CN110954523B - Two-photon scanning structure light microscopic imaging method and device - Google Patents
Two-photon scanning structure light microscopic imaging method and device Download PDFInfo
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
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- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0036—Scanning details, e.g. scanning stages
- G02B21/0048—Scanning details, e.g. scanning stages scanning mirrors, e.g. rotating or galvanomirrors, MEMS mirrors
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N2021/6463—Optics
Abstract
The invention provides a two-photon scanning structure light microscopic imaging method and a device, which are characterized in that laser is modulated into exciting light of which the light intensity periodically changes along with time according to a sine function, and a sample to be imaged is scanned by using the modulated exciting light to form scanning structure light excitation; collecting a fluorescence signal generated by scanning and exciting a sample to be imaged by exciting light to obtain a non-sinusoidal fluorescence structure light image corresponding to the fluorescence signal, and extracting a frequency component of the fluorescence structure light image; the super-resolution image of the sample to be imaged is obtained according to the superposition reconstruction of the frequency components of the fluorescence structure light images in different directions, the method of the embodiment can realize higher resolution imaging than a linear structure light two-photon super-resolution microscope without carrying out fluorescence saturation excitation and high-power accessory STED light, the resolution is improved by 3 times or even higher than the diffraction limit, and therefore the requirement of dozens of nanometers or even higher for two-photon fluorescence imaging can be met, and the resolution of the two-photon fluorescence image is improved.
Description
Technical Field
The invention relates to the technical field of optical microscopic imaging, in particular to a two-photon scanning structure optical microscopic imaging method and device.
Background
In recent years, with the rapid development of photoelectric device development technologies such as high-intensity laser and high-sensitivity detectors and related fields such as the development of novel fluorescent probes, the super-resolution microscopic imaging technology has achieved remarkable and enormous achievement, and the diffraction limit which is once considered to be insurmountable is no longer an obstacle to obtaining high-resolution information of biological samples. The super-resolution microscopic imaging technology changes the traditional optical microscopic imaging, breaks the diffraction limit of Abbe, and provides a unique platform for optical research of biological imaging nano-structure. Several methods are currently available to solve the diffraction limit well: photo-activated positioning microscope (PALM), stochastically optically reconstructed microscope (STORM), Stimulated emission depletion (STED) microscope, and Stimulated emission depletion (STED) and structured light illumination microscope (SIM) technologies. The resolution of the methods of PLAM, STORM and STED can reach 10nm, but in order to obtain super-resolution images, the methods of PLAM and STORM need to obtain thousands of raw images on average, the imaging speed is limited, the methods can only be used for fixing cells generally, and the real-time detection and imaging of living biological cells are still difficult to achieve up to now. The STED imaging speed depends on the scanning speed and can meet the requirement of live cell imaging, however, the STED optical power density is 4 to 6 orders of magnitude higher than the STORM optical power density, so that the phototoxicity and optical damage to the live cells are serious, and the high-power STED light can realize fluorescence erasure and simultaneously increase the photobleaching of fluorescent molecules, which also limits the application of STED in live cell imaging. The SIM belongs to wide-field microscopic imaging, does not need scanning, has high imaging speed, no special mark, low requirement on the power of exciting light (the laser power density of the SIM is 2-3 orders of magnitude lower than that of a STORM/PALM technology), and small photobleaching and photodamage degrees. SIM therefore has unique advantages in live cell imaging. The resolution of a linear SIM can be increased by a factor of 2 at most on the basis of the diffraction limit.
A scanning structure light microscopic technology (2P-SPIM) based on two photons utilizes femtosecond laser to generate a two-photon excitation effect on a sample, an intensity modulator is used for modulating the pulse peak value of the femtosecond laser to change along with time according to a sine function, the scanning of a galvanometer is combined, two-photon fluorescence with sine change of intensity is generated on the sample, then a linear structure light reconstruction algorithm is used for obtaining a super-resolution image, and the resolution ratio is improved by 2 times at most than the diffraction limit. Moreover, there are currently bifocals basedScanning structured light microscopy of sub-or control of the temporal modulation frequency omegatGreater than the system transfer function cutoff frequency
(ωtDepending on the EOM and scan speed, the aim is to bring the generated harmonics outside the effective two-photon transfer function); or the modulation pattern is controlled to be in a sine open form to avoid harmonic generation, but because the harmonic generation is associated with the resolution, the improvement of the resolution is limited while the harmonic generation is inhibited, so that the fluorescent light needs to meet the sine intensity distribution when the two-photon scanning structure light microscopy technology in the prior art realizes super-resolution, and the improvement of the resolution does not exceed 2 times of diffraction limit (namely the resolution of linear scanning structure light) at most, and the requirement of people on obtaining super-resolution images with tens of nanometers or even higher resolution cannot be met.
