CN215678048U - Super-resolution fluorescence microscopic imaging measurement system based on multiple modulation technology - Google Patents
Super-resolution fluorescence microscopic imaging measurement system based on multiple modulation technology Download PDFInfo
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- 238000001514 detection method Methods 0.000 claims description 25
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Abstract
A super-resolution fluorescence microscopic imaging measurement system based on multiple modulation technology belongs to the technical field of fluorescence microscopic imaging, and the multiple modulation technology is introduced into the super-resolution fluorescence microscopic imaging system, so that the limit of the original optical far-field diffraction limit on the limit resolution of an optical system is broken, the limit of the optical resolution is easily exceeded with the help of fluorescent molecules, and the nanoscale resolution is achieved. The method can reduce continuous light consumption and the acting time of the sample in the system, does not change the intensity of the light consumption, can ensure and improve the spatial resolution of the system, simultaneously reduces phototoxicity caused by the light consumption, realizes super-resolution fluorescence microscopic imaging and widens the application field of the super-resolution fluorescence microscopic imaging.
Description
Technical Field
The utility model belongs to the technical field of fluorescence microscopic imaging, and particularly relates to a super-resolution fluorescence microscopic imaging measurement system based on a multiple modulation technology.
Background
In the research of life science, physics and material science, an optical microscope has been regarded as an intuitive detection means, and becomes an indispensable part for the research of living cells and organisms. The structure of proteins in mitochondria of animal cells in the field of life science has been resolved at present, but the protein localization, protein dynamic behavior and electron transfer process have not been studied by technical means. The traditional microscopic imaging technology obviously cannot meet the research of the problem, so that the spatial resolution of the microscopic imaging is required to be improved to obtain clearer images so as to accurately position the protein.
In 1994 hel and Wichmann proposed stimulated emission depletion microscopy and for the first time "broken through" the diffraction limit of far-field optical microscopy. This technique requires two light sources, excitation light and loss light. The former irradiates a fluorescence-labeled sample to make it emit light; the light-emitting device forms a hollow bread aperture which is used for quenching exciting light to form excited state fluorescent molecules around a light spot, so that the effective fluorescent light-emitting area is reduced, and a light-emitting point smaller than the diffraction limit is obtained. The resolution of the stimulated emission depletion microscopy imaging system is directly determined by the size of the fluorescent spot focused on the excitation. The type of a laser light source mainly depends on in the prior art is that pulsed light is used as exciting light; the continuous light acts as the dissipated light. However, continuous light acts as dissipated light, and the sample is easy to damage on the research sample after long-time action, so that phototoxicity is induced. Although the decrease of the intensity of the continuous light can reduce the phototoxicity, the quenching degree of excited state phosphor particles around the excitation light is also reduced, and the spatial resolution of the system is affected.
Disclosure of Invention
The utility model aims to provide a super-resolution fluorescence microscopic imaging measurement system for reducing continuous phototoxicity. Continuous light in the super-resolution fluorescence microscope is simultaneously modulated by the acousto-optic modulator and the chopper, so that the action time of the continuous light and a research object is reduced while the continuous light can better quench excited state fluorescent molecules around exciting light. By using the method, the intensity of the continuous light can be even further improved, and the fluorescent molecules can be further quenched without damaging a research object. In the system, the continuous laser in the system is modulated by the acousto-optic modulator and the chopper together, so that the time of the continuous laser acting on a sample is shortened, and the phototoxicity of the continuous laser acting on the sample is reduced. The acousto-optic modulator and the chopper are synchronized with the femtosecond pulses output by the femtosecond laser together through a synchronous modulation system. When the system is applied to a dissipative light path in a super-resolution fluorescence system, the time of the dissipative light acting on a sample can be shortened, and the phototoxicity of the dissipative light is reduced. The total intensity of the thus modulated dissipated light is only 25% of the original. While the intensity of the dissipated light does not change on the time scale of quenching fluorescence.
The laser detection device comprises a laser generating and adjusting part I, a detection part II and a receiving part III, wherein the laser generating and adjusting part I, the detection part II and the receiving part III are positioned on an optical platform at the same height; the laser generating and adjusting part I, the detecting part II and the receiving part III are sequentially arranged from right to left.
