulse laser signals through the scanning mirror 126 coincides with the rear image plane of the objective lens 128. The relayed image produced by the pulsed laser signal scan is scaled to the sample according to the magnification of objective lens 128. The focus of the pulse laser signal on the sample can generate a fluorescence signal and a second harmonic signal, the generated fluorescence signal and the generated second harmonic signal are collected through the objective lens 128 and reflected by the dichroic mirror 127 and enter a collection optical fiber bundle to be collected, wherein the objective lens 128 is a limited far micro objective lens 128, and a second optical path sequentially comprises the objective lens 128 and the dichroic mirror 127 for collecting and transmitting the fluorescence signal and the second harmonic signal; wherein the scanning galvanometer may be a scanning galvanometer based on mechanical, electromechanical or microelectromechanical principles, as is the case in the various embodiments below.
On the basis of the above embodiments, the first light path in the adsorbable microscope detecting device provided by the embodiment of the present invention includes a first light ray, a second light ray, a third light ray, a fourth light ray and a fifth light ray that are sequentially connected, where:
The pulse laser signal is incident to a polarization spectroscope through a collimating lens and an analyzer in a first light path to form first light;
after forming the first light, the pulse laser signal is reflected by the polarization spectroscope and then transmitted to the plane reflector by the second quarter wave plate to form the second light;
After forming a second light ray, the pulse laser signal is reflected by the plane reflecting mirror and then transmitted to the scanning galvanometer again through the second quarter wave plate, the polarization spectroscope and the third quarter wave plate to form a third light ray;
after forming a third light ray, the pulse laser signal is reflected by the scanning galvanometer and then transmitted to the polarization spectroscope again to form a fourth light ray;
After forming the fourth light, the pulse laser signal is reflected by the polarization spectroscope and then transmitted to the scanning mirror and the dichroic mirror to form a fifth light;
The second light path sequentially comprises an objective lens for collecting and transmitting fluorescent signals and second harmonic signals and a dichroic mirror. FIG. 3 is a schematic cross-sectional view of an adsorbable microscope detection device according to another embodiment of the present invention, wherein the schematic cross-sectional view is related to an optical path in a micro microscope probe, as shown in FIG. 3, when a first optical path in the adsorbable microscope detection device according to the embodiment of the present invention processes a pulse laser signal, the pulse laser signal forms a fifth ray when passing through an optical device of the first optical path, and the fifth ray is formed by folding back the optical path, wherein a collimating lens 140, a polarization analyzer 141 and a polarization beam splitter 142 are arranged in the optical path for forming the first ray, a plane mirror 144, a plane mirror 143, a polarization beam splitter 142, a third quarter wave plate 145 and a scanning beam splitter 146 are arranged in the optical path for forming the third ray, and a scanning beam splitter 146, a third quarter wave splitter 145 and a polarization beam splitter 142 are arranged in the optical path for forming the fourth ray, and polarization beam splitters 149 and 148 are arranged in the optical path for forming the fifth ray;
The pulse laser signal is emitted from the optical fiber, and then enters the polarization analyzer 141 after being collimated by the collimating lens 140, so that the collimated light is changed into linear polarized light, and the polarization direction of the linear polarized light is perpendicular to the transmission polarization direction of the polarization spectroscope 142, so that the linear polarized light is reflected after entering the polarization spectroscope 142 and is emitted from the right side of the polarization spectroscope 142, and a first light is formed; after the first light is formed, the emergent linear polarized light enters a second quarter wave plate 143, wherein the fast axis direction of the second quarter wave plate forms an angle of +/-45 degrees with the linear polarized light polarization direction, so that the linear polarized light is changed into circular polarized light after passing through the second quarter wave plate and enters a plane reflector 144 to form the second light; after forming the second light, the circularly polarized light is reflected by the plane mirror 144, returns along the original light path and is changed into linearly polarized light by the second quarter wave plate 143, and the polarization direction of the linearly polarized light is consistent with the transmission polarization direction of the polarization spectroscope 142, so that the linearly polarized light directly transmits through the polarization spectroscope 142 after entering the polarization spectroscope 142 for the second time, and enters the third quarter wave plate 145 positioned at the left side of the polarization spectroscope 142, wherein the fast axis direction of the third quarter wave plate forms an angle of +/-45 degrees with the polarization direction of the linearly polarized light, and