Therefore, the prior art is awaiting further improvement.
Disclosure of Invention
In view of the defects in the prior art, the invention provides a two-photon scanning structure light microscopic imaging method and a two-photon scanning structure light microscopic imaging device, which overcome the limitation that the resolution ratio of the existing two-photon scanning structure light is improved by no more than 2 times of diffraction limit at most, can not meet the requirement of people on acquisition of two-photon fluorescence super-resolution images with the resolution ratio of dozens of nanometers or even higher, and limit the application of two-photon microscopy.
In a first aspect, the present embodiment discloses a two-photon scanning structure optical microscopic imaging method, including:
modulating laser into exciting light with light intensity periodically changing according to a sine function along with time according to a preset modulation function, and scanning and exciting a sample to be imaged by using the modulated exciting light;
collecting a fluorescence signal generated by scanning and exciting a sample to be imaged by exciting light to obtain a fluorescence structure light image group corresponding to the fluorescence signal; wherein the fluorescent structured light image group comprises: a plurality of fluorescent structured light images; the orientation and the phase of the excitation light patterns corresponding to the fluorescence structure light images are different;
and extracting frequency components in each fluorescence structure light image, resetting and superposing the frequency components of which the corresponding exciting light patterns are in the same orientation and different phases, and reconstructing a super-resolution image of the sample to be imaged according to the superposed value group of the frequency components in each orientation.
Optionally, the step of acquiring a fluorescence signal generated by scanning and exciting the sample to be imaged by the excitation light to obtain a fluorescence structured light image group corresponding to the fluorescence signal includes:
and acquiring a fluorescence structure light image group corresponding to the excitation light patterns with different orientations and different phases by changing the period and the phase of the preset modulation function.
Optionally, the step of scanning and exciting the sample to be imaged by using the modulated laser includes:
and scanning the sample to be imaged point by point longitudinally, after one longitudinal scanning is finished, performing stepping scanning once along one transverse direction of the sample to be imaged, and repeatedly executing the steps of scanning point by point and scanning transversely by stepping until the sample to be imaged is scanned.
Optionally, the step of acquiring a fluorescence signal generated by the sample to be imaged after the excitation light scanning to obtain a fluorescence structured light image group corresponding to the fluorescence signal includes:
and acquiring and recording fluorescent signals generated after the sample to be imaged is scanned by exciting light point by point to obtain a fluorescent structure light image group corresponding to the fluorescent signals, wherein the fluorescent signals are non-sinusoidal structure light.
Optionally, the modulation function satisfies the following formula:
wherein I is the light intensity of the exciting light on the imaging surface of the sample to be imaged, omegamTheta is the included angle between the stripes in the fluorescence structure light image and the horizontal direction, t is the scanning time in the X direction, and h is the modulation frequencyRepresents the step number scanned in the Y direction when the scanning galvanometer scans,
is the initial phase.
Optionally, the step of obtaining a super-resolution image of the sample to be imaged according to superposition reconstruction of the frequency components of each fluorescence structure light image includes:
and carrying out inverse Fourier transform on the superposed value group of the superposed frequency components to obtain a super-resolution image of the reconstructed fluorescence signal.
In a second aspect, the present embodiment further discloses an apparatus for two-photon scanning structured light microscopic imaging, including:
a laser for generating laser light;
the intensity modulator is used for modulating the laser into exciting light of which the light intensity periodically changes along with time according to a sine function;
the scanning galvanometer is used for controlling the exciting light to scan the sample to be imaged;
the detector is used for collecting a fluorescence signal generated after the sample to be imaged is scanned by the exciting light to obtain a fluorescence structure light image group corresponding to the fluorescence signal; wherein the fluorescent structured light image group comprises: a plurality of fluorescent structured light images; the orientation and the phase of the excitation light patterns corresponding to the fluorescence structure light images are different;
the computing terminal is used for extracting the frequency components in each fluorescence structure light image, resetting and superposing the frequency components corresponding to the same orientation and different phases, and reconstructing a super-resolution image of the sample to be imaged according to the superposed value group of the frequency components in each orientation;
the intensity modulator, the scanning galvanometer, the detector and the computing terminal are connected.
Optionally, a spatial filter is further disposed between the intensity modulator and the scanning galvanometer;
the spatial filter includes: a first lens, a diaphragm and a second lens.
Optionally, a convex lens, a tube lens, a first optical filter and a dichroic mirror are further disposed in a light path between the scanning galvanometer and the detector, and an objective lens is further disposed in a light path between the sample to be imaged and the detector;
the back focal plane of the convex lens is superposed with the front focal plane of the tube lens; the back focal plane of the tube lens is superposed with the front focal plane of the objective lens; the first optical filter and the dichroic mirror are used for filtering and reflecting exciting light respectively.
