CN111202499A - Rapid and efficient self-adaptive optical compensation stimulated Raman scattering imaging system and method - Google Patents
Rapid and efficient self-adaptive optical compensation stimulated Raman scattering imaging system and method Download PDFInfo
- Publication number
- CN111202499A CN111202499A CN202010125026.4A CN202010125026A CN111202499A CN 111202499 A CN111202499 A CN 111202499A CN 202010125026 A CN202010125026 A CN 202010125026A CN 111202499 A CN111202499 A CN 111202499A
- Authority
- CN
- China
- Prior art keywords
- light
- reflected
- module
- lens
- deformable mirror
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
- A61B5/0075—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence by spectroscopy, i.e. measuring spectra, e.g. Raman spectroscopy, infrared absorption spectroscopy
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/0033—Features or image-related aspects of imaging apparatus classified in A61B5/00, e.g. for MRI, optical tomography or impedance tomography apparatus; arrangements of imaging apparatus in a room
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
-
- G—PHYSICS
- G02—OPTICS
- G02F—OPTICAL DEVICES OR ARRANGEMENTS FOR THE CONTROL OF LIGHT BY MODIFICATION OF THE OPTICAL PROPERTIES OF THE MEDIA OF THE ELEMENTS INVOLVED THEREIN; NON-LINEAR OPTICS; FREQUENCY-CHANGING OF LIGHT; OPTICAL LOGIC ELEMENTS; OPTICAL ANALOGUE/DIGITAL CONVERTERS
- G02F1/00—Devices or arrangements for the control of the intensity, colour, phase, polarisation or direction of light arriving from an independent light source, e.g. switching, gating or modulating; Non-linear optics
- G02F1/35—Non-linear optics
-
- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01S—DEVICES USING THE PROCESS OF LIGHT AMPLIFICATION BY STIMULATED EMISSION OF RADIATION [LASER] TO AMPLIFY OR GENERATE LIGHT; DEVICES USING STIMULATED EMISSION OF ELECTROMAGNETIC RADIATION IN WAVE RANGES OTHER THAN OPTICAL
- H01S3/00—Lasers, i.e. devices using stimulated emission of electromagnetic radiation in the infrared, visible or ultraviolet wave range
- H01S3/30—Lasers, i.e. devices using stimulated emission of electromagnetic radiation in the infrared, visible or ultraviolet wave range using scattering effects, e.g. stimulated Brillouin or Raman effects
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
- G01N2021/0106—General arrangement of respective parts
- G01N2021/0112—Apparatus in one mechanical, optical or electronic block
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A90/00—Technologies having an indirect contribution to adaptation to climate change
- Y02A90/10—Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- Nonlinear Science (AREA)
- General Physics & Mathematics (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Surgery (AREA)
- Animal Behavior & Ethology (AREA)
- Heart & Thoracic Surgery (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Electromagnetism (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Medical Informatics (AREA)
- Immunology (AREA)
- Optics & Photonics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Radiology & Medical Imaging (AREA)
- Plasma & Fusion (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention discloses a stimulated Raman scattering imaging system and a method for rapid and efficient adaptive optical compensation. The two half-wave plates and the polarization beam splitter are arranged in front of the output end of the laser, the laser emits two beams which pass through the half-wave plates and the polarization beam splitter, the first beam is incident to the reflecting mirror after sequentially passing through the acousto-optic modulator and the beam expanding module, the second beam is incident to the dichroic mirror after passing through the beam expanding module and the phase delay module, the second beam after being reflected is combined with the second beam reflected by the dichroic mirror at intervals and incident to two areas of the deformable mirror, the two beams are reflected into one beam, the beam is focused by the scanning module incident to the microscope objective, and scattered beams generated by the transmission of an experimental sample are received and detected. The invention optimizes and adjusts the compensation phase value of the deformable mirror, so that a focusing light spot with stronger central light intensity can be formed in the sample after the phase compensation is carried out on the light beam, the nonlinear effect is better excited, and the imaging quality of the deep inside the scattering medium is improved.
Description
Technical Field
The invention belongs to an optical compensation scattering imaging system and method in the field of optical microscopic imaging, and particularly relates to a stimulated Raman scattering imaging system and method for rapid and efficient adaptive optical compensation, which are applied to non-invasive deep penetration unmarked optical microscopic imaging.
Background
In the field of biomedical optics, optical scattering is a major factor that limits the quality of optical imaging. Most optical techniques for deep tissue imaging (e.g., confocal laser imaging, two-photon microscopy, and optical coherence tomography) primarily utilize non-scattered photon (i.e., ballistic photon) imaging. The number of ballistic photons decays exponentially with depth, thus limiting the optical focus range to depths of 1 mm.
The adaptive optical technology applied to astronomy in the past provides a new technical support for realizing deep biological tissue imaging.
The existing non-invasive adaptive optogenetic technology is based on an accurate phase correction technology of adaptive optics or a coherent light adaptive technology to perform phase compensation, so that a distorted phase is corrected in a sample, and good light beam focusing is formed, so that a specific substance marked in the sample is excited, the specific substance absorbs photon energy to a certain degree, and a fluorescence signal with another specific wavelength is emitted.