the linearly polarized light is changed into circularly polarized light after passing through the third quarter wave plate and then enters the scanning vibrating mirror 146 to form third light; after forming the third light, the circularly polarized light reflected by the scanning galvanometer 146 enters the third quarter wave plate 145 again to become linearly polarized light, wherein the polarization direction of the linearly polarized light is perpendicular to the transmission polarization direction of the polarizing beam splitter 142, so that the polarized light is reflected on the light splitting surface of the polarizing beam splitter 142 and exits from the lower part of the polarizing beam splitter 142 to form a fourth light; after forming the fourth light, the emergent collimated light enters a scanning mirror for converging, enters an objective lens 149, and the focus of the collimated light is positioned at the relay image plane to form a fifth light; scanning the scanning galvanometer 146 on the XY axis causes the pulsed laser signal focal point to scan across the relay image plane. The pulse laser signal is transmitted through a parallel plate as the dichroic mirror 148, and the dichroic mirror 148 distinguishes the pulse laser signal from a fluorescence signal and a second harmonic signal generated by excitation of the autofluorescent substance according to wavelength, transmits the pulse laser signal, and reflects the fluorescence signal and the second harmonic signal. The relay image plane generated by converging the pulse laser signals through the scanning mirror 147 coincides with the rear image plane of the objective lens 149. The relayed image produced by the pulsed laser signal scan is scaled to the sample according to the magnification of the objective lens 149. The focal point of the pulse laser signal on the sample can generate a fluorescence signal and a second harmonic signal, the generated fluorescence signal and the generated second harmonic signal are collected through the objective lens 149 and reflected by the dichroic mirror 148 to enter the collection optical fiber bundle for collection, wherein the objective lens 149 is a finite far micro objective lens 149, and the second optical path sequentially comprises the objective lens 149 and the dichroic mirror 148 for collecting and conducting the fluorescence signal and the second harmonic signal.
On the basis of the above embodiments, the adsorbable microscope detecting device provided by the present invention further includes an electrically adjustable curvature lens, and fig. 4 is a schematic cross-sectional structure diagram of a combined adsorbable microscope detecting device provided by another embodiment of the present invention, where, as shown in fig. 4, the electrically adjustable curvature lens 120 is located between the collimating lens 121 and the analyzer 122, and the pulsed laser signal transmits the collimating lens 121, the electrically adjustable curvature lens 120, the analyzer 122, the polarizing beam splitter 123, and the first quarter wave plate 124 to the scanning galvanometer 125 to form a new first light. I.e. the optical elements in the new first light ray, comprise in order a collimator lens 121, an electrically tunable curvature lens 120, an analyzer 122, a polarizing beamsplitter 123, a first quarter wave plate 124 and a scanning galvanometer 125. The electrically adjustable curvature lens 120 is arranged such that the surface of the electrically adjustable curvature lens 120 generates corresponding bending by applying voltage or current to the electrically adjustable curvature lens 120, so as to generate different optical powers for the parallel light emitted from the straight lens 121. The specific light path is: the laser signal is emitted from the optical fiber, passes through the collimating lens 121 and then is parallel incident to the electrically adjustable curvature lens 120, corresponding focal power is generated by the electrically adjustable curvature lens 120 according to the loaded voltage or current signal, and the emitted converging or diverging light passes through the optical element in the first optical path to form new first light, second light and third light, and is transmitted to the objective lens 128 and then is converged on the sample. The focal power change introduced by the electrically adjustable curvature lens 120 can enable the focal point of the laser signal emitted from the opening of the objective lens 128 to move up and down in the depth direction, and the response speed of the electrically adjustable curvature lens 120 is very high, and the scanning frequency is in the order of KHz, so that rapid scanning imaging in the depth direction can be realized. The electrically adjustable curvature lens 120 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 generate any shift of the focal point behind the objective lens 128, thereby realizing three-dimensional stereoscopic imaging. When the device is specifically used, the electrically adjustable curvature lens 120 is complementary to the zoom motor, the position of the objective lens 128 is adjusted by the zoom motor, after coarse adjustment to the corresponding depth position, the system is switched to a zoom scanning mode of the electrically adjustable curvature lens 120, and rapid three-dimensional imaging is performed on a sample, wherein when the adsorbable microscope detection device is not provided with the zoom motor, the zoom adjustment can be performed only by the electrically adjustable curvature lens 120.