The scanning light emitted by the scanning galvanometer is incident into the tube lens after passing through the convex lens, parallel light is emitted by the tube lens to be incident into the first optical filter, the first optical filter filters out exciting light, the filtered exciting light is incident into the dichroic mirror, and the dichroic mirror reflects the exciting light and transmits a fluorescent signal.
Optionally, an emission filter and a third lens are disposed in a light path between the dichroic mirror and the detector;
the emission optical filter receives the fluorescent signal transmitted from the dichroic mirror, reflects the excitation light and the stray light in the fluorescent signal and transmits the fluorescent signal;
and the third lens is used for receiving the fluorescent signal emitted by the emission filter and focusing and imaging the fluorescent signal on the detector.
Compared with the prior art, the embodiment of the invention has the following advantages:
according to the method provided by the embodiment of the invention, laser is modulated into exciting light with light intensity periodically changing according to a sine function along with time, and the modulated exciting light is used for scanning and exciting a sample to be imaged; collecting a fluorescence signal generated by scanning and exciting a sample to be imaged by exciting light to obtain a non-sinusoidal fluorescence structure light image corresponding to the fluorescence signal; and respectively acquiring fluorescence structure light images when the excitation light patterns are in different orientations and different phases, separating frequency components in each fluorescence structure light image, resetting and superposing the frequency components in the same direction, and reconstructing super-resolution images of fluorescence signals in all directions according to superposed value groups in all directions. In the embodiment, all frequency components which are separated from the non-sinusoidal fluorescence structure light image and are positioned in the same direction and contain higher harmonics are reset and then overlapped, and the super-resolution image of the sample to be imaged is reconstructed according to the overlapped value group of the frequency components in each orientation, so that the method of the embodiment can realize two-photon microscopic super-resolution imaging without fluorescence saturation excitation and high-power STED additional light, more importantly, the resolution ratio of the non-sinusoidal fluorescence structure light is improved by 3 times or even higher than the diffraction limit, and the two-photon fluorescence structure light imaging with the resolution ratio of dozens of nanometers or even higher can be realized.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments described in the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a flow chart of the method steps for two-photon scanning structured light microscopy imaging in an embodiment of the present invention;
FIG. 2 is a graph of the intensity of a femtosecond laser modulated by an intensity modulator according to a sine function along with time in the embodiment of the invention;
FIG. 3 is a schematic structural diagram of a two-photon scanning structure optical microscopic imaging device according to an embodiment of the present invention;
FIG. 4 is a schematic diagram of the light pattern of the excitation light stripe structure in an embodiment of the present invention;
fig. 5 is a graph of a point spread function in an embodiment of the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer and clearer, the present invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
In order to realize super-resolution imaging, the two-photon scanning structured light illumination microscopy in the prior art must avoid harmonic waves of fluorescence patterns generated by a two-photon nonlinear effect (the harmonic waves cause non-sinusoidal distribution of fluorescence intensity) to obtain sine-distribution fluorescence structured light patterns, and utilizes a constant-amplitude sine stripe structured light reconstruction algorithm to reconstruct super-resolution images, so that the final resolution is improved by 2 times of diffraction limit at most, namely about one-half wavelength (about 120 nanometers), and the requirement of super-resolution image imaging of dozens of nanometers cannot be met. In order to realize the two-photon structure light imaging with the resolution of dozens of nanometers or even higher, the embodiment discloses a nonlinear two-photon scanning structure light microscopic imaging method and device, which realize the two-photon scanning structure light super-resolution microscopic imaging under the unsaturated excitation condition, achieve the improvement of the resolution by 3 times of diffraction limit, and even higher, namely the resolution of 74nm, further improve the imaging resolution, and meet the requirements of the two-photon structure light imaging with the resolution of dozens of nanometers or even higher.
In a first aspect, this embodiment discloses a two-photon scanning structure light microscopic imaging method, as shown in fig. 1, the method includes:
and step S1, modulating the laser into exciting light with light intensity periodically changing according to a sine function along with time according to a preset modulation function, and scanning the sample to be imaged by using the modulated exciting light.
In this step, a laser is first used to emit laser light, and in one embodiment, the laser may be a titanium sapphire femtosecond laser, which can be used to realize two-photon excitation of the fluorescent substance.
In order to obtain a plurality of imaging images of a sample to be imaged on different phases in different directions, the step of modulating the laser into the exciting light with the light intensity periodically changing according to a sine function along with time comprises the following steps:
the laser is modulated into exciting light with light intensity periodically changing according to a sine function along with time according to a preset modulation function pair, and fluorescent images with different phases in the same direction are obtained by changing the phase of the modulation function.
In this embodiment, an intensity modulator is used to adjust laser with a preset modulation function, so that the light intensity of the modulated laser sinusoidally and periodically changes with time, wherein the intensity modulator is an electro-optic modulator or an acousto-optic modulator. As shown in fig. 2, the waveform diagram of the modulated excitation light with the intensity periodically changing according to the sine function with time is shown, and the function expression of the preset modulation function satisfies:
where I is the intensity of the excitation light on the imaging plane of the sample to be imaged, ωmIn order to preset modulation frequency according to requirements, theta is an included angle between the stripes in the fluorescent structure light image and the horizontal direction, t is the scanning time in the X direction, h represents the scanning step number in the Y direction during scanning of the scanning galvanometer,
is the initial phase.
In order to realize better laser transmission, as shown in fig. 3, a first half-wave plate 2 is further disposed in an optical path between the laser 1 and the intensity modulator 3, and the first half-wave plate 2 is configured to modulate a polarization direction of laser light emitted by the laser 1, so that the polarization direction of the laser light matches a polarization direction of the intensity modulator 3.
The step of scanning the sample to be imaged with modulated excitation light comprises:
and scanning the sample to be imaged point by point longitudinally, after one longitudinal scanning is finished, scanning the sample to be imaged transversely in a stepping mode, and repeatedly executing the steps of scanning point by point and scanning transversely step by step until the sample to be imaged is scanned completely.
In one embodiment, the scanning galvanometer is used for controlling the exciting light to scan a sample to be imaged, and specifically, the method comprises the following steps:
firstly, the galvanometer in the Y direction starts to carry out point-by-point scanning in the longitudinal direction, and after the point-by-point scanning of the galvanometer in the Y direction is finished, the galvanometer in the Y direction returns to the original position, and meanwhile, the galvanometer in the X direction carries out step scanning in the transverse direction, namely, the longitudinal position of an excitation light point is adjusted.
And circularly executing the point-by-point scanning and the step scanning, and correspondingly scanning the sample to be imaged point-by-point and step scanning until the sample to be imaged is scanned.
Exciting a fluorescence signal, wherein the fluorescence signal is non-sinusoidal structured light, the scanning galvanometer controls exciting light to scan point by point along the longitudinal direction of a sample to be imaged, when the longitudinal bottom end is scanned, the step scanning is carried out along the transverse direction of the sample to be imaged, then the longitudinal point by point scanning is carried out, and the steps of the longitudinal point by point scanning and the transverse step scanning are repeatedly executed until the whole sample to be imaged is scanned, so that the whole sample to be imaged excites the fluorescence signal. It is contemplated that the scanning of the sample to be imaged may also be performed in a transverse point-by-point scan, a longitudinal step scan.
S2, collecting a fluorescence signal generated by scanning and exciting a sample to be imaged by exciting light to obtain a fluorescence structure light image group corresponding to the fluorescence signal; wherein the fluorescent structured light image group comprises: a plurality of fluorescent structured light images; the orientation and phase of the excitation light patterns corresponding to the respective fluorescence structure light images are different.
As the fluorescent substance is distributed in the sample to be imaged, when the sample to be imaged is scanned by the exciting light, the fluorescent substance in the sample to be imaged generates a two-photon excitation effect under the action of the exciting light point and generates a fluorescent signal. The exciting light point excites the sample to be imaged to generate a two-photon signal, and the fluorescence signal excited by the whole sample to be imaged is obtained after the scanning galvanometer scans the whole sample to be imaged.
Referring to fig. 3, each light beam is scanned point by the scanning galvanometer 9 along the longitudinal direction of the sample 15 to be imaged, after one line of scanning along the longitudinal direction of the sample 15 to be imaged is finished, the scanning galvanometer 9 is scanned step by step, that is, moved to the next position along the transverse direction of the sample, and the next longitudinal position is scanned point by point, that is, scanned in the second line, and the above steps are repeated, so that the whole sample 15 to be imaged can be scanned, and fluorescence information excited in the whole area of the sample 15 to be imaged can be obtained. The corresponding excitation light stripe structure light pattern is shown in figure 4. The detector 18 records the fluorescence signal simultaneously point by point at the start of the scan and also at the completion of the scan of the entire sample, i.e. records an image, and stores it in the computing terminal 19.
The image obtained through calculation and recording in the above steps is a non-sinusoidal fluorescent structured light image, the non-sinusoidal fluorescent structured light image contains frequency information, and a fluorescent structured light image group with the same orientation phase and the same orientation but different phases can be obtained by changing the period and the phase of the modulation function. The fluorescent structured light image group comprises a plurality of fluorescent structured light images, and each fluorescent structured light can have the same orientation but different phases of the corresponding excitation patterns or can correspond to different orientations but the same phases of the corresponding laser patterns.
And step S3, extracting the frequency components in each fluorescence structure light image, resetting and superposing the frequency components corresponding to the same orientation and different phases, and reconstructing a super-resolution image of the sample to be imaged according to the superposed value group of the frequency components in each orientation.
After the fluorescence structured light images with different orientations and the same orientation and different phases are obtained in step S2, the frequency components of the fluorescence signals in the fluorescence structured light images are separated, the separated frequency components are reset, the reset frequency components are integrated to obtain a superposition value group of the frequency components in the same direction, and inverse fourier transform is performed to obtain a super-resolution image of the sample to be imaged.
Further, the step of obtaining a super-resolution image of the sample to be imaged according to the superposition reconstruction of the frequency components of each fluorescence structure light image includes:
and performing inverse Fourier transform on the fluorescence image corresponding to the superposed frequency value group to obtain a super-resolution image of the reconstructed fluorescence signal.
Specifically, as shown in FIG. 3, the modulated laser light is passed through a scanning galvanometer9 scanned to a sample 15 to be imaged to form a sinusoidal fringe excitation, for example as shown in fig. 4, to form a non-sinusoidal fluorescent structured light pattern containing all frequency information of higher harmonics, i.e. a high-order harmonic
Where k is the spatial frequency, wtIs the temporal modulation frequency, by varying the phase of the modulation function of the intensity modulator 3, fringe patterns of different phases in one orientation are obtained. In one embodiment, referring to fig. 4, the phase difference between adjacent images is 2 pi/5, a super-resolution image in the same direction is reconstructed by a reconstruction algorithm, in order to obtain a super-resolution image with uniform directions, different phase patterns with constant phase difference (2 pi/5) are respectively obtained in three orientations of 0 degree, 120 degrees and 240 degrees, and then the super-resolution image with uniform directions is reconstructed by the reconstruction algorithm.
The frequency s% (k) constituting this non-sinusoidal fluorescent structured light pattern is defined in the embodiments of the present invention,
the components meet the equations (4) and (5-9), and meanwhile, the saturated excitation is not needed in the embodiment, the exciting light power is similar to the traditional two-photon excitation power, special dyes are not needed, and common two-photon dyes are only needed, so that the technology can be directly used for a two-photon microscope to realize super-resolution imaging.
Another important invention is to provide a WS reconstruction algorithm, which is as follows:
the excitation structured light (i.e., excitation light) is assumed to be:
wherein
m＝1,2,…,M。
M must be 5 or more when imaging two-photon structured light because high frequency information is generated when separating high frequency information
An item.
After passing through the optical system, the intensity image recorded and formed point by the detector 18:
wherein Iex is the intensity of excitation light, h2pExcitation point spread function, s (x) sample structure, hemThe launch point spread function, x is the sample location.
Fourier transform is performed on the above formula (2), and each frequency component s% (k), s% (k-omega) is subjected to Fourier transformt)，s％(k+ωt)，s％(k-2ωt) And s% (k + 2. omega.)t) Separation and translation are carried out:
adding the signal spectra to obtain:
thus, can obtain
Multiplying each shifted spectrum by
And summed, to give:
therefore, the first and second electrodes are formed on the substrate,
the same can be obtained:
in this embodiment, the frequency components in the same orientation and different phases are separated first, the separated frequency components are reset, the reset separated frequency components are superimposed, and finally the superimposed values of the frequency components obtained in each orientation are subjected to inverse fourier transform, so that the super-resolution image of the sample to be imaged can be obtained.
By changing the modulation function of the intensity modulator 3, the direction of the sinusoidal illumination light pattern on the sample to be imaged is rotated (i.e., the orientation of the excitation light pattern is rotated), and the above operation is repeated, so that the resolution in the other direction of the sample can be improved. By analogy, the imaging resolution of the scanning area in each direction in the sample plane can be improved. Finally, the frequency spectrums in all directions are linearly added and subjected to inverse Fourier change to reconstruct a final super-resolution image, and the highest resolution is improved by about 3 times or even higher than the diffraction limit.
With reference to fig. 5, the image reconstruction algorithm provided in this embodiment simulates the point spread functions of the wide-field fluorescence microscope, the linear SIM, the two-photon fluorescence SIM, the three-photon fluorescence SIM, and the four-photon fluorescence SIM, and the resolutions thereof respectively reach 210nm, 112nm, 74nm, 53nm, and 43nm, theoretically, the optical nonlinear order is large enough, and the patented technology can reach the resolution capability of infinitesimal.
