CN211086090U - Nanometer precision optogenetic control device - Google Patents
Nanometer precision optogenetic control device Download PDFInfo
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- CN211086090U CN211086090U CN201921954481.4U CN201921954481U CN211086090U CN 211086090 U CN211086090 U CN 211086090U CN 201921954481 U CN201921954481 U CN 201921954481U CN 211086090 U CN211086090 U CN 211086090U
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Abstract
The utility model discloses a nanometer precision optogenetic controlling device, including light source one, light source two, light source three, light detector, quarter wave plate, speculum one, first dichroic mirror, second dichroic mirror, third dichroic mirror, mirror system, speculum two, speculum three, focus objective, sample platform shake. The utility model discloses to the control problem of the optogenetic of traditional method under being difficult to realize subcellular size, a nanometer precision optogenetic control device has been proposed. The basic principle of the device is that the size of a light genetic light spot is compressed by superposition of two beams of light, so that the light spot reaches the nanometer level, and further, the movement control among organelles with smaller sizes is realized, and the device has great significance for the research of subcellular organelle structures. And simultaneously, the utility model discloses utilize and move back activation light simultaneous control excitation light and activation light, system architecture is comparatively simple, builds easily.
Description
Technical Field
The utility model relates to a method and a system for optogenetic technology optogenetic manipulation, in particular to a nano-precision optogenetic manipulation device.
Background
The optogenetic technology combines optical technology and genetic technology, and controls the activation and the closing of photosensitive protein on a cell membrane structure through the irradiation of light with specific wavelength, so as to control cells, usually neurons, in living tissues and further control the behavior of organisms. As a novel biotechnology, the optogenetic technology provides a non-invasive, reversible and effective means for researching cell functions, and realizes the control and monitoring of the activities of individual cells. These cells are genetically manipulated to express specific membrane proteins for controlling the opening and closing of ion channels.
The current optogenetic technology has realized the manipulation of cells and the observation of intercellular activities, for example, Wu YI et al, in 2009, journal NATURE, "a genetic encoded photosynthetic random of motility cells, realized the manipulation of cellular activities with 10um illumination spots, but the current optogenetic technology has realized the activities of organelles of relatively smaller size: the control and observation of the vesicular secretion of Golgi apparatus is impossible, which hinders the further development of biology towards deeper fields.
Disclosure of Invention
The utility model discloses to the control problem of the optogenetic of traditional method under being difficult to realize subcellular size, a nanometer precision optogenetic control device has been proposed. The basic principle of the device is that the size of a light genetic light spot is compressed by superposition of two beams of light, so that the light spot reaches the nanometer level, and further the control of the movement between organelles with smaller sizes is realized.
A nanometer precision optogenetic control device comprises a first light source, a second light source, a third light source, a light detector, a quarter wave plate, a first reflecting mirror, a first dichroic mirror, a second dichroic mirror, a third dichroic mirror, a vibrating mirror system, a second reflecting mirror, a third reflecting mirror, a focusing objective lens and a sample stage.
The light source emits deactivation light, the deactivation light passes through the quarter-wave plate, is totally reflected by the reflecting mirror and then penetrates through the first dichroic mirror; the second light source emits active light, and after being reflected by the first dichroic mirror, the active light and the deactivated light emitted by the first light source are combined to form a combined light I; and the light source III emits exciting light, the exciting light is reflected by the second dichroic mirror and then combined with the combined light to form a combined light II, the combined light II passes through the third dichroic mirror, is totally reflected by the second reflecting mirror and the third reflecting mirror through the vibrating mirror system and enters the focusing objective lens, and finally forms a focusing light spot near the sample stage.
And the galvanometer system controls the scanning of the focusing light spots in the direction of the sample stage.
And the sample table is provided with an organelle sample.
The organelle sample is subjected to fluorescence calibration and genetic manipulation in advance; the genetic manipulation treatment is to lead the surfaces of organelles of the organs to express corresponding membrane proteins by introducing specific photosensitive protein genes, and particularly refers to the influence of activating prefrontal cortex by a photopheresis method on the c-Dos table of a brain area, which is published in journal of university of Yangzhou (agricultural and life science edition) of Liu Mey et al.
The quarter-wave plate changes the polarization state of the deactivation light emitted by the first light source into circularly polarized light, a circular hollow focusing light spot is formed on the focusing plane of the focusing objective lens, and the inner diameter of the hollow focusing light spot is in a nanometer magnitude.
The activation light can open the cation channel on the cell membrane, and the deactivation light closes the cation channel on the cell membrane; and closing the cation channel in the superposed region of the activated light focusing spot and the hollow deactivated light focusing spot, opening the cation channel only in the hollow region of the deactivated light focusing spot, and compressing the region of the optogenetic operation to nanometer level to realize the optogenetic operation under the size of the subcellular.
The exciting light excites the fluorescent substance to emit fluorescence, and the deactivating light inhibits the excitation of the fluorescent substance; on the focusing plane, the fluorescent substance does not emit light in the superposed area of the exciting light focusing spot and the hollow deactivation light focusing spot, only emits light in the hollow area of the deactivation light focusing spot, and the size of the spot is smaller than that of the exciting light focusing spot, thereby reaching the nanometer level.
Preferably, the activating light has a wavelength of 470 nm, a pulse of 80 MHz, a pulse width of 1 picosecond or less, and a power of 1 megawatt.
Preferably, the excitation light has a wavelength of 488 nm, a pulse of 80 MHz, a pulse width of 1 picosecond or less, and a power of 1 megawatt.
Preferably, the deactivation light has a wavelength of 590 nm, a pulse of 80 MHz, a pulse width of 600 picoseconds or more and a power of 250 megawatts, and can simultaneously close the cation channel opened by the activation light and the fluorescence excited by the excitation light.
Preferably, the first dichroic mirror exhibits high transmittance for de-activated light and high reflectance for activated light; the second dichroic mirror is high in transmittance for de-activation light and high in reflectance for excitation light; the third dichroic mirror is high in transmittance for incident laser from the plane of incidence directions of the de-excitation light, the activation light and the excitation light, and high in reflectance for incident laser and fluorescence from the plane of the other incidence direction; the high transmittance refers to the transmittance of more than 98 percent; the high reflectivity means that the reflectivity is more than 98%, specifically 98-99.9%.
Preferably, the galvanometer system, the quarter-wave plate and the focusing plane of the focusing objective form a conjugate plane, so that the laser transmission direction can be controlled with high precision, and the optogenetic manipulation can be effectively ensured.
Preferably, the focusing objective lens is an immersion flat field achromatic objective lens, has a numerical aperture of more than 1.05, and has high light receiving capacity and spatial resolution.
Compared with the prior art, the utility model discloses following profitable technological effect has:
1. the deactivation light is used for controlling the excitation light and the activation light at the same time, so that the system is simple in structure and easy to build;
2. the system can generate nanometer-level focusing light spots by using a light beam superposition principle, can realize optogenetic operation on a nanometer scale, and has great significance for the research of subcellular organelle structures.
Drawings
Fig. 1 is a schematic diagram of the nano-precision optogenetic manipulation device of the present invention.
Wherein: 1. a first light source; 2. a second light source; 3. a third light source; 4. a light detector; 5. a quarter wave plate; 6. a first reflecting mirror; 7. a first dichroic mirror; 8. a second dichroic mirror; 9. a third dichroic mirror; 10. a galvanometer system; 11. a second reflecting mirror; 12. a third reflector 13 and a focusing objective lens; 14. a sample stage.
Fig. 2 is a schematic diagram of two light spots superposed and compressed to obtain a nanometer-scale light spot.
FIG. 3 is a schematic diagram showing the display of the Golgi apparatus detected in the photodetector for both illumination and non-illumination conditions of the second light source.
Detailed Description
The embodiments of the present invention will be described in detail below with reference to the accompanying drawings, but the present invention is not limited thereto.
Fig. 1 is a schematic diagram of the apparatus for a method and a system for nano-precision optogenetic manipulation of the present invention, which includes: the device comprises a first light source 1, a second light source 2, a third light source 3, a light detector 4, a quarter-wave plate 5, a first reflecting mirror 6, a first dichroic mirror 7, a second dichroic mirror 8, a third dichroic mirror 9, a vibrating mirror system 10, a second reflecting mirror 11, a reflecting mirror 12, a focusing objective 13 and a sample stage 14.
The light source III 3 emits exciting light with the wavelength of 488 nanometers, so that fluorescent substances in the Golgi apparatus can emit fluorescence; the light source II 2 emits activating light with the wavelength of 470 nanometers, photosensitive protein in the Golgi apparatus can be activated, and a cation channel is opened; the light source I1 emits deactivation light with the wavelength of 590 nanometers, so that the fluorescent substance on the surface of the Golgi apparatus is deactivated and cannot emit fluorescence, activated photosensitive protein is deactivated, and a cation channel is closed.