However, the above methods (including confocal laser imaging, two-photon microscopy, etc.) require the sample to be fluorescently labeled in advance. Although some fluorochromes have proven to be harmless at present, most materials are still not applicable to living samples due to their toxicity, either long-term or short-term.
The stimulated Raman scattering technology utilizes the Raman spectrum of a substance, generates a stimulated Raman scattering signal through two beams of light beams with specific frequency difference and the specific Raman spectrum adapted to the substance, obtains a scattering optical signal with original frequency and periodically changed intensity, and only collects and amplifies an emergent scattering signal with the same modulation frequency through a phase-locked amplifier. Because the stimulated Raman scattering belongs to the nonlinear effect, a more obvious required signal can be generated only for a focus part, so that the optical section effect is realized, and images with different depths can be obtained without section. Due to the sensitivity of the raman spectra of different substances, background noise that is often generated by fluorescent staining can be avoided and high resolution imaging can be achieved.
Although stimulated raman scattering imaging enables label-free imaging, the resulting signal is still not strong enough due to its weak nonlinear effects themselves. In samples with more severe scattering, a sufficiently good focus may not be formed to obtain the desired signal. Ensuring good signal intensity and imaging speed while completing label-free high-resolution imaging is also an urgent problem to be solved in current biological applications.
Disclosure of Invention
In order to solve the problems existing in the background technology, the invention aims to provide a stimulated Raman scattering imaging system and a stimulated Raman scattering imaging method for rapid and efficient adaptive optical compensation, and the problem that a spatial light modulator in adaptive optics applied to biomedicine traditionally consumes a long time is solved by using a deformable mirror with a high image refresh rate.
The method utilizes the quick refresh rate of the deformable mirror and combines the principle of the stimulated Raman scattering technology to perform partition processing on the deformable mirror, respectively perform phase modulation on two beams of light required by the stimulated Raman scattering to different degrees, and the incident wavefront is matched with the scattering condition through a self-adaptive algorithm, so that the quality of light beam focusing is improved, the intensity of a stimulated Raman scattering signal is improved, and the resolution of unmarked imaging is improved. Thereby improving the efficiency of the stimulated raman scattering technique.
In order to achieve the purpose, the technical scheme of the invention comprises the following steps:
a stimulated Raman scattering imaging system based on rapid and efficient adaptive optical compensation comprises:
the system comprises a laser, a half-wave plate, a polarization beam splitter, an acousto-optic modulator, a beam expanding module, a reflecting mirror, a dichroic mirror, a phase delay module, a beam combining module, a deformable mirror, a scanning module, a microscope objective, an experimental sample and a light intensity detection module; the two half-wave plates and the two polarization beam splitters are arranged in front of the output end of the laser, the laser emits two beams of light beams with different wavelengths, the two beams of light beams are adjusted to be in the same polarization direction through the respective half-wave plates and the respective polarization beam splitters, the first beam of light beam is emitted from the polarization beam splitters, then sequentially passes through the acousto-optic modulator and the beam expanding module and is incident to the reflecting mirror, the second beam of light beam is sequentially passed through the beam expanding module and the phase delay module and is incident to the dichroic mirror, the first beam of light beam is reflected by the reflecting mirror, then is transmitted by the dichroic mirror and is incident to two areas of the deformable mirror through the beam combining module at intervals together with the second beam of light reflected by the dichroic mirror, then is reflected to the beam combining module through the deformable mirror to form a beam of light beam, the light beam is incident to the microscope objective for focusing through the scanning module, the experimental sample is positioned on the focal, the experimental sample is stimulated by Raman reflection to generate a nonlinear signal, and the nonlinear signal is received by the light intensity detection module to be detected.
Each beam expanding module comprises a front beam expanding module lens and a rear beam expanding module lens; the front beam expanding module lens and the rear beam expanding module lens are sequentially arranged behind the polarization beam splitter along an optical axis, and two beams of light beams emitted by the laser are sequentially expanded to the same diameter after passing through the respective beam expanding modules.
The phase delay module comprises an emergent reflector, a front deflection reflector, a rear deflection reflector and an incident reflector which are sequentially arranged along a light path, wherein a second light beam which is not modulated by the acousto-optic modulator is incident to the emergent reflector and is reflected by the emergent reflector, the front deflection reflector, the rear deflection reflector and the incident reflector in sequence and then is emitted to the dichroic mirror; the distance between the front deflection reflector and the exit reflector and the distance between the rear deflection reflector and the incident reflector can be adjusted, so that different delay effects are achieved.
Two beams emitted by the laser are pulse beams, and the pulses of the two beams are synchronous through different delay adjustment of the phase delay module.
The beam combining module comprises a left beam splitter and a right beam splitter; the connecting line between the left beam splitter and the right beam splitter is placed in parallel to the reflecting surface of the deformable mirror, the left beