CN117007571A - High-resolution CARS microscopic imaging system based on two-dimensional optical lattice - Google Patents
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Abstract
The invention provides a high-resolution coherent anti-Stokes Raman scattering (coerentanti-StokesRamanscattering, CARS) microscopic imaging system based on a two-dimensional optical lattice. The method is characterized in that: the device forms a two-dimensional optical lattice with a specific transmission direction in a view field range through multi-beam coherent superposition, the two-dimensional optical lattice is periodically arranged to be used as detection light and pumping light respectively, laser with specific wavelength is used as Stokes light to form full-field illumination in the view field range, biomolecules in a sample to be detected are excited at each focal point position of the optical lattice light field to generate CARS spectrum signals, chemical specificity and imaging contrast are provided, a multi-focus scanning mode is adopted to realize a multi-focus scanning high-resolution CARS microscopic imaging method for acquiring three-dimensional spatial distribution image information of biomolecules in cells in situ in real time on the premise of not introducing exogenous markers, and the device has the characteristics of non-marking, high time and spatial resolution and the like, and has wide application prospects in a plurality of research fields such as biology, medicine and life science.
Description
Technical Field
The invention relates to a high-resolution CARS microscopic imaging system based on a two-dimensional optical lattice, which realizes rapid biomolecular imaging of a non-invasive and non-invasive multi-focus light source without introducing exogenous substances, and belongs to the field of biophotonics.
Background
The 21 st century is the century of life science. The cells are the basic units of life structures and functions, and the intensive research on the cells is the key to revealing the life phenomenon mystery, modifying life and convincing diseases. At present, one of the hot spots of life science research is to elucidate the basic activity law of single cells at the molecular level, which requires the development of a systematic and intensive study of the physical, chemical properties and functions of a wide variety of biomolecules in cells.
With the deep research of living single cells, large-environment living cells for developing life activities are required to be used as test tubes, so that various biological molecules in the single cells can be rapidly and accurately identified under the condition of avoiding influencing the properties of the living cells and the microenvironments of the living cells as far as possible, and the biological molecules can be accurately and quantitatively analyzed to acquire the physical and chemical characteristics and functional information of the living cells. Therefore, the research and development of the spectral analysis and microscopic imaging technology for carrying out real-time, in-situ and dynamic detection on the vital activity of the single cell on the molecular level can rapidly acquire the spatial distribution information and the functional information of the biological molecules in the living cell without introducing exogenous markers, and the research and development of the method has important significance in researching the influence of the external environment on the properties, the functions and the actions of the biological molecules in the living cell on the vital activity process of the single cell, and further searching the properties, the functions, the interaction dynamic processes and the like of the biological molecules in the living cell.
In the far-field fluorescence microscopy imaging technology, a plurality of far-field super-resolution fluorescence microscopy imaging methods which break through the optical diffraction limit have been explored. The methods of stimulated emission loss (Stimulated Emission Depletion, STED) microscopy, fluorescence saturation structure illumination microscopy (Structured Illumination Microscopy, SIM) and the like have achieved remarkable success in improving the spatial resolution of fluorescence microscopy imaging, and the spatial resolution below tens of nanometers is obtained.
However, the above-described techniques rely to a large extent on the characteristic fluorescent molecules of a specially designed exogenous fluorescent label, which are usually exogenous, and thus necessarily affect the movement and metabolic processes of biomolecules within the cell, thereby directly affecting the self-properties and vital activities of the cell, and even generating phototoxicity and photobleaching.
The CARS microscopic imaging technology is a non-invasive, non-damaging and non-marking microscopic imaging technology for providing imaging contrast based on the inherent vibration fingerprint spectrum of the sample molecules to be detected, and has the advantages of high sensitivity, time resolution and the like. At present, CARS microscopic imaging technology has become one of non-marked microscopic imaging tools widely used in many research fields such as biology, medicine, material science and the like, and is widely applied to living cells and biological tissue microscopic imaging research works. However, as the spatial resolution of the CARS microscopic imaging technique is still within the optical diffraction limit of the far-field optical microscope, there is an increasing need to further increase the spatial resolution of the CARS microscopic imaging technique. Unlike the mechanism of super-resolution fluorescence microscopy imaging techniques that rely on specially designed fluorophore properties, an entirely new challenge is presented to achieve non-labeled super-resolution microscopy imaging techniques based on CARS spectra.