The sample table 14 is loaded with a Golgi apparatus sample, the organelle sample is processed by genetic manipulation in advance, and a specific photosensitive protein gene is introduced to express a corresponding photosensitive protein ChR2 on the surface of the organelle for controlling the opening and closing of a cation channel on a cell membrane; and then the Golgi apparatus is processed by fluorescence labeling, so that only the 488 nm exciting light emitted by the light source III 3 can excite the surface fluorescent substance to emit fluorescence.
470-nanometer activation light emitted by the light source II 2 is totally reflected by the first dichroic mirror 7, is totally reflected by the second dichroic mirror 8 and the light source III 3 to emit 488-nanometer excitation light combined beams, then passes through the third dichroic mirror 9, is totally reflected by the vibrating mirror system 10, the reflecting mirror II 11 and the reflecting mirror III 12, enters the focusing objective lens 13 to be focused, and finally forms a 1.5-micrometer focusing light spot I near the sample stage 14, and the focusing light spot I can excite the fluorescent substance on the surface of the organelle sample to emit light to activate the photosensitive protein ChR2 in the organelle, so that a cation channel is opened.
The 590 nanometer deactivation light emitted by the first light source 1 is changed into circularly polarized light through the quarter-wave plate 5, is totally reflected by the reflector 6, is totally reflected by the first dichroic mirror 7 and the activated photosynthetic beam emitted by the second light source, is totally reflected by the second dichroic mirror 8 and the excited photosynthetic beam emitted by the third light source, then passes through the third dichroic mirror 9, enters the focusing objective lens 13 for focusing after passing through the vibrating mirror system 10, the second reflector 11 and the third reflector 12, and finally forms a hollow focusing light spot II with the diameter of 3 micrometers near the sample stage 14, wherein the diameter of the middle hollow circular area is 500 nanometers.
As shown in fig. 2, the left picture is a focused spot one with a diameter of 1.5 μm formed near the sample stage 14 after the combination of 470 nm of activating light and 488 nm of exciting light, the focused spot one can excite the fluorescent substance on the surface of the golgi to emit fluorescence, and activate the photosensitive protein ChR2, and open the cation channel; the middle picture is a focusing light spot II with an outer diameter of 3 microns and an inner diameter of 500 nanometers, which is formed by the deactivation light with a wavelength of 590 nanometers near the sample stage 14; and the right picture is the focused light spot after the focused light spot I and the focused light spot II are superposed, the fluorescent substance on the surface of the Golgi apparatus in the superposed region is de-excited and can not emit fluorescence, the activated photosensitive protein ChR2 is de-activated, and the cation channel is closed. And after superposition, the size of the light spot is effectively compressed to form a focusing light spot of 500 nanometers, so that the optogenetic operation under the subcellular size is realized.
In this embodiment, the fluorescence emitted by the fluorescent substance exits to the focusing objective 13, is totally reflected by the third reflecting mirror 12 and the second reflecting mirror 11, is totally reflected by the third dichroic mirror 9 through the galvanometer system 10, and finally enters the optical detector 4, and the optical detector 4 receives the optical information and presents the received information in the form of an image.
As shown in the left picture of fig. 3, the schematic diagram of the golgi display detected by the light detector 4 when the first light source and the third light source are turned on and the second light source is turned off is shown: only the part irradiated by the laser in the Golgi apparatus appears fluorescence; the right picture is a schematic representation of the display of the golgi detected by the light detector 4 when the three light sources are turned on: fluorescence also appears around the outside of the Golgi apparatus except the laser irradiated part in the Golgi apparatus; the 470 nm activating light emitted by the light source two 2 opens the cation channel in the Golgi apparatus, so that the fluorescence labeled cation passes through the cell membrane to reach the outside of the Golgi apparatus, and the optogenetic operation is realized.
In this example, UP L SAPO 100XS from Olympus was selected as the focusing objective lens 9.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and are not limited. Although the present invention has been described in detail with reference to the embodiments, those skilled in the art will understand that modifications and equivalent substitutions can be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, which should be covered by the spirit and scope of the present invention, and which should be covered by the scope of the claims of the present invention.