On the basis of the above embodiments, the adsorbable microscope detecting device according to the present invention further includes an electrically adjustable curvature lens, and fig. 5 is a schematic cross-sectional structure diagram of a combined adsorbable microscope detecting device according to another embodiment of the present invention, where, as shown in fig. 5, the electrically adjustable curvature lens 150 is located between the collimating lens 140 and the analyzer 141, and the pulse laser signal transmits the collimating lens 140, the electrically adjustable curvature lens 150, and the analyzer 141 to be incident on the polarizing beam splitter 142, so as to form a new first light. I.e. the optical elements in the new first light ray, comprise in order a collimator lens 140, an electrically adjustable curvature lens 150, an analyzer 141 and a polarizing beamsplitter 142. The electrically adjustable curvature lens 150 is arranged such that the surface of the electrically adjustable curvature lens 150 generates corresponding bending by applying voltage or current to the electrically adjustable curvature lens 150, so as to generate different optical powers for the parallel light emitted from the straight lens 121. The specific light path is: the laser signal is emitted from the optical fiber, passes through the collimating lens 121 and then is parallel incident to the electrically adjustable curvature lens 150, corresponding focal power is generated by the electrically adjustable curvature lens 150 according to the loaded voltage or current signal, and the emitted converging or diverging light passes through the optical element in the first light path to form new first light, second light, third light, fourth light and fifth light, and then is transmitted to the objective lens 149 and then is converged on the sample. The focal power change introduced by the electrically adjustable curvature lens 150 can enable the focal point of the laser signal emitted from the opening of the objective lens 149 to move up and down in the depth direction, and the response speed of the electrically adjustable curvature lens 150 is very high, and the scanning frequency is in the order of KHz, so that rapid scanning imaging in the depth direction can be realized. The electrically adjustable curvature lens 150 is equivalent to a parallel plate glass when no voltage or current signal is applied, and does not have optical power to a laser signal and does not cause any shift of a focal point after the objective lens 149, thereby realizing three-dimensional stereoscopic imaging. In specific use, the electrically adjustable curvature lens 150 is complementary to the zoom motor 13, the position of the objective lens 149 is adjusted by the zoom motor 13, and after coarse adjustment to the corresponding depth position, the system is switched to the zoom scanning mode of the electrically adjustable curvature lens 150 to perform rapid three-dimensional imaging on the sample, wherein when the adsorbable microscope detection device is not provided with the zoom motor 13, the zoom adjustment can be performed only by the electrically adjustable curvature lens 150.
On the basis of the above embodiments, the movement device in the adsorbable microscope detection device provided by the embodiment of the invention includes a fixed bracket and a limiting block, as shown in fig. 1, wherein:
the fixed bracket 131 is fixed on the side wall of the adsorption shell, and the limiting block 132 is in sliding connection with the fixed bracket 131 relatively and is used for driving the micro-microscope probe to move up and down. Namely, the moving device in the adsorbable microscope detecting device provided by the embodiment of the invention comprises a fixed bracket 131 and a limiting block 132, and is used for driving the micro-microscope probe to move up and down.
On the basis of the above embodiments, the motion device in the adsorption device for setting a micro-microscope probe according to the embodiment of the present invention further includes a probe fixing frame, as shown in fig. 1, the probe fixing frame 133 is detachably and fixedly connected to the limiting block 132, and the micro-microscope probe is detachably fixed to the limiting block 132 through the probe fixing frame 133.
On the basis of the above embodiments, the suction cup in the adsorbable microscope detecting device provided by the embodiment of the invention further includes an adsorption port, and the adsorption port is communicated with the sealing port and is used for being adsorbed on a living body to be detected through the external adsorption space. In the adsorbable microscope detecting device provided by the embodiment of the invention, the sucking disc arranged on the base comprises the sucking mouth besides the sealing mouth, and the sealing mouth is communicated with the sucking mouth, as shown in fig. 2, an inner space 1114 of the sucking disc is formed, when the cover glass is sealed and fixed on the sealing mouth, the inner space 1114 of the sucking disc forms an outer sucking space 1115, and the adsorbable microscope detecting device is adsorbed on a life body to be detected through the sucking mouth and the outer sucking space 1115.