The laser generating and adjusting part I consists of a femtosecond laser 1, a total reflection mirror I2, a solid laser group 3, a lambda/4 wave plate 4, a lambda/2 wave plate 5, a vortex phase plate 6, an acoustic-optical modulator 7, a frequency doubling bandwidth compressor, a picosecond optical parameter amplifier 8, a total reflection mirror II 9 and a dichroic mirror I10, wherein the femtosecond laser 1 is provided with a port a, and the acoustic-optical modulator 7 is provided with a port b; the solid laser group 3 consists of a 532nm solid laser, a 561nm solid laser and a 647nm solid laser; the femtosecond laser 1, the solid laser group 3, the frequency doubling bandwidth compressor and the picosecond optical parametric amplifier 8 are arranged from left to right; the solid laser group 3, the lambda/4 wave plate 4, the lambda/2 wave plate 5, the vortex phase plate 6, the acousto-optic modulator 7 and the dichroic mirror I10 are sequentially arranged from front to back and are positioned on the same horizontal line; the total reflection mirror I2, the frequency doubling bandwidth compressor, the picosecond optical parametric amplifier 8 and the total reflection mirror II 9 are sequentially arranged from front to back and are positioned on the same horizontal line; the port a of the femtosecond laser 1 is connected with the port b of the acousto-optic modulator 7.
Laser generated by the femtosecond laser 1 is projected through a dichroic mirror I10 through a frequency doubling bandwidth compressor, a picosecond optical parametric amplifier 8 and a total reflection mirror II 9 in sequence after being reflected by the total reflection mirror I2; laser generated by the solid laser group 3 is refracted by the lambda/4 wave plate 4, the lambda/2 wave plate 5, the vortex phase plate 6, the acousto-optic modulator 7 and the dichroic mirror I10.
The detection part II consists of a vibrating mirror 11, a dichroic mirror II 12, a reflective objective lens 13 and a sample stage 14, wherein the vibrating mirror 11, the dichroic mirror II 12, the reflective objective lens 13 and the sample stage 14 are sequentially arranged from back to front and are positioned on the same horizontal line.
The receiving part III consists of a computer 15, a signal integrator 16, a photomultiplier 17, a spectrometer 18 and a holophote III 19, wherein the computer 15 is provided with a port c and a port d; the signal integrator 16 is provided with a port e and a port f; the photomultiplier 17 is provided with a port g; the spectrometer 18 is provided with a port h; the signal integrator 16 and the spectrometer 18 are arranged in the left and right directions and are positioned in front of the computer 15; the photomultiplier 17 is fixedly connected in front of the spectrometer 18; the total reflection mirror III 19 is arranged between the computer 15 and the spectrometer 18, and the received incident light is reflected to the spectrometer 18 through the total reflection mirror III 19; the port h of the spectrometer 18 is connected with the port c of the computer 15; the port e of the signal integrator 16 is connected to the port d of the computer 15; the port g of the photomultiplier 17 is connected to the port f of the signal integrator 16.
The femtosecond laser generated by the femtosecond laser 1 in the laser generating and adjusting part I passes through the total reflection mirror I2, the frequency doubling bandwidth compressor, the picosecond optical parameter amplifier 8 and the total reflection mirror II 9 and enters the detection part II through the dichroic mirror I10; laser generated by the solid laser group 3 in the laser generating and adjusting part I passes through the lambda/4 wave plate 4, the lambda/2 wave plate 5, the vortex phase plate 6 and the acoustic-optical modulator 7, and then is reflected by the dichroic mirror I10 to enter the detection part II; the laser entering the detection part II is reflected by the vibrating mirror 11, transmitted by the dichroic mirror II 12, converged on the sample stage 14 by the reflective objective lens 13, reflected by the reflective objective lens 13 and the dichroic mirror II 12 to access the image on the sample stage 14 to the receiving part III, and then introduced into the spectrometer 18 by the reflecting mirror 19 in the receiving part III, converted into an electric signal by the photomultiplier 17, and received by the signal integrator 16 and the computer 15.
The utility model introduces multiple modulation technology into the super-resolution fluorescence microscopic imaging system, can reduce continuous light consumption and action time of a sample in the system, does not change the intensity of the light consumption, can ensure and improve the spatial resolution of the system, simultaneously reduces phototoxicity caused by the light consumption and the light scattering, realizes super-resolution fluorescence microscopic imaging and widens the application field of the super-resolution fluorescence microscopic imaging.