Numerous research teams at home and abroad develop in-depth theoretical and experimental research work in improving spatial resolution of CARS microscope, and creatively propose various design schemes. These designs have disadvantages such as: based on the principle of energy level exhaustion, a switching mechanism is introduced in the CARS nonlinear optical process. Most of the related research works remain in the theoretical research and numerical simulation stages, and are not yet confirmed by experimental verification.
Based on the existing CARS spectrum detection and microscopic imaging technology and the theory and experimental study of the fluorescence microscopic imaging technology, the invention provides a multi-vector light field coherent superposition regulation mechanism, which forms a two-dimensional optical lattice formed by periodically arranged optical unit cell light fields with high density, high intensity and excitation volume breaking through the optical diffraction limit in the field of view of an objective lens as a multi-focus excitation light source. Excitation of specific biomolecules within the cell at each focal point produces a CARS spectral signal, providing chemical specificity and imaging contrast. By adopting the multi-focus scanning mode, the multi-focus scanning super-resolution CARS microscopic imaging system with two-dimensional high resolution can rapidly and accurately identify biomolecules in cells in a non-invasive and non-invasive mode and acquire two-dimensional distribution image information in situ in real time on the premise of not introducing exogenous markers.
Disclosure of Invention
A CARS microscopic imaging system for acquiring high-resolution multi-focus rapid scanning imaging in a non-invasive and atraumatic manner based on a two-dimensional optical lattice light field.
The purpose of the invention is realized in the following way:
the tunable picosecond lasers 1, 2 and 3 in the system respectively emit Stokes light, detection light and pump light with specific wavelengths. The detection light passes through the 1/2 wave plate 4 and the one-dimensional micro displacement table 6 and then is combined with the pump light passing through the 1/2 wave plate 5 into one beam of light at the beam combining lens 7. The laser after beam combination enters a spatial light modulator 12 through lenses 8 and 9, a polarization beam splitter prism 10 and a 1/4 wave plate 11, the spatial light modulator 12 generates specific light beams, the specific light beams enter an objective lens 15 through a beam expanding and shaping system 14, and the specific light beams are irradiated to a three-dimensional nano object stage 16. Stokes light passes through the beam expanding and shaping system 17, passes through the dichroic mirror 19 and enters the objective lens 18, and is irradiated to the three-dimensional nano-stage 16. The CARS signal finally generated on the three-dimensional nano-stage 16 is received from the objective lens 18 by the EMCCD camera 21 through the dichroic mirror 19 and the short-wave pass filter 20 and is transmitted back to the computer 22, and the measurement result is given by the computer 22.
The optical lattice light field is composed of a plurality of vector light fields which are incident at different angles, and periodically arranged optical unit cell light fields generated by mutual superposition in the same area. The detection light and the pump light respectively form two-dimensional optical lattices with different symmetry, period and transverse-longitudinal ratio in the focal plane of the sample to be detected, and the two-dimensional optical lattices meet the phase matching requirement. In space, the wave vector and intensity of the incident vector field respectively have different effects on the formation of the optical lattice field. In condensed state physics, the relationship between the reciprocal original vector of the reciprocal lattice and the wave vector can be expressed as:
b n ＝k 0 -k n ,n＝1,…,D (1)
according to the above, a desired two-dimensional optical lattice satisfying phase matching can be produced by controlling the number of superimposed optical fields and the wave vector direction. The original unit cell wave vector a of the direct lattice is defined first n Let a= [ a ] 1 ,…,a D ]. And the original unit cell wave vector b of reciprocal lattice n Let b= [ B ] 1 ,…,b D ]. From b' i ·a j ＝2πδ ij (i.e., the same subscript to the right equals 2pi, otherwise the right result is 0, where b' i I.e., the reciprocal vector) can be obtained:
k 0 ＝A·β/4π (2)
the wave vector set { k) can be obtained according to the formulas (1) and (2) n And contains information about the direction and periodicity of the crystal lattice. In combination with the above, the direct primitive cell vector a of different cell shapes is selected according to design requirements n Sum wave vector k n To generate a two-dimensional optical lattice that satisfies the phase matching.