The embodiment of the invention also provides an adsorption type three-dimensional nonlinear laser scanning microscope, fig. 6 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. 6, the laser scanning microscope comprises:
Fluorescence collection device 56, air extraction device 52, scan collection controller 531, femtosecond pulse laser 541, fiber optic coupling module 542 and adsorbable microscope detection device 51 that the above-described embodiment provided, fluorescence collection device 56 and fiber optic coupling module 542 all are connected with adsorbable microscope detection device 51 fiber optic communication, fluorescence collection device 56 and adsorbable microscope detection device 51 all are connected with scan collection controller 531 electricity, air extraction device 52 and adsorbable microscope detection device 51 electricity are connected, 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 adsorbable microscope detection device;
The adsorbable 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 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 scanning galvanometer in 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 adsorbable 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 adsorbable 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 is formed for detection, 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, 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, and is connected with an air extraction pipeline, an air extraction valve is arranged in the air extraction pipeline, the air extraction valve is electrically connected with the air extraction device 52, the air extraction flow of the air extraction pipeline is controlled by adjusting the opening and closing of the air extraction valve, so that air extraction control of an external adsorption space is realized, negative pressure in the external adsorption space is further adjusted, the adsorption device is adsorbed on tissues such as skin and intestines and stomach of a living body through the action of atmospheric pressure, the adsorption three-dimensional nonlinear laser scanning microscope can comprise a two-photon scanning microscope, a multi-photon 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 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 collection controller, a femtosecond pulse laser, an optical fiber coupling module and an adsorbable 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 miniature microscope probe and a cover glass, the three-dimensional scanning of the laser scanning microscope is realized, the self-fluorescent substances in cells are excited by the femtosecond pulse laser to obtain a multiphoton fluorescence signal and a second harmonic signal, 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, wherein the adsorbable microscope detection device can avoid the vibration influence of life body activity on the miniature microscope probe, so as to avoid the vibration influence on the imaging quality, the laser scanning microscope can realize various imaging modes including XY imaging, XZ imaging and 3D imaging, wherein the XY imaging is realized at a certain depth of a cell structure, the X Z imaging is carried out at a certain depth, and the X Z imaging is carried out at a certain depth of a surface layer, and the X imaging is carried out at a certain depth of a surface layer, and 3D imaging is carried out at a certain depth, and a certain depth is formed by a certain imaging depth and a 3D imaging device is convenient.
On the basis of the above embodiments, the adsorption device for setting a micro microscope probe provided by the embodiment of the present invention further includes a motor, where the motor is disposed in the inner enclosed space, and the adsorption device includes:
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. The adsorption device for setting the micro-microscope probe is also 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 adsorption apparatus 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, in which:
An accommodating space is arranged in the first shell 1111, and the suction cup and the cover glass are arranged in the accommodating space, so that the first shell 1111 and the second shell 1112 are detachably and fixedly connected. That is, the outer housing of the adsorption type device for setting a micro-microscope probe according to the embodiment of the present invention includes two parts, namely, a first housing 1111 and a second housing 1112, the first housing 1111 has a receiving space for setting a suction cup and a cover glass, that is, the suction cup hole in the above embodiment is also provided at the bottom of the first housing 1111, the suction cup is embedded in the suction cup hole of the first housing 1111, and the cover glass and the second housing 1112 are combined to form an outer adsorption space and an inner closed space in the above embodiment, wherein the first housing 1111 and the second housing 1112 can be detachably and fixedly connected by screws, so that the assembly, the disassembly and the replacement of components of the whole device are facilitated.
On the basis of the above embodiments, fig. 7 is a schematic structural diagram of a fluorescence collection device according to an embodiment of the present invention, as shown in fig. 7, 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. 6, 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. 6, 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.
On the basis of the above embodiments, the number of the adsorbable microscope detection devices in the adsorbable three-dimensional nonlinear laser scanning microscope provided by the embodiment of the invention is multiple. 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 adsorbable microscope detection devices in an optical fiber communication way, namely, a plurality of detection devices are integrated in one adsorbable 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 adsorbable microscope detection device respectively, 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 adsorbable microscope detection device through the adjustable-length adjusting optical fiber in an optical fiber transmission manner, so that the detection device can be 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 can be adjusted, the application in various occasions can be 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 is illustrated in fig. 8, as shown in fig. 8, the adsorbable 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 adsorbable microscope detecting device 51 through optical fibers, 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. 9 is a schematic diagram of detecting skin tissue of a human chest by using an adsorption three-dimensional nonlinear laser scanning microscope according to an embodiment of the present invention, as shown in fig. 9, the adsorbable microscope detecting device 51 is adsorbed on the human chest by using an air extracting function of the air extracting device 52, wherein a scan 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 adsorbable microscope detecting device 51 by optical fiber transmission, wherein the working principle of the adsorption three-dimensional nonlinear laser scanning microscope is the same as that of the above embodiments, and the description is omitted.