It is worth noting that the image reconstruction algorithm provided by the embodiment of the invention is not only suitable for two-photon fluorescence super-resolution microscopic imaging of structured light illumination, but also suitable for second harmonic structured light super-resolution microscopic imaging, and the same principle is also suitable for multi-photon and higher harmonic structured light super-resolution imaging. Meanwhile, the method is also suitable for two-photon fluorescence super-resolution microscopic imaging under the illumination of wide-field structured light.
On the basis of the above method, the embodiment also discloses a two-photon scanning structured light microscopic imaging apparatus, which is shown in fig. 3 and includes:
a laser 1 for generating laser light;
the intensity modulator 3 is used for modulating the laser into exciting light with light intensity periodically changing according to a sine function along with time;
the scanning galvanometer 9 is used for controlling the exciting light to scan the sample to be imaged;
the detector 18 is used for collecting a fluorescence signal generated after the sample to be imaged is scanned by the exciting light to obtain a fluorescence structure light image group corresponding to the fluorescence signal; wherein the fluorescent structured light image group comprises: a plurality of fluorescent structured light images; the orientation and the phase of the excitation light patterns corresponding to the fluorescence structure light images are different;
the computing terminal 19 is configured to extract frequency components in each fluorescent structured light image, reset and superimpose the frequency components corresponding to the same orientation and different phases, and reconstruct a super-resolution image of the sample to be imaged according to the superimposed value group of the frequency components in each superimposed orientation;
wherein, the intensity modulator 3, the scanning galvanometer 9, the detector 18 and the computing terminal 19 are connected. The intensity modulator 3 is an electro-optic modulator or an acousto-optic modulator.
The intensity modulator 3, the scanning galvanometer 9 and the detector 18 are connected with a computing terminal 19. The computing terminal 19 controls the intensity modulator 3 to change the modulation function, so that the transmitted laser is modulated into exciting light with the light intensity changing sinusoidally along with the time, and controls the scanning speed and the scanning range of the scanning galvanometer 9, and the computing terminal 19 is also used for controlling the detector 18 to acquire fluorescent signals point by point.
A first half-wave plate 2 is arranged in front of the intensity modulator 3, and a first reflecting mirror 4 and a second half-wave plate 5 are respectively arranged between the intensity modulator 3 and the scanning galvanometer 9. The first half-wave plate 2 and the second half-wave plate 5 are respectively used for modulating the polarization direction of incident light, and the first reflecting mirror 4 is used for adjusting the propagation path of the laser light.
Specifically, in order to achieve a better laser transmission effect, as shown in fig. 3, a spatial filter is further disposed between the intensity modulator 3 and the scanning galvanometer 9; the spatial filter is used for filtering and stray light. The spatial filter includes: a first lens 6, a diaphragm 7 and a second lens 8.
Further, a convex lens 10, a tube lens 11, a first optical filter 12 and a dichroic mirror 13 are further arranged in a light path between the scanning galvanometer 9 and the detector 18, and an objective lens 14 is further arranged in a light path between the sample 15 to be imaged and the detector 18; the back focal plane of the convex lens 10 is superposed with the front focal plane of the tube lens 11; the back focal plane of the tube lens 11 coincides with the front focal plane of the objective lens 14; the first optical filter 12 and the dichroic mirror 13 are used for filtering and reflecting the excitation light, respectively.
Scanning light emitted by the scanning galvanometer 9 enters the tube mirror 11 after passing through the convex lens 10, parallel light emitted by the tube mirror 11 enters the first optical filter 12, the first optical filter 12 filters out exciting light, the filtered exciting light enters the dichroic mirror 13, and the dichroic mirror 13 reflects the exciting light and transmits a fluorescent signal.
The parallel light is emitted after passing through the tube mirror 11, and respectively passes through the first optical filter 12, and is focused on an imaging surface of a sample 15 to be imaged through the objective lens 14 after being reflected by the dichroic mirror 13. The first filter 12 filters out light noise other than the excitation light, and the dichroic mirror 13 reflects the excitation light and transmits the fluorescence signal. For the excitation light beam, the scanning focus point on the front focal plane of the tube mirror 11 is conjugated with the scanning focus point on the imaging plane of the sample 15 to be imaged, when the scanning galvanometer 9 scans, the scanning point on the sample 15 to be imaged scans point by point along the longitudinal direction of the sample 15, and the fluorescent substance generates a two-photon excitation effect under the action of the excitation light point and generates fluorescence. After the longitudinal point-by-point scanning of the sample 15 to be imaged is finished, the sample 15 to be imaged is scanned transversely in a stepping manner, that is, the transverse position of the excitation light array point on the sample 15 to be imaged is adjusted. The point-by-point scanning and the step scanning are cyclically performed until the scanning of the area to be imaged on the sample 15 to be imaged is completed.
