CN110836876B - Super-resolution microscopy method and system based on saturated pumping-stimulated radiation detection - Google Patents
Super-resolution microscopy method and system based on saturated pumping-stimulated radiation detection Download PDFInfo
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Abstract
The invention discloses a super-resolution microscopic method and a system based on saturated pump-stimulated radiation detection, belonging to the field of super-resolution microscopic imaging and nonlinear optics, wherein the system comprises a light source module, a detection module and a processing module, a sample is placed between the light source module and the detection module, and the light source module comprises a first light source for emitting exciting light, a second light source for emitting reading light, a modulation device, a dichroic mirror and a scanning galvanometer; the detection module comprises a photoelectric detector; the photoelectric detector is connected with the phase-locked amplifier, and the phase-locked amplifier is in communication connection with the processing module. On the basis of confocal microscopy, through introducing saturated exciting light and carrying out time domain restriction to the exciting light, make the sample can twine fluorescence higher harmonic signal, simultaneously, read laser through introducing another bundle of, make the fluorescence that originally will send launch with the form of stimulated emission light, survey demodulation stimulated emission light higher harmonic signal through the phase-locked at last, can obtain final imaging result.
Description
Technical Field
The invention relates to the field of super-resolution microscopic imaging and nonlinear optics, in particular to a super-resolution microscopic method and system based on saturated pumping-stimulated radiation detection.
Background
Optical microscopy allows in vivo imaging of biological tissues and cells, which has the advantage that electron microscopy, etc., is incomparable with some other methods. Although the imaging resolution cannot reach the level of electron microscopy, the optical microscopy can observe tissues and cells of a living body on the premise of not causing serious damage to a biological sample, so that various biological phenomena are presented visually. In various optical microscopy methods, confocal fluorescence microscopy can improve the axial slicing capability and the transverse resolution of the traditional far-field optical microscopy, and meanwhile, specific imaging of various components or structures in tissues and cells, such as gene expression, change of different ion concentrations, change of cell membrane potential and the like, can be realized by means of various specific fluorescent probes.
However, confocal microscopy has limited improvement in lateral resolution due to the effects of diffraction limit. Therefore, in order to overcome the limit of diffraction limit, various fluorescence nano-microscopy (super-resolution microscopy) has been proposed and widely used in recent years, and among them, the saturation phenomenon in the optical effect has been attracting much attention and application. Fluorescence saturation can introduce strong nonlinear effect between the excitation and the radiation of fluorescence, and then the resolution of the microscope system is effectively improved. Based on this principle, stimulated emission depletion (STED) microscopy suppresses fluorescence radiation in a partial region by means of a saturation effect, and reduces the effective light-emitting area of the fluorescence radiation, thereby improving the resolution capability of the system. To date, STED has been applied to a variety of fluorescent dyes and fluorescent proteins. However, since another laser beam with very high power is required to be introduced to suppress the fluorescence radiation, bleaching of the fluorescent dye and destruction of the biological sample are caused, and the system structure for realizing the STED is also complicated, the STED has many limitations in practical application. In addition, saturation excitation (SAX) fluorescence microscopy is another super-resolution microscopy based on the saturation effect of fluorescence. By combining the time-domain modulation of the saturated excitation and the excitation light, higher-order harmonic signals emitted by the sample can be demodulated by means of phase-locked detection, and the higher-order harmonic signals carry higher-frequency spatial information of the sample, so that an imaging result with higher resolution can be obtained. The laser power required by the method is obviously lower than that of STED, the system structure is relatively simple, but because the intensity of the signal is weakened along with the increase of the order of the harmonic signal, the existing method can only detect the second-order (a few parts to three orders) signal of the sample, and the method is limited to carry out higher-resolution microscopic imaging.
Disclosure of Invention
The invention aims to provide a super-resolution microscopic method based on saturated pump-stimulated radiation detection, which can obtain higher-resolution microscopic imaging by virtue of a saturation effect in nonlinear optics.
It is another object of the present invention to provide a super-resolution microscopy system based on saturated pump-stimulated emission detection, which can be used to implement the above super-resolution microscopy method.