The generation of the CARS signal results from the system outputting near infrared wavelength laser pulses of a specific center frequency as stokes light for full-field illumination, probe light in the form of a two-dimensional optical lattice, and pump light, respectively, using wavelength tunable picosecond lasers 1, 2, and 3. The energy conservation condition can be realized by adjusting the central wavelength of the laser output. The laser beam is incident at a specific position in the back focal plane of the large numerical aperture objective lens 15, and is coherently superimposed in a sample to be detected in the field range at different angles through the objective lens 15 to form different types of two-dimensional optical lattice multi-focus excitation light sources meeting phase matching. Near-infrared wavelength laser pulses as stokes light are imaged by a beam expanding and shaping system 17 at the back focal plane of another large numerical aperture objective 18, forming full field illumination over the entire field of view of the objective 18. The multi-focus excitation light source formed by superposition of the two-dimensional lattice light fields and the stokes light of full-field illumination form a composite light field for generating CARS signals. The two-dimensional optical lattice in a specific propagation direction enables the total optical field to simultaneously meet the condition of momentum conservation of four-wave mixing, and the CARS signal is finally detected.
From the principle of CARS nonlinear optical process, it can be known that the intensity of CARS signal is proportional to the third-order nonlinear polarization of the sample to be measured, which includes a resonance part (χ (3)R ) And a non-resonant portion (χ) (3)NR ) As shown in formula (3):
the simultaneous non-resonant background (Nonresonant Background, NRB) noise accompanying the CARS signal greatly reduces the detection sensitivity, spectral resolution, and image contrast of the system. Especially under broadband excitation conditions, NRB noise with a high intensity tends to drown out the weaker CARS signal of the biomolecule of interest, which is detrimental to accurate recognition of the biomolecule.
The center wavelength of the laser pulses output from the picosecond pulse lasers 1 and 3 is adjusted, and when the frequency difference between stokes light and pump light is consistent with the vibration frequency of the vibration mode of the specific substance molecules, the vibration mode of the substance molecules in the light field range of each unit cell is enhanced in resonance. Because the time-coincident pump light and stokes light pulses interact with the sample, a molecular vibrational state is created that contains both resonant and non-resonant portions. The non-resonant state has a flat response characteristic in the frequency domain and an instantaneous peak response characteristic in the time domain. The transition including an actual vibrational state in the resonant state has a longer dephasing time (about several picoseconds to about several tens of picoseconds in liquids and solids, and about several hundred picoseconds in gases) than in the non-resonant state. Thus, in CARS spectral analysis and microscopy imaging techniques, strong NRB noise is present at the beginning of the time domain waveform received by the detector, masking the useful CARS signal.
The time delay between the pump light and Stokes light laser pulse and the detection light laser pulse which arrive at the same time is precisely controlled based on the one-dimensional micro-displacement table 6, so that the time resolution method under the condition of multi-focus excitation is realized. By selecting the optimal time window, on the premise of avoiding useful signal loss as much as possible, NRB noise accompanying CARS spectrum signals is effectively restrained, so that the purposes of improving the spectrum resolution, the detection sensitivity and the imaging contrast of the system are achieved. The laser pulse output by the picosecond pulse laser 2 is used as detection light to mix with a resonance enhanced molecular vibration mode, forward CARS spectrum signals generated by excitation of each unit cell light field are collected by an objective lens 18, laser and background noise are eliminated through a short-wave pass filter 20, and the signals are detected and received by an EMCCD camera 21. Based on the CARS spectrum signals obtained by detection, chemical specificity and imaging contrast are provided, the three-dimensional nano object stage 16 is regulated to realize rapid scanning of a two-dimensional optical lattice light field in a sample to be detected, and a two-dimensional high-resolution image of specific molecules in the sample to be detected is obtained
Drawings
FIG. 1 (System content)
Fig. 2 is a simulation result of a two-dimensional bravais regular hexagonal basic optical lattice. When the included angle between the incident beams is 120 degrees, the regular hexagonal basic optical lattice generated by coherent superposition is shown in fig. 2, and the direction of the incident beams is shown by an arrow in the figure. From simulation results, the narrowest part of the optical field intensity of the single optical unit cell has a full width at half maximum (Full Width at Half Maximum, FWHM) of 290nm.