Drawings
FIG. 1 is a schematic structural diagram of a super-resolution fluorescence microscopic imaging measurement system based on a multiple modulation technology;
FIG. 2 is a schematic view of the structure of a laser generating and adjusting section I;
FIG. 3 is a schematic structural view of a detection section II;
FIG. 4 is a schematic view of the structure of a receiving section III;
FIG. 5 is a system synchronization timing diagram;
wherein: the laser detector comprises a laser generating and adjusting part I, a laser generating and adjusting part II, a detection part III, a receiving part 1, a femtosecond laser 2, a total reflection mirror I3, a solid laser group 4, a lambda/4 wave plate 5, a lambda/2 wave plate 6, a vortex phase plate 7, an acoustic optical modulator 8, a frequency multiplication bandwidth compressor, a picosecond optical parametric amplifier 9, a total reflection mirror II 10, a dichroic mirror I11, a vibrating mirror 12, a dichroic mirror II 13, a reflective objective lens 14, a sample stage 15, a computer 16, a signal integrator 17, a photomultiplier 18, a spectrometer 19 and a total reflection mirror III.
Detailed Description
The utility model is described below with reference to the drawings.
As shown in FIG. 1, the present invention comprises a laser generating and adjusting part I, a detecting part II and a receiving part III, wherein the laser generating and adjusting part I, the detecting part II and the receiving part III are positioned on an optical platform at the same height; the laser generating and adjusting part I, the detecting part II and the receiving part III are sequentially arranged from right to left; the femtosecond laser generated by the femtosecond laser 1 in the laser generating and adjusting part I passes through the total reflection mirror I2, the frequency doubling bandwidth compressor, the picosecond optical parameter amplifier 8 and the total reflection mirror II 9 and enters the detection part II through the dichroic mirror I10; laser generated by the solid laser group 3 in the laser generating and adjusting part I passes through the lambda/4 wave plate 4, the lambda/2 wave plate 5, the vortex phase plate 6 and the acoustic-optical modulator 7, and then is reflected by the dichroic mirror I10 to enter the detection part II; the laser entering the detection part II is reflected by the vibrating mirror 11, transmitted by the dichroic mirror II 12, converged on the sample stage 14 by the reflective objective lens 13, reflected by the reflective objective lens 13 and the dichroic mirror II 12 to access the image on the sample stage 14 to the receiving part III, and then introduced into the spectrometer 18 by the reflecting mirror 19 in the receiving part III, converted into an electric signal by the photomultiplier 17, and received by the signal integrator 16 and the computer 15.
As shown in fig. 2, the laser generating and adjusting part i comprises a femtosecond laser 1, a total reflection mirror i 2, a solid laser group 3, a lambda/4 wave plate 4, a lambda/2 wave plate 5, a vortex phase plate 6, an acousto-optic modulator 7, a frequency doubling bandwidth compressor, a picosecond optical parametric amplifier 8, a total reflection mirror ii 9 and a dichroic mirror i 10, wherein the femtosecond laser 1 is provided with a port a, and the acousto-optic modulator 7 is provided with a port b; the solid laser group 3 consists of a 532nm solid laser, a 561nm solid laser and a 647nm solid laser; the femtosecond laser 1, the solid laser group 3, the frequency doubling bandwidth compressor and the picosecond optical parametric amplifier 8 are arranged from left to right; the solid laser group 3, the lambda/4 wave plate 4, the lambda/2 wave plate 5, the vortex phase plate 6, the acousto-optic modulator 7 and the dichroic mirror I10 are sequentially arranged from front to back and are positioned on the same horizontal line; the total reflection mirror I2, the frequency doubling bandwidth compressor, the picosecond optical parametric amplifier 8 and the total reflection mirror II 9 are sequentially arranged from front to back and are positioned on the same horizontal line; the port a of the femtosecond laser 1 is connected with the port b of the acousto-optic modulator 7; laser generated by the femtosecond laser 1 is projected through a dichroic mirror I10 through a frequency doubling bandwidth compressor, a picosecond optical parametric amplifier 8 and a total reflection mirror II 9 in sequence after being reflected by the total reflection mirror I2; laser generated by the solid laser group 3 is refracted by the lambda/4 wave plate 4, the lambda/2 wave plate 5, the vortex phase plate 6, the acousto-optic modulator 7 and the dichroic mirror I10.