Fig. 10 is a schematic diagram of simultaneous detection of skin tissues of a human body by using multiple detection devices of the adsorption type three-dimensional nonlinear laser scanning microscope provided by the embodiment of the invention, as shown in fig. 10, by using the air extraction function of the air extraction device 52, the multiple detection devices of the adsorption type microscope 51 are respectively and simultaneously adsorbed on the face, chest and legs of the human body, 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 collection device, 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 type 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 type 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. 11 is a schematic diagram of an adsorption three-dimensional nonlinear laser scanning microscope for detecting animal skin tissue, as shown in fig. 11, the adsorbable microscope detecting device 51 can be adsorbed on the skin tissue of a living body through the air suction function of the air suction 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, 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 both connected with the adsorbable microscope detecting device 51 through optical fiber transmission, 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. 12 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. 12, 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 adsorbable 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. 13 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. 13, a display 55 integrated on a box cover is integrated with a box body in which each module is installed, so that the whole equipment can be conveniently moved, and a workplace can be conveniently replaced, and the display 55 can be externally placed on the box body when in use, so that a worker can conveniently obtain information on the display. 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 fluorescence and a tissue with non-SHG (Second Harmonic Generation ) signal activity, 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 application has been described in connection with the embodiments of the present application, it will be understood by those skilled in the art that the present application is not limited to the preferred embodiments of the present application, and various modifications and variations can be made thereto by those skilled in the art, based on the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle 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 (9)
1. An adsorbable microscope detection device, comprising:
Adsorption shell and set up in micro-microscope probe in the adsorption shell, the adsorption shell inboard is provided with the motion, micro-microscope probe set up in on the motion, the motion is used for driving micro-microscope probe reciprocates, wherein:
the motion device with micro-microscope probe all set up in the absorption casing, be provided with first light path and second light path in the micro-microscope probe, wherein:
The first optical path is used for receiving the pulse laser signal and transmitting and outputting the pulse laser signal to an autofluorescent substance in a living body cell;
The second light path is used for collecting and conducting fluorescent signals and second harmonic signals generated by excitation of the autofluorescent substances;
The first light path comprises a first light ray, a second light ray and a third light ray which are sequentially connected, wherein:
The pulse laser signal transmits a collimating lens, an analyzer, a polarization spectroscope and a first quarter wave plate in the first light path to a scanning galvanometer to form the first light;
After the first light ray is formed, the pulse laser signal is reflected by the scanning galvanometer and then transmitted to the polarization beam splitter again to form the second light ray;
after the second light ray is formed, the pulse laser signal is reflected by the polarization spectroscope and then transmitted to a scanning mirror and a dichroic mirror to an objective lens to form the third light ray; wherein, the focus of the objective lens is positioned at the relay image plane; the objective lens is a limited far objective lens;
the second light path sequentially comprises the objective lens and the dichroic mirror for collecting and conducting the fluorescence signal and the second harmonic signal.
2. The adsorbable microscope detection device of claim 1, wherein the adsorption housing comprises an outer housing, a base, and a cover slip, the base is provided with a suction cup, the suction cup is embedded into a suction cup hole formed in the bottom of the outer housing, and the outer housing is detachably connected with the base through magnetic 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 moving device and the micro microscope probe are arranged in the inner space, and the micro microscope probe is aligned to the cover glass in the forward direction.