FIG. 3 is a simulation result of a two-dimensional Bragg square-based optical lattice, and the direction of an incident beam is shown by an arrow in the figure. The FWHM of the narrowest part of the optical field intensity of a single optical unit cell is 249nm.
Fig. 4 is a simulation result of a two-dimensional composite optical lattice obtained by a highly symmetric operation. Through carrying out vertical rotation and mirror image overturning highly symmetrical operation on incident laser, 8 incident laser beams have high symmetry among each other, a two-dimensional composite optical lattice simulation result generated by coherent superposition is shown in fig. 4, and the direction of the incident laser beams is shown by an arrow in the figure. The FWHM of the narrowest part of the optical field intensity of a single optical unit cell is 240nm.
FIG. 5 is a schematic diagram of the configuration of the optical paths of the excitation light fields for implementing the CARS nonlinear optical process, and in order to implement the condition of conservation of momentum, in the present invention, laser beams with different wavelengths are modulated in phase and intensity by a spatial light modulator to form a laser beam with a specific transmission direction. Each beam is incident at a specific position in the circular area of the rear focal plane of the large numerical aperture objective lens 15, and a specific two-dimensional optical lattice is generated by coherent superposition of the objective lens 15 at different angles in the sample to be tested in the field of view. By controlling the number of incident light beams and adjusting the incidence position of each light beam at the back focal plane of the objective lens 15, two-dimensional optical lattice light fields of two specific transmission directions overlapping each other are formed within the field of view as probe light and pump light, respectively. Near-infrared wavelength laser pulses as stokes light are imaged by a beam shaping and coupling system at the back focal plane of another large numerical aperture objective 18, forming full field illumination over the entire field of view of the objective 18.
Fig. 6 is a phase matching condition for a two-dimensional optical lattice light field to implement a CARS nonlinear optical process. The wave vectors of the pumping light, the detecting light, the Stokes light and the CARS signal are K respectively P 、K Pr 、K S And K AS . The energy transmission direction of each two-dimensional optical lattice light field at the focal plane of the sample to be detected and the phase matching condition of the CARS spectrum signal generated by the two-dimensional optical lattice light field excited substance molecules in the specific transmission direction are shown in the figure. Therefore, by adjusting the incidence positions of the probe light and the pump light on the rear focal plane of the objective lens 15, the energy propagation direction of the two-dimensional optical lattice generated by the probe light and the pump light after passing through the objective lens 15 can be changed, so that phase matching is realized, and the purpose of meeting the condition of conservation of momentum is achieved.
Detailed Description
The invention is further illustrated below in conjunction with specific examples. It will be understood that terms, such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
The system consists of tunable picosecond lasers 1, 2 and 3;1/2 wave plates 4, 5; a one-dimensional micro-displacement table 6; a beam combiner 7; lenses 8, 9; a polarization beam splitter prism 10; a 1/4 wave plate 11; a spatial light modulator 12; a mask plate 13; a beam expanding and shaping system 14, 17; objective lenses 15, 18; a three-dimensional nano-stage 16; a dichroic mirror 19; a short-wave pass filter 20; an EMCCD camera 21 and a computer 22. The tunable picosecond lasers 1, 2 and 3 in the system respectively emit stokes light, detection light and pump light with specific wavelengths. The detection light passes through the 1/2 wave plate 4 and the one-dimensional micro displacement table 6 and then is combined with the pump light passing through the 1/2 wave plate 5 into one beam of light through the beam combining mirror 7. The laser after beam combination enters a spatial light modulator 12 through lenses 8 and 9, a polarization beam splitter prism 10 and a 1/4 wave plate 11, the spatial light modulator 12 generates specific light beams, the specific light beams enter an objective lens 15 through a beam expanding and shaping system 14, and the specific light beams are irradiated to a three-dimensional nano object stage 16. Stokes light enters an objective lens 18 through a beam expanding and shaping system 17 and a dichroic mirror 19, and irradiates a three-dimensional nano-object stage 16. The CARS signal finally generated on the three-dimensional nano-stage 16 is received from the objective lens 18 by the EMCCD camera 21 through the dichroic mirror 19 and the short-wave pass filter 20 and is transmitted back to the computer 22, and the measurement result is given by the computer 22.