Claims (7)
1. A nanometer precision optogenetic control device comprises a first light source, a second light source, a third light source, a light detector, a quarter wave plate, a first reflecting mirror, a first dichroic mirror, a second dichroic mirror, a third dichroic mirror, a vibrating mirror system, a second reflecting mirror, a third reflecting mirror, a focusing objective lens and a sample stage;
the light source emits deactivation light, the deactivation light passes through the quarter-wave plate, is totally reflected by the reflecting mirror and then penetrates through the first dichroic mirror; the second light source emits active light, and after being reflected by the first dichroic mirror, the active light and the deactivated light emitted by the first light source are combined to form a combined light I; the light source III emits exciting light, the exciting light is reflected by the second dichroic mirror and then combined with the combined light to form a combined light II, the combined light II passes through the third dichroic mirror, then is totally reflected by the second reflecting mirror and the third reflecting mirror through the vibrating mirror system to enter the focusing objective lens, and finally forms a focusing light spot near the sample stage;
the galvanometer system controls the scanning of the focusing light spots in the direction of the sample stage;
a organelle sample is placed on the sample table;
the organelle sample is subjected to fluorescence calibration and genetic manipulation in advance; the genetic manipulation treatment is to express corresponding membrane protein on the surface of an organelle of the gene ChR2 photosensitive protein by introducing the gene ChR2 photosensitive protein;
the quarter-wave plate changes the polarization state of the deactivation light emitted by the first light source into circularly polarized light, a circular hollow focusing light spot is formed on a focusing plane of the focusing objective lens, and the inner diameter of the hollow focusing light spot is in a nanometer order;
the activation light can open the cation channel on the cell membrane, and the deactivation light closes the cation channel on the cell membrane; closing a cation channel in a superposition area of the activated light focusing spot and the hollow deactivated light focusing spot, opening the cation channel only in the hollow area of the deactivated light focusing spot, and compressing the area of the optogenetic operation to a nanometer level to realize the optogenetic operation under the size of the subcell;
the exciting light excites the fluorescent substance to emit fluorescence, and the deactivating light inhibits the excitation of the fluorescent substance; on the focusing plane, the fluorescent substance does not emit light in the superposed area of the exciting light focusing spot and the hollow deactivation light focusing spot, only emits light in the hollow area of the deactivation light focusing spot, and the size of the spot is smaller than that of the exciting light focusing spot, thereby reaching the nanometer level.
2. The nanometer-precision optogenetic manipulation device of claim 1, wherein: the light source I emits deactivation light with wavelength of 590 nanometers, pulse of 80 MHz, pulse width of more than or equal to 600 picoseconds and power of 250 megawatts, and can simultaneously close a cation channel opened by the activation light and fluorescence excited by the excitation light.
3. The nanometer-precision optogenetic manipulation device of claim 1, wherein: the wavelength of the activating light emitted by the light source II is 470 nanometers, the pulse is 80 MHz, the pulse width is less than or equal to 1 picosecond, and the power is 1 megawatt.
4. The nanometer-precision optogenetic manipulation device of claim 1, wherein: the wavelength of exciting light emitted by the light source III is 488 nanometers, the pulse is 80 MHz, the pulse width is less than or equal to 1 picosecond, and the power is 1 megawatt.
5. The nanometer-precision optogenetic manipulation device of claim 1, wherein: the first dichroic mirror is high in transmittance for de-activation light and high in reflectance for activation light; the second dichroic mirror is high in transmittance for de-activation light and high in reflectance for excitation light; the third dichroic mirror is high in transmittance for incident laser from the plane of incidence directions of the de-excitation light, the activation light and the excitation light, and high in reflectance for incident laser and fluorescence from the plane of the other incidence direction; the high transmittance refers to the transmittance of more than 98 percent; the high reflectivity means that the reflectivity is more than 98%, specifically 98-99.9%.
6. The nanometer-precision optogenetic manipulation device of claim 1, wherein: the galvanometer system, the quarter-wave plate and the focusing plane of the focusing objective form a conjugate plane, so that the laser transmission direction can be controlled at high precision, and the optogenetic operation is effectively ensured.
7. The nano-precision optogenetic manipulation device of claim 1, wherein the focusing objective is an immersion field achromatic objective, having a numerical aperture of 1.05 or more, and having high light-receiving capacity and spatial resolution.
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| CN201921954481.4U CN211086090U (en) | 2019-11-13 | 2019-11-13 | Nanometer precision optogenetic control device |
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| CN201921954481.4U CN211086090U (en) | 2019-11-13 | 2019-11-13 | Nanometer precision optogenetic control device |
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