As shown in fig. 3, the detecting part ii is composed of a vibrating mirror 11, a dichroic mirror ii 12, a reflective objective 13 and a sample stage 14, wherein the vibrating mirror 11, the dichroic mirror ii 12, the reflective objective 13 and the sample stage 14 are sequentially arranged from back to front and are located on the same horizontal line.
As shown in fig. 4, the receiving part iii is composed of a computer 15, a signal integrator 16, a photomultiplier 17, a spectrometer 18 and a holophote iii 19, wherein the computer 15 is provided with a port c and a port d; the signal integrator 16 is provided with a port e and a port f; the photomultiplier 17 is provided with a port g; the spectrometer 18 is provided with a port h; the signal integrator 16 and the spectrometer 18 are arranged in the left and right directions and are positioned in front of the computer 15; the photomultiplier 17 is fixedly connected in front of the spectrometer 18; the total reflection mirror III 19 is arranged between the computer 15 and the spectrometer 18, and the received incident light is reflected to the spectrometer 18 through the total reflection mirror III 19; the port h of the spectrometer 18 is connected with the port c of the computer 15; the port e of the signal integrator 16 is connected to the port d of the computer 15; the port g of the photomultiplier 17 is connected to the port f of the signal integrator 16.
The laser generating and adjusting part I is connected with the detecting part II through a dichroic mirror 10; the detection part II is connected with the receiving part III through a dichroic mirror 12. The vibrating mirror 11, the dichroic mirror 12, the reflective objective 13 and the sample stage 14 in the detection part II are positioned right left of the laser generation and regulation part I; the mirror 19, the spectrometer 18, the photomultiplier 17, the signal integrator 16 and the computer 15 of the receiving section iii are located directly to the left of the detection section ii.
Respectively connecting by using special cables: the port a of the femtosecond laser 1 is connected with the port of the acousto-optic modulator 7; the port h of the spectrometer 18 is connected with the port c of the computer 15; the port e of the signal integrator 16 is connected with the port d of the computer 15; port g of the photomultiplier 17 is connected to port f of the signal integrator.
Preliminarily adjusting the center height of each optical device: the femtosecond laser 1, the total reflection mirror I2, the solid laser group 532/561/647nm3, the lambda/4 wave plate 4, the lambda/2 wave plate 5, the vortex phase plate 6, the acousto-optic modulator 7, the frequency doubling bandwidth compressor, the picosecond optical parametric amplifier 8, the total reflection mirror II 9 and the dichroic mirror 10 are positioned on the same horizontal central line; the galvanometer 11, the dichroic mirror 12, the reflective objective 13 and the sample stage 14 are positioned on the same horizontal central line; the computer 15, the signal integrator 16, the photomultiplier 17, the spectrometer 18 and the holomirror 19 are located on the same horizontal centerline. Accurately adjusting the multi-dimensional positions of the centers of the optical devices: the positions of each lens, the galvanometer 11 and the reflective objective 13 in the detection part II are adjusted, and stable fluorescence is formed in the detection part. Fine tuning the height, left-right and front-back position, tilt angle and pitch of all the equipment and the frame ensures that spectra with evenly distributed intensity values in the vertical and horizontal directions occur.
Example (b):
as shown in fig. 5, continuous light (dissipated light in the system) is first modulated to mhz by the acousto-optic modulator, and the continuous light (dissipated light) and femtosecond excitation light are simultaneously irradiated onto the sample through the time sequence control system, so as to obtain a super-resolution fluorescence signal. Then, the chopper is used for chopping the frequency of 1 KHz, and the continuous light adjusted by the acousto-optic modulator is secondarily modulated. Further reducing the time of application of the continuous light to the sample. Under the combined action of the acousto-optic modulator and the chopper, the action time of continuous light acting on a sample can be reduced by 75 percent under the condition of ensuring that the intensity of the continuous light is not changed, and the phototoxicity is further reduced.