In the system, the tunable picosecond lasers 2 and 3 respectively emit probe light and pump light with specific wavelengths. The probe light and the pump light respectively pass through the 1/2 wave plates 4 and 5 to obtain specific polarized light beams. The detection light is combined with the pump light into one beam of light through a beam combining mirror 7 after passing through a one-dimensional micro-displacement table 6. The laser beam after the beam combination passes through lenses 8 and 9, and enters a spatial light modulator 12 controlled by a computer 22 through a polarization splitting prism 10 and a 1/4 wave plate 11. The computer 22 controls the spatial light modulator 12 to generate a light beam with a specific transmission direction, and the light beam passes through the customized mask 13 and enters the objective lens 15 through the beam expanding and shaping system 14. Incidence at different locations on the back pupil plane produces beams with different wave vector directions that interfere to form a two-dimensional optical lattice of a particular propagation direction, which ultimately impinges on the sample of the three-dimensional nano-stage 16. The two-dimensional optical lattice light field superposition of a specific transmission angle can form a dense multi-focus light source in a focal plane by utilizing symmetrical operation according to requirements, and the size of the light source breaks through the limit of diffraction limit.
In the system, a light beam emitted by the tunable picosecond laser 1 enters an objective lens 18 through a beam expanding and shaping system 17 and a dichroic mirror 19, so as to irradiate on a three-dimensional nano-object stage 16 as stokes light of full-field illumination. The three-dimensional nano-stage 16 is adjusted to realize rapid scanning of the two-dimensional optical lattice light field in the sample to be detected, and a high-resolution image of specific molecules in the sample to be detected is obtained. CARS signals generated by two-dimensional optical lattice light fields corresponding to detection light and pumping light with specific wavelengths on the three-dimensional nano object stage 16 and Stokes light with full-field illumination, wherein the CARS signals start from the objective lens 18, pass through the dichroic mirror 19 and the short-wave pass filter 20, are finally received by the EMCCD camera 21 and are transmitted back to the computer 22, and the computer 22 gives measurement results
In the system, laser pulses output by the picosecond pulse laser 2 are used as detection light and are mixed with a molecular vibration mode with enhanced resonance, and a light pulse time delay system formed by the one-dimensional micro displacement table 6 is regulated to effectively separate resonance CARS signals and non-resonance background signals with different time response characteristics in a time domain, so that the influence of the non-resonance signals is eliminated, and the detection sensitivity and the image contrast of the system are effectively improved. Finally, forward CARS spectral signals generated by the excitation of each unit cell field are collected by a microscope objective 18, and laser and background noise are further eliminated by a short-wave pass filter 20.
The above examples are provided for the purpose of describing the present invention only and do not limit the scope of the present invention. The scope of the invention is defined by the appended claims. Various equivalent substitutions and modifications may be made without departing from the spirit and principles of the present invention, and it is intended to be within the scope of the present invention.