In order to achieve the above object, the super-resolution microscopy method based on saturated pump-stimulated emission detection provided by the invention comprises the following steps:
1) the first light source emits light with a wavelength of lambda1The excitation light is time-domain modulated to load a time frequency omega1；
2) The second light source emits light with a wavelength of lambda2The reading light of (1);
3) combining the excitation light with the time frequency and the reading light to form combined light;
4) scanning a sample by using beam combining light to enable the sample to emit stimulated radiation signal light;
5) the stimulated radiation signal light enters the photoelectric detector after being collected and is converted into an electric signal;
6) demodulating the electrical signal to have a frequency k · ω1And processing the demodulated signal components to obtain the super-resolution image.
Among the above-mentioned technical scheme, on confocal microscopy's basis, through introducing saturated exciting light and carrying out the time domain restriction to the exciting light, make the sample can produce fluorescence higher harmonic signal, simultaneously, through introducing another bundle of reading laser, make the fluorescence that originally will send launch with the form of stimulated emission light, survey through the phase-locked and demodulate stimulated emission light higher harmonic signal at last, can obtain final imaging result.
In the invention, because the detected higher harmonic signals are transmitted by the sample and carry the high-frequency spatial information of the sample, the resolution ratio can be further improved on the basis of the traditional confocal microscopy, and the super-resolution imaging is realized. Meanwhile, due to the fact that another beam of reading laser is introduced, the original fluorescent signal can be converted into the stimulated radiation signal, the intensity of stimulated radiation is obviously higher than the fluorescent light intensity, the signal intensity of the system is effectively improved, higher-order harmonic signals can be obtained, and therefore imaging quality is improved and higher imaging resolution is achieved.
The preferable scheme is that in the step 1), the electro-optical modulator (EOM) is used for time-domain modulation of the exciting light, so that the exciting light is loaded with the time frequency omega1. The method comprises the steps of firstly, using a beam-reducing mirror with proper multiplying power to reduce exciting light to enable the size of a light spot to meet the incident requirement of an electro-optic modulator, then rotating the electro-optic modulator and the direction of a polarization fractional prism positioned in front of the electro-optic modulator to enable the directions of a fast axis and a slow axis of the electro-optic modulator to be matched, and finally using the beam-expanding mirror with corresponding multiplying power to restore the size of the light spot to the initial size.
Another preferred scheme is that in step 1), the excitation light is modulated in time domain by using an acousto-optic modulator (AOM), so that the excitation light is loaded with a time frequency ω1. Firstly, a polarization beam splitter prism positioned in front of an acousto-optic modulator is adjusted to enable the polarization direction of linearly polarized light to be the specific direction required by the acousto-optic modulator, then a beam shrinking mirror with proper multiplying power is used for shrinking exciting light to enable the size of a light spot to meet the incidence requirement of the acousto-optic modulator, and finally the beam expanding mirror with corresponding multiplying power is used for restoring the size of the light spot to the original size.
Another preferred embodiment is the time frequency ω in step 1)1The frequency range is 10 kHz-100 kHz, which not only accords with the modulation frequency range which can be realized by the current modulator, but also can avoid the influence of low-frequency background noise under the condition of low frequency; and k in the step 6) is a positive integer larger than 1.
Still another preferred solution is to demodulate the electrical signal in step 6) with a lock-in amplifier.
In order to achieve the other object, the invention provides a method based on saturated pump-stimulated radiationThe super-resolution microscope system comprises a light source module, a detection module and a processing module, wherein a sample is placed between the light source module and the detection module, the light source module comprises a first light source for emitting exciting light, a second light source for emitting reading light, a first light source for modulating the exciting light in time domain and loading an upper time frequency omega1The modulating device, the dichroic mirror which combines the exciting light and the reading light beam and the scanning galvanometer which controls the light beam to scan the sample; the detection module comprises a photoelectric detector for detecting optical signals emitted by the sample; the photoelectric detector is connected with a phase-locked amplifier for demodulating the frequency k.omega from the electric signal of the photoelectric detector1The signal component of (a); and the processing module processes the demodulated signal components to obtain a super-resolution image.
And focusing the combined excitation light and reading light on a sample, and scanning the sample by controlling a scanning galvanometer to obtain stimulated radiation signal light emitted by the sample. The signal light is collected and filtered and then sent to a photoelectric detector, then is converted into an electric signal and then is input to a phase-locked amplifier, and the phase-locked amplifier demodulates the electric signal to obtain the signal light with the frequency of k omega1And (k is a positive integer greater than 1), and then the signals are processed by the processing module to form a result image. Finally, the limit of diffraction limit in optical microscopy is overcome, and super-resolution fluorescence microscopic imaging is realized.