An emission filter 16 and a third lens 17 are further arranged in a light path between the dichroic mirror 13 and the detector 18;
the emission filter 16 receives the fluorescent signal transmitted from the dichroic mirror 13, reflects the excitation light and the stray light in the fluorescent signal, and transmits the fluorescent signal;
the third lens 17 is configured to receive the fluorescence signal emitted by the emission filter and focus the fluorescence signal on the detector 18.
In one embodiment, the dichroic mirror 13 is disposed between the objective lens 14 and the sample 15 to be imaged, the dichroic mirror 13 is highly reflective for pulsed laser light and highly transmissive for fluorescent light, and the angle between the dichroic mirror 13 and each light beam is 45 ° or 135 °. The light path of the fluorescence transmitted by the dichroic mirror 13 is sequentially provided with an emission filter 16 and a third lens 17, and finally the fluorescence is received by a detector 18. The emission filter 16 is a band pass filter, highly reflective of the excitation light, and only allows the fluorescence signal to pass through.
According to the method provided by the embodiment of the invention, laser is modulated into exciting light with light intensity periodically changing according to a sine function along with time, and the modulated exciting light is used for scanning and exciting a sample to be imaged; collecting a fluorescence signal generated by scanning and exciting a sample to be imaged by exciting light to obtain a non-sinusoidal fluorescence structure light image corresponding to the fluorescence signal; and respectively acquiring all frequency components including higher harmonics corresponding to the fluorescent signals in the fluorescent structure light image when the exciting light is in different phases, resetting and superposing the frequency components in the same direction, and reconstructing a super-resolution image of the fluorescent signals in each direction according to the superposed frequency value group in each direction. In the embodiment, the frequency components of the fluorescent signals in the same direction are overlapped to obtain the superposed value groups of the frequency components of the fluorescent signals in different directions, and the super-resolution image of the sample to be imaged is obtained according to the superposed value groups of the frequency components of the fluorescent signals, so that the method of the embodiment can realize two-photon microscopic super-resolution imaging without fluorescence saturation excitation and additional high-power STED light, more importantly, the resolution ratio of the method is improved by 3 times or even higher than the diffraction limit by using non-sinusoidal fluorescent structure light, and the two-photon fluorescent structure light imaging with the resolution ratio of dozens of nanometers or even higher can be realized.
Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. This invention is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.
It will be understood that the invention is not limited to the precise arrangements described above and shown in the drawings and that various modifications and changes may be made without departing from the scope thereof. The scope of the invention is only limited by the appended claims
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (7)
1. A two-photon scanning structure light microscopic imaging method is characterized by comprising the following steps:
modulating laser into exciting light with light intensity periodically changing according to a sine function along with time according to a preset modulation function, and scanning and exciting a sample to be imaged by adopting a scanning galvanometer by using the modulated exciting light;
collecting a fluorescence signal generated by scanning and exciting a sample to be imaged by exciting light to obtain a fluorescence structure light image group corresponding to the fluorescence signal; wherein the fluorescent structured light image group comprises: a plurality of fluorescent structured light images; the orientation and the phase of the excitation light patterns corresponding to the fluorescence structure light images are different;
extracting frequency components in each fluorescence structure light image, resetting and superposing the frequency components of which the corresponding exciting light patterns are in the same orientation and different phases, and reconstructing a super-resolution image of the sample to be imaged according to the superposed value group of the frequency components in each orientation;
the fluorescent signal is non-sinusoidal structured light; the non-sinusoidal fluorescent structure light pattern contains all frequency information of higher harmonics;
the method for obtaining the super-resolution image of the sample to be imaged according to the superposition reconstruction of the frequency components of each fluorescence structure light image comprises the following steps:
carrying out inverse Fourier transform on the superposed value group of the superposed frequency components to obtain a super-resolution image of the reconstructed fluorescence signal; reconstructing a super-resolution image with consistent directions by an image reconstruction algorithm; the reconstruction algorithm is to perform Fourier transform on an intensity image which is recorded and formed point by the detector, and separate and translate each frequency component to obtain a super-resolution image in the same orientation;
the step of acquiring a fluorescence signal generated by scanning and exciting a sample to be imaged by excitation light to obtain a fluorescence structure light image group corresponding to the fluorescence signal comprises the following steps: obtaining a fluorescence structure light image group corresponding to the excitation light patterns with different orientations and different phases by changing the period and the phase of the preset modulation function;
the preset modulation function satisfies the following formula:
wherein I is the light intensity of the exciting light on the imaging surface of the sample to be imaged, omegamFor modulating frequency, theta is the included angle between the stripes in the fluorescence structure light image and the horizontal direction, and t isThe time of scanning in the X direction, h represents the number of scanning steps in the Y direction when the scanning galvanometer scans,
is the initial phase.