Claims (4)
1. A high-resolution coherent anti-Stokes Raman scattering (corherenti-StokesRamanscattering, CARS) microscopic imaging system based on a two-dimensional optical lattice. The method is characterized in that: the system consists of a two-dimensional optical lattice light field regulation system, a CARS signal acquisition system and a non-resonance background signal elimination system. The system consists of tunable picosecond lasers 1, 2 and 3;1/2 wave plates 4, 5; a one-dimensional micro-displacement table 6; a beam combiner 7; lenses 8, 9; a polarization beam splitter prism 10; a 1/4 wave plate 11; a spatial light modulator 12; a mask plate 13; a beam expanding and shaping system 14, 17; objective lenses 15, 18; a three-dimensional nano-stage 16; a dichroic mirror 19; a short-wave pass filter 20; an EMCCD camera 21 and a computer 22. The tunable picosecond lasers 1, 2 and 3 in the system respectively emit stokes light, detection light and pump light with specific wavelengths. The detection light passes through the 1/2 wave plate 4 and the one-dimensional micro displacement table 6 and then is combined with the pump light passing through the 1/2 wave plate 5 into one beam of light at the beam combining lens 7. The laser after beam combination enters a spatial light modulator 12 through lenses 8 and 9, a polarization beam splitter prism 10 and a 1/4 wave plate 11, the spatial light modulator 12 generates specific light beams, the specific light beams enter an objective lens 15 through a beam expanding and shaping system 14, and the specific light beams are irradiated to a three-dimensional nano object stage 16. Stokes light enters an objective lens 18 through a beam expanding and shaping system 17 and a dichroic mirror 19, and irradiates a three-dimensional nano-object stage 16. The CARS signal finally generated on the three-dimensional nano-stage 16 is received from the objective lens 18 by the EMCCD camera 21 through the dichroic mirror 19 and the short-wave pass filter 20 and is transmitted back to the computer 22, and the measurement result is given by the computer 22.
2. The two-dimensional optical lattice light field manipulation system of claim 1. The method is characterized in that: the tunable picosecond lasers 2 and 3 emit probe light and pump light of specific wavelengths, respectively. The probe light and the pump light respectively pass through the 1/2 wave plates 4 and 5 to obtain specific polarized light beams. The detection light is combined with the pump light into one beam of light through a beam combining mirror 7 after passing through a one-dimensional micro-displacement table 6. The laser beam after the beam combination passes through lenses 8 and 9, and enters a spatial light modulator 12 controlled by a computer 22 through a polarization splitting prism 10 and a 1/4 wave plate 11. The beam generated by the spatial light modulator 12 in a specific transmission direction passes through the customized mask plate 13 and enters the objective lens 15 through the beam expanding and shaping system 14. The light beams with specific transmission directions are irradiated on the rear pupil surface of the objective lens 15 at different incident positions, the light beams are focused at specific angles, and two-dimensional optical lattices meeting phase matching with different symmetry, period and transverse-longitudinal ratio are respectively formed in the focal plane of the sample to be detected, so that a two-dimensional optical lattice field is irradiated on the three-dimensional nano object stage 16. The two-dimensional optical lattice light field superposition of a specific transmission angle can form a dense multi-focus light source in a focal plane according to requirements, and the size of the light source breaks through the limit of diffraction limit.
3. The CARS signal acquisition system of claim 1 wherein: the beam emitted by the tunable picosecond laser 1 controlled by the computer 22 enters the objective 18 through the beam expanding and shaping system 17 and the dichroic mirror 19, and is irradiated to the three-dimensional nano-stage 16 as stokes light of full-field illumination. The two-dimensional optical lattice light field and the stokes light of full-field illumination respectively corresponding to the detection light and the pump light with specific wavelengths on the three-dimensional nano-stage 16 generate CARS signals. The three-dimensional nano-stage 16 is adjusted to realize rapid scanning of the two-dimensional optical lattice light field in the sample to be detected, and high-resolution image signals of specific molecules in the sample to be detected are obtained. The signal passes from the objective lens 18 through the dichroic mirror 19 and the short-wave pass filter 20, and the resulting CARS signal is received by the EMCCD camera 21 and transmitted back to the computer 22, where the measurement results are given by the computer 22.
4. The non-resonant background signal cancellation system of claim 1, wherein: the laser pulse output from the picosecond pulse laser 2 is mixed as detection light with a resonance-enhanced molecular vibration mode. By adjusting an optical pulse time delay system formed by the one-dimensional micro-displacement platform 6, resonance CARS signals with different time response characteristics and non-resonance background signals are effectively separated in a time domain, so that the influence of the non-resonance signals is eliminated, and the detection sensitivity and the image contrast of the system are effectively improved. Finally, forward CARS spectral signals generated by the excitation of each unit cell field are collected by a microscope objective 18, further eliminating laser and background noise via a short-pass filter 20.
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