Preferably, the electro-optical modulator is connected to a signal generator for generating a square wave signal, which signal generator simultaneously generates the external reference signal required by the lock-in amplifier. The same signal generator generates the frequency k.omega1And the external reference signal is input into the phase-locked amplifier to demodulate a signal with a corresponding frequency.
Preferably, a first collimating lens, a first polarization splitting prism and a beam shrinking mirror are sequentially arranged between the first light source and the modulation device along a light path, and the beam expanding mirror is arranged between the modulation device and the dichroic mirror; and a second collimating lens, a second polarization splitting prism, an 1/2 wave plate, a 1/4 wave plate and a reflecting mirror for reflecting the light beams onto the dichroic mirror are sequentially arranged between the second light source and the dichroic mirror along a light path.
The first light source and the second light source are both lasers, so that the fluorescent sample is excited, and the original fluorescent signal light is converted into stimulated radiation light signal light. The first collimating lens and the second collimating lens are used for collimating laser light emitted by the laser. The first polarization beam splitter prism and the second polarization beam splitter prism are used for adjusting the polarization state of the laser to be linearly polarized light. 1/2 wave plate and 1/4 wave plate are used to polarization modulate the laser light to be circularly polarized light at the entrance pupil of the objective lens. The reflector is combined with the dichroic mirror to combine two laser beams with different wavelengths.
Preferably, a scanning lens, a field lens and an illumination objective lens are sequentially arranged between the scanning galvanometer and the sample; and a collection objective lens and an optical filter are sequentially arranged between the sample and the photoelectric detector. The scanning galvanometer comprises two galvanometers which are respectively used for scanning light beams in the x direction and the y direction; the scanning mirror and the field lens are used for laser scanning imaging; the illumination objective lens is used for focusing the laser to illuminate the sample for imaging; the collection objective lens is used for collecting stimulated emission light emitted by the sample; the filter is used before the photodetector to filter out noise light other than the stimulated emission light.
Preferably, the modulation device is an electro-optical modulator, and the excitation light is subjected to time domain modulation and then loaded with a certain time frequency omega1。
Preferably, the modulation device is an acousto-optic modulator, and the exciting light is subjected to time domain modulation and then loaded with a certain time frequency omega1。
Compared with the prior art, the invention has the beneficial effects that:
(1) compared with the current SAX and STED super-resolution microscopy, the invention converts the fluorescence signal into the stimulated radiation light by introducing the second beam of reading laser, effectively improves the signal intensity, gets rid of the limitation of weak signal intensity when detecting higher harmonic signals, can further improve the imaging resolution and improve the imaging quality, does not need to use high-power laser to inhibit fluorescence, effectively lightens the photobleaching and phototoxic action on a sample, and can be applied to the field of super-resolution optical microscopy imaging.
(2) Compared with the current confocal microscopy, the super-resolution imaging is realized through saturation excitation and phase-locked detection; stimulated radiation light is used as signal light, so that the imaging signal-to-noise ratio of the microscope system is effectively improved; the requirements on the photoelectric detector and the scanning dwell time are reduced, the system cost is reduced, and the imaging speed is increased.
Drawings
FIG. 1 is a schematic view of a super-resolution microscope system according to example 1 of the present invention;
FIG. 2 is a graph showing the relationship between the excitation light intensity and the radiation light intensity of rhodamine 6G (a commonly used fluorescent dye in microscopy), wherein the graph gradually changes from linear to nonlinear with the increase of the excitation light intensity;
FIG. 3 is a schematic view of a super-resolution microscope apparatus according to example 2 of the present invention;
FIG. 4 is a schematic contrast diagram of an embodiment of the present invention, wherein the solid line is the point spread function curve of the microscope system under unsaturated excitation; the dotted line is a point spread function curve of the microscope system under saturation excitation;
fig. 5 is a schematic diagram of the triplet energy level system and absorption, spontaneous emission and stimulated emission of fluorescent molecules.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described with reference to the following embodiments and accompanying drawings.