2. The two-photon scanning structured light microscopy imaging method according to claim 1, wherein the step of scanning excitation of the sample to be imaged with modulated excitation light comprises:
and scanning the sample to be imaged point by point along the longitudinal direction, when one longitudinal scanning is finished, performing stepping scanning once along the transverse direction of the sample to be imaged, then performing longitudinal point by point scanning, and repeatedly executing the steps of point by point scanning and transverse stepping scanning until the sample to be imaged is scanned completely.
3. The two-photon scanning structure light microscopic imaging method according to claim 1, wherein the step of acquiring a fluorescence signal generated by a sample to be imaged being excited by an excitation light scanning to obtain a fluorescence structure light image group corresponding to the fluorescence signal comprises:
and acquiring and recording the fluorescent signals generated by scanning and exciting the sample to be imaged by the exciting light point by point to obtain a fluorescent structure light image group corresponding to the fluorescent signals.
4. A two-photon scanning structure light microscopic imaging device is characterized by comprising a laser, an intensity modulator, a scanning galvanometer and a detector which are arranged in sequence;
a laser for generating laser light;
the intensity modulator is used for modulating the laser into exciting light of which the light intensity periodically changes according to a sine function along with time according to a preset modulation function;
the scanning galvanometer is used for controlling the modulated exciting light to scan the sample to be imaged;
the detector is used for collecting a fluorescence signal generated after the sample to be imaged is scanned by the exciting light to obtain a fluorescence structure light image group corresponding to the fluorescence signal; wherein the fluorescent structured light image group comprises: a plurality of fluorescent structured light images; the orientation and the phase of the excitation light patterns corresponding to the fluorescence structure light images are different;
the computing terminal is used for extracting frequency components in each fluorescence structure light image, resetting and superposing the frequency components corresponding to the excitation light patterns in the same orientation and different phases, and reconstructing a super-resolution image of the sample to be imaged according to the superposed value group of the frequency components in each orientation;
the intensity modulator, the scanning galvanometer and the detector are respectively connected with the computing terminal;
the fluorescent signal is non-sinusoidal structured light; the non-sinusoidal fluorescent structure light pattern contains all frequency information of higher harmonics;
the method for obtaining the super-resolution image of the sample to be imaged according to the superposition reconstruction of the frequency components of each fluorescence structure light image comprises the following steps:
carrying out inverse Fourier transform on the superposed value group of the superposed frequency components to obtain a super-resolution image of the reconstructed fluorescence signal; reconstructing a super-resolution image with consistent directions by an image reconstruction algorithm; the reconstruction algorithm is to perform Fourier transform on an intensity image which is recorded and formed point by the detector, and separate and translate each frequency component to obtain a super-resolution image in the same orientation;
the step of acquiring a fluorescence signal generated by a sample to be imaged after the excitation light scanning to obtain a fluorescence structure light image group corresponding to the fluorescence signal comprises the following steps: acquiring a fluorescence structure light image group corresponding to the excitation light patterns with different orientations and different phases by changing the period and the phase of the preset modulation function;
the preset modulation function satisfies the following formula:
wherein I is the laser on the imaging surface of the sample to be imagedLuminous intensity, omegamTheta is the angle between the stripe in the fluorescent structure light image and the horizontal direction, t is the time of X-direction scanning, h represents the scanning step number in the Y direction during the scanning of the scanning galvanometer,
is the initial phase.
5. The two-photon scanning structured light microscopic imaging device according to claim 4, wherein a spatial filter is provided between said intensity modulator and said scanning galvanometer;
the spatial filter includes: a first lens, a diaphragm and a second lens.
6. The two-photon scanning structure light microscopic imaging device according to claim 4, wherein a convex lens, a tube lens, a first optical filter and a dichroic mirror are further arranged in a light path between the scanning galvanometer and the detector, and an objective lens is further arranged in a light path between the sample to be imaged and the detector;
the back focal plane of the convex lens is superposed with the front focal plane of the tube lens; the back focal plane of the tube lens is superposed with the front focal plane of the objective lens; the first optical filter and the dichroic mirror are respectively used for filtering and reflecting exciting light;
scanning light emitted by the scanning galvanometer is incident into the tube lens after passing through the convex lens, parallel light is emitted by the tube lens to be incident into the first optical filter, the first optical filter filters exciting light, the filtered exciting light is incident into the dichroic mirror, the dichroic mirror reflects the exciting light and transmits a fluorescent signal.
7. The two-photon scanning structure light microscopic imaging device according to claim 6, wherein an emission filter and a third lens are disposed in a light path between the dichroic mirror and the detector;
the emission optical filter receives the fluorescent signal transmitted from the dichroic mirror;
and the third lens is used for receiving the fluorescent signal transmitted by the emission filter and focusing and imaging the fluorescent signal on the detector.
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