Claims (1)
1. A super-resolution fluorescence microscopic imaging measurement system based on multiple modulation technology is characterized in that: the laser detection device comprises a laser generating and adjusting part (I), a detection part (II) and a receiving part (III), wherein the laser generating and adjusting part (I), the detection part (II) and the receiving part (III) are positioned on an optical platform at the same height; the laser generating and adjusting part (I), the detecting part (II) and the receiving part (III) are arranged in sequence from right to left; the laser generating and adjusting part (I) consists of a femtosecond laser (1), a total reflection mirror I (2), a solid laser group (3), a lambda/4 wave plate (4), a lambda/2 wave plate (5), a vortex phase plate (6), an acousto-optic modulator (7), a frequency doubling bandwidth compressor, a picosecond optical parametric amplifier (8), a total reflection mirror II (9) and a dichroic mirror I (10), wherein the femtosecond laser (1) is provided with a port a, and the acousto-optic modulator (7) is provided with a port b; the solid laser group (3) consists of a 532nm solid laser, a 561nm solid laser and a 647nm solid laser; the femtosecond laser (1), the solid laser group (3), the frequency doubling bandwidth compressor and the picosecond optical parametric amplifier (8) are arranged from left to right; the solid laser group (3), the lambda/4 wave plate (4), the lambda/2 wave plate (5), the vortex phase plate (6), the acousto-optic modulator (7) and the dichroic mirror I (10) are sequentially arranged from front to back and are positioned on the same horizontal line; the total reflection mirror I (2), the frequency doubling bandwidth compressor, the picosecond optical parametric amplifier (8) and the total reflection mirror II (9) are sequentially arranged from front to back and are positioned on the same horizontal line; the port a of the femtosecond laser (1) is connected with the port b of the acousto-optic modulator (7); laser generated by the femtosecond laser (1) is projected through the dichroic mirror I (10) through the light reflected by the total reflection mirror I (2) sequentially by the frequency doubling bandwidth compressor, the picosecond optical parametric amplifier (8) and the total reflection mirror II (9); laser generated by the solid laser group (3) is refracted through a lambda/4 wave plate (4), a lambda/2 wave plate (5), a vortex phase plate (6), an acousto-optic modulator (7) and a dichroic mirror I (10); the detection part (II) consists of a vibrating mirror (11), a dichroic mirror II (12), a reflective objective lens (13) and a sample stage (14), wherein the vibrating mirror (11), the dichroic mirror II (12), the reflective objective lens (13) and the sample stage (14) are sequentially arranged from back to front and are positioned on the same horizontal line; the receiving part (III) consists of a computer (15), a signal integrator (16), a photomultiplier (17), a spectrometer (18) and a total reflection mirror III (19), wherein the computer (15) is provided with a port c and a port d; the signal integrator (16) is provided with a port e and a port f; a port g is arranged on the photomultiplier (17); the spectrometer (18) is provided with a port h; the signal integrator (16) and the spectrometer (18) are arranged at the left and the right and are positioned in front of the computer (15); the photomultiplier (17) is fixedly connected in front of the spectrometer (18); the total reflection mirror III (19) is arranged between the computer (15) and the spectrometer (18), and the received incident light is reflected to the spectrometer (18) through the total reflection mirror III (19); the port h of the spectrometer (18) is connected with the port c of the computer (15); the port e of the signal integrator (16) is connected with the port d of the computer (15); the port g of the photomultiplier (17) is connected with the port f of the signal integrator (16); the femtosecond laser generated by the femtosecond laser (1) in the laser generating and adjusting part (I) passes through the holophote I (2), the frequency doubling bandwidth compressor, the picosecond optical parametric amplifier (8) and the holophote II (9) and enters the detection part (II) through the dichroic mirror I (10); laser generated by a solid laser group (3) in the laser generating and adjusting part (I) passes through a lambda/4 wave plate (4), a lambda/2 wave plate (5), a vortex phase plate (6) and an acousto-optic modulator (7) and then is reflected by a dichroic mirror I (10) to enter a detection part (II); laser entering the detection part (II) is reflected by a vibrating mirror (11), transmitted by a dichroic mirror II (12), converged on a sample stage (14) by a reflective objective lens (13), reflected by the reflective objective lens (13) and the dichroic mirror II (12) to connect an image on the sample stage (14) into a receiving part (III), introduced into a spectrometer (18) by a reflecting mirror (19) in the receiving part (III), converted into an electric signal by a photomultiplier (17), and received by a signal integrator (16) and a computer (15).
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