Example 1
As shown in FIG. 1, the super-resolution microscope system based on the saturated pump-stimulated emission detection with the time-domain modulation by the electro-optical modulator of the present embodiment includes a laser (wavelength λ `)1)1, laser 2 (wavelength λ)2) A first collimating lens 3, a second collimating lens 4, a first polarization beam splitter prism 5, a second polarization beam splitter prism 6, a beam reducer 7, an electro-optical modulator 8, a beam expander 9, an 1/2 wave plate 10, a 1/4 wave plate 11, a 1/4 wave plate 12, a reflector 13, a dichroic mirror 14, a scanning galvanometer 15, a scanning mirror 16, a field lens 17, an illumination objective lens 18, a sample 19, a collection objective lens 20, an optical filter 21, a photoelectric detector 22, a signal generator 23, a lock-in amplifier 24, a collection card and a computer 25.
The system shown in fig. 1 is adopted to realize super-resolution imaging based on saturated pump-stimulated emission detection, and the process is as follows:
(1) the laser 1 emits light with a wavelength lambda1Exciting light is collimated by a first collimating lens 3 and then converted into linearly polarized light by a first polarization splitting prism 5;
(2) the excitation light is contracted by a beam contraction mirror 7, then the angles of a polarization beam splitter prism 5 and an electro-optic modulator 8 are rotated to match the fast axis and the slow axis of the polarization beam splitter prism and the electro-optic modulator, a signal generator 23 generates a square wave signal with the frequency of 50kHz and inputs the square wave signal to the electro-optic modulator, at the moment, the time frequency of 50kHz is loaded after the excitation light is modulated by the electro-optic modulator, and the modulated excitation light is expanded to the size of a light spot after initial collimation by a beam expansion mirror 9;
(3) the exciting light passes through the dichroic mirror 14 and then passes through the 1/4 wave plate 12 for polarization modulation, and the angle of the wave plate is rotated to make the exciting light become circularly polarized light at the entrance pupil surface of the illumination objective lens 18;
(4) the laser 2 emits light with a wavelength lambda2The reading light is collimated by a second collimating lens 4 and then converted into linearly polarized light by a second polarization beam splitter prism 6;
(5) the reading light sequentially passes through 1/2 wave plate 11 and 1/4 wave plate 12, and the angles of the two wave plates are rotated to make the reading light become circularly polarized light at the entrance pupil surface of the illumination objective lens 18;
(6) the reading light is reflected by a reflecting mirror 13 and a dichroic mirror 14 in sequence, and is combined with the excitation light beam by adjusting the angles of the two mirrors;
(7) the two beams of laser after being combined pass through a scanning galvanometer 15, a scanning mirror 16 and a field lens 17, and are finally focused by an illumination objective lens 18 to realize the scanning of a sample 19, and the specific scanning range and the scanning speed are controlled by the scanning galvanometer 15;
(8) stimulated radiation light emitted by the sample 19 is collected by the collection objective lens 20 and filtered by the optical filter 21 and then enters the photoelectric detector 22;
(9) the photodetector 22 converts the obtained optical signal into an electrical signal and inputs the electrical signal into the lock-in amplifier 24, the lock-in amplifier demodulates a signal component with a frequency of 100kHz or 150kHz from the signal, and an external reference signal required by the lock-in amplifier is also generated by the signal generator 23;
(10) the demodulated signals are input into an acquisition card and a computer 25 to form an imaging result image.
Example 2
As shown in fig. 3, the present embodiment of the super-resolution microscope system based on the saturated pump-stimulated emission detection, which performs time-domain modulation by an acousto-optic modulator, has the same structure as that of embodiment 1 except that the modulation device is an electro-optic modulator 28. And will not be described in detail herein.