3. An adsorbable microscope detection device, comprising:
Adsorption shell and set up in micro-microscope probe in the adsorption shell, the adsorption shell inboard is provided with the motion, micro-microscope probe set up in on the motion, the motion is used for driving micro-microscope probe reciprocates, wherein:
the motion device with micro-microscope probe all set up in the absorption casing, be provided with first light path and second light path in the micro-microscope probe, wherein:
The first optical path is used for receiving the pulse laser signal and transmitting and outputting the pulse laser signal to an autofluorescent substance in a living body cell;
The second light path is used for collecting and conducting fluorescent signals and second harmonic signals generated by excitation of the autofluorescent substances;
The first light path comprises a first light ray, a second light ray, a third light ray, a fourth light ray and a fifth light ray which are sequentially connected, wherein:
The pulse laser signal is incident to a polarization spectroscope through a collimating lens and an analyzer in the first light path to form the first light; specifically, the collimated light enters the polarization analyzer after being collimated by the collimating lens, so that the collimated light is changed into linearly polarized light, and the polarization direction of the linearly polarized light is perpendicular to the transmission polarization direction of the polarization spectroscope, so that the linearly polarized light enters the polarization spectroscope, is reflected and exits from the right side of the polarization spectroscope, and forms first light;
After the first light ray is formed, the pulse laser signal is reflected by the polarization spectroscope and then transmitted to a plane reflector through a second quarter wave plate to form the second light ray; specifically, the fast axis direction of the second quarter wave plate forms an angle of +/-45 degrees with the polarization direction of the linear polarized light, so that the linear polarized light is changed into circular polarized light after passing through the second quarter wave plate and is incident on the plane reflector to form second light;
after the second light ray is formed, the pulse laser signal is reflected by the plane reflecting mirror and then transmitted to the scanning vibrating mirror again through the second quarter wave plate, the polarization spectroscope and the third quarter wave plate to form the third light ray; specifically, the circularly polarized light is reflected by the plane reflecting mirror, returns to be changed into linearly polarized light by the second quarter wave plate along the original light path, and the polarization direction of the linearly polarized light is consistent with the transmission polarization direction of the polarization spectroscope, so that the linearly polarized light directly transmits through the polarization spectroscope after entering the polarization spectroscope for the second time, enters a third quarter wave plate positioned at the left side of the polarization spectroscope, wherein the fast axis direction of the third quarter wave plate forms an angle of +/-45 degrees with the polarization direction of the linearly polarized light, and the linearly polarized light is changed into circularly polarized light after passing through the third quarter wave plate and then enters the scanning galvanometer to form third light;
After the third light ray is formed, the pulse laser signal is reflected by the scanning galvanometer and then transmitted to the polarization beam splitter again to form the fourth light ray; specifically, circularly polarized light reflected by the scanning galvanometer enters the third quarter wave plate again to become linearly polarized light, wherein the polarization direction of the linearly polarized light is perpendicular to the transmission polarization direction of the polarization spectroscope, so that the polarized light is reflected on the light splitting surface of the polarization spectroscope and exits from the lower part of the polarization spectroscope to form fourth light;
after the fourth light ray is formed, the pulse laser signal is reflected by the polarization spectroscope and then transmitted to a scanning mirror and a dichroic mirror to an objective lens to form the fifth light ray; specifically, the emergent collimated light enters a scanning mirror for converging, enters an objective lens, and forms a fifth ray, wherein the focus of the collimated light is positioned at a relay image plane;
The second light path sequentially comprises the objective lens and the dichroic mirror, wherein the objective lens is used for collecting and conducting the fluorescent signals and the second harmonic signals.
4. The adsorbable microscope detection device of claim 1, further comprising an electrically tunable curvature lens positioned between the collimating lens and the analyzer, the pulsed laser signal transmitting the collimating lens, the electrically tunable curvature lens, the analyzer, the polarizing beamsplitter, the first quarter wave plate to the scanning galvanometer to form a new first light ray.
5. The adsorbable microscopic examination device of claim 3 further comprising an electrically tunable curvature lens positioned between the collimating lens and the analyzer, the pulsed laser signal transmitted through the collimating lens, the electrically tunable curvature lens, and the analyzer to the polarizing beamsplitter to form a new first light ray.
6. The adsorbable microscope detection device of claim 1 or 3, wherein the movement device comprises a fixed bracket and a stopper, wherein:
the fixed support is fixed on the side wall of the adsorption shell, and the limiting block is in relative sliding connection with the fixed support and is used for driving the micro microscope probe to move up and down.
7. The adsorbable microscope detection device of claim 2, wherein the suction cup further comprises a suction port in communication with the sealing port for suction onto a living being under test through the external suction space.
8. An adsorption type three-dimensional nonlinear laser scanning microscope, comprising:
The fluorescence collection device, the air exhaust device, the scanning acquisition controller, the femtosecond pulse laser, the optical fiber coupling module and the adsorbable microscope detection device according to any one of claims 1-7, wherein the fluorescence collection device and the optical fiber coupling module are in optical fiber communication connection with the adsorbable microscope detection device, the fluorescence collection device and the adsorbable microscope detection device are electrically connected with the scanning acquisition controller, and the air exhaust device is electrically connected with the adsorbable 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 adsorbable microscope detection device;
The adsorbable microscope detection device is used for outputting the pulse laser signal to an autofluorescent substance in a living body cell after receiving the pulse laser signal, acquiring a fluorescent signal and a second harmonic signal generated after excitation of the autofluorescent substance, and outputting the fluorescent signal and the second harmonic signal 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 a scanning galvanometer in 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 external adsorption space of the adsorbable microscope detecting device so as to form negative pressure in the external adsorption space.
9. The adsorptive three-dimensional nonlinear laser scanning microscope of claim 8, 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.
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