The system shown in fig. 3 is adopted to realize super-resolution imaging based on saturated pump-stimulated emission detection, and the process is as follows:
(1) the laser 1 emits light with a wavelength lambda1Exciting light is collimated by a first collimating lens 3 and then converted into linearly polarized light by a first polarization splitting prism 5;
(2) exciting light is contracted by a beam contraction mirror 7, then is subjected to acousto-optic modulator 28 to obtain multi-level diffraction spots, first-level diffraction light (the time frequency is 10kHz) is taken, an external modulation signal required by the electro-optic modulator is generated by a signal generator 23 and is input to the electro-optic modulator, and the modulated exciting light is expanded to the size of the spots after initial collimation through a beam expansion mirror 9;
(3) the exciting light passes through the dichroic mirror 14 and then passes through the 1/4 wave plate 12 for polarization modulation, and the angle of the wave plate is rotated to make the exciting light become circularly polarized light at the entrance pupil surface of the illumination objective lens 18;
(4) the laser 2 emits light with a wavelength lambda2The reading light is collimated by a second collimating lens 4 and then converted into linearly polarized light by a second polarization beam splitter prism 6;
(5) the reading light sequentially passes through 1/2 wave plate 11 and 1/4 wave plate 12, and the angles of the two wave plates are rotated to make the reading light become circularly polarized light at the entrance pupil surface of the illumination objective lens 18;
(6) the reading light is reflected by a reflecting mirror 13 and a dichroic mirror 14 in sequence, and is combined with the excitation light beam by adjusting the angles of the two mirrors;
(7) the two beams of laser after being combined pass through a scanning galvanometer 15, a scanning mirror 16 and a field lens 17, and are finally focused by an illumination objective lens 18 to realize the scanning of a sample 19, and the specific scanning range and the scanning speed are controlled by the scanning galvanometer 15;
(8) stimulated radiation light emitted by the sample 19 is collected by the collection objective lens 20 and filtered by the optical filter 21 and then enters the photoelectric detector 22;
(9) the photodetector 22 converts the obtained optical signal into an electrical signal and inputs the electrical signal into the lock-in amplifier 24, the lock-in amplifier demodulates a signal component with the frequency of 20kHz or 30kHz from the signal, and an external reference signal required by the lock-in amplifier is also generated by the signal generator 23;
(10) the demodulated signals are input into an acquisition card and a computer 25 to form an imaging result image.
Example 3
The embodiment provides a super-resolution microscopy method based on saturated pump-stimulated radiation detection, which comprises the following steps:
s1 the first light source emits light with wavelength of lambda1The exciting light of (2) is subjected to time domain modulation, so that the exciting light is loaded with a time frequency of 50 kHz; the modulation device is an electro-optic modulator or an acousto-optic modulator.
S2 second light source emitting light with wavelength of lambda2The reading light of (1).
S3 combines the excitation light having the temporal frequency and the readout light to form combined light.
S4 scans the sample with the combined beam light to cause the sample to emit stimulated emission signal light.
The stimulated emission signal light of S5 enters the photoelectric detector after being collected and is converted into an electric signal. And a backward detection mode is adopted, and the stimulated radiation signal light is collected by using a collection objective lens.
S6 demodulates the signal component with frequency of 100kHz or 150kHz from the electric signal, and the demodulated signal component is processed to obtain super-resolution image with improved signal intensity and resolution. The electrical signal may be demodulated using a lock-in amplifier.
The principle of the above embodiment is as follows:
based on the traditional confocal microscopy and the saturation effect in nonlinear optics, the limit of diffraction limit in optical microscopy is overcome, and super-resolution fluorescence microscopic imaging is realized. When the sample is excited in non-saturation, as shown in the first half of the curve in fig. 2, the radiation intensity of the sample and the excitation intensity are in a linear relationship, and the Optical Transfer Function (OTF) of the system is shown in the solid line in fig. 4, so that only the sample information in a limited frequency range can be obtained. When the saturation excitation is adopted, as shown in the second half of the curve in fig. 2, the radiation intensity gradually tends to a certain fixed intensity along with the increase of the excitation intensity, the introduction of the nonlinearity widens the passband of the microscope system, higher-frequency information of a sample can be obtained and extracted, and the corresponding OTF is shown as a dotted line in fig. 4, thereby realizing the improvement of the system resolution.
Meanwhile, because the intensity of the high-order signal is rapidly attenuated along with the increase of the signal order, in practical application, only the second-order harmonic signal can be effectively detected under normal conditions, and the higher-order signal is basically covered by various noises such as background noise, detector noise and the like, so that the actual resolution improvement effect is very limited. Therefore, the present invention introduces the second beam of laser light as the reading light to generate the stimulated emission light. As shown in fig. 5, after being excited by excitation light with a wavelength λ 1, fluorescent molecules will transition from a ground state to an excited state, and then return to the ground state by means of fluorescence radiation, if another laser beam with a wavelength λ 2 is introduced at the same time, the molecules in the excited state can emit excited radiation light with the same wavelength λ 2 in the form of excited radiation and return to the ground state, and the original fluorescent signal is converted into an excited radiation signal, and the intensity of the excited radiation light is much higher than that of the fluorescence, so that detection of a higher-order harmonic signal can be realized, and the resolution of the microscope system is effectively improved.
In addition, the invention has strong advantage in improving the signal-to-noise ratio due to the high signal intensity of the stimulated emission light. For conventional fluorescence detection, it is often necessary to cooperate with expensive detectors with high quantum efficiency and low noise to achieve better imaging effect, and meanwhile, in order to obtain sufficient signal intensity, the dwell time of a single point in scanning imaging is relatively long, which limits the overall imaging speed; in the invention, the requirements of the detection of the stimulated emission light on the photoelectric detector are relaxed, and the rapid low-cost and high-quality super-resolution microscopic imaging can be realized by matching with the shorter single-point retention time.
The invention can realize the super-resolution microscopic imaging with high signal-to-noise ratio, simultaneously improves the imaging speed of scanning imaging and reduces the system cost.
Claims (8)
1. A super-resolution microscopic method based on saturated pump-stimulated emission detection is characterized by comprising the following steps:
1) the first light source emits light with a wavelength of lambda1The excitation light is time-domain modulated to load a time frequency omega1(ii) a Time frequency omega110 kHz-100 kHz; enabling the sample to generate a fluorescence higher harmonic signal;
2) the second light source emits light with a wavelength of lambda2The original fluorescent signal is converted into a stimulated radiation signal and emitted;
3) combining the excitation light with the time frequency and the reading light to form combined light;
4) scanning a sample by using the beam combining light to enable the sample to emit stimulated radiation signal light;
5) the stimulated radiation signal light enters the photoelectric detector after being collected and is converted into an electric signal;
6) demodulating the frequency k.omega from the electric signal by means of phase-locked detection1The demodulated signal components are processed to obtain a super-resolution image, and k is a positive integer greater than 1.
2. The super-resolution microscopy method according to claim 1, wherein:
in the step 1), the electro-optical modulator is used for carrying out time domain modulation on the exciting light, so that the exciting light is loaded with a time frequency omega1。
3. The super-resolution microscopy method according to claim 1, wherein:
in the step 1), the time domain modulation is carried out on the exciting light by using an acousto-optic modulator, so that the exciting light is loaded with a time frequency omega1。
4. The super-resolution microscopy method according to claim 1, wherein:
and 6) demodulating the electric signal by using a phase-locked amplifier.
5. A super-resolution microscopic system based on a super-resolution microscopic method of saturated pump-stimulated emission detection comprises a light source module, a detection module and a processing module, wherein a sample is placed between the light source module and the detection module, and the super-resolution microscopic system is characterized in that:
the light source module comprises a first light source emitting exciting light, a second light source emitting reading light, a time domain modulation for the exciting light and an upper time frequency omega1The modulating device, the dichroic mirror which combines the exciting light and the reading light beam and the scanning galvanometer which controls the light beam to scan the sample; time frequency omega1The frequency is 10 kHz-100 kHz, so that a sample can generate a fluorescence higher harmonic signal; the wavelength of the reading light emitted by the second light source is lambda2Converting the original fluorescence signal into a stimulated radiation signal and emitting the stimulated radiation signal; a first collimating lens, a first polarization beam splitter prism and a beam shrinking mirror are sequentially arranged between the first light source and the modulation device along a light path, and a beam expanding mirror is arranged between the modulation device and the dichroic mirror;
a second collimating lens, a second polarization splitting prism, an 1/2 wave plate, a 1/4 wave plate and a reflecting mirror for reflecting light beams onto the dichroic mirror are sequentially arranged between the second light source and the dichroic mirror along a light path;
the detection module comprises a photoelectric detector for detecting optical signals emitted by the sample;
the photoelectric detector is connected with a phase-locked amplifier, and the frequency of the electric signal of the photoelectric detector is demodulated into k.omega in a phase-locked detection mode1The signal component of (a);
and the processing module processes the demodulated signal components to obtain a super-resolution image.
6. The super-resolution microscopy system of claim 5, wherein:
the modulation device is connected with a signal generator for generating square wave signals, and the signal generator simultaneously generates an external reference signal required by the phase-locked amplifier.
7. The super-resolution microscopy system according to any one of claims 5 to 6, wherein:
the modulation device is an electro-optical modulator, and the exciting light is subjected to time domain modulation and then loaded with a certain time frequency omega1。
8. The super-resolution microscopy system according to any one of claims 5 to 6, wherein:
the modulation device is an acousto-optic modulator, and the exciting light is subjected to time domain modulation and then loaded with a certain time frequency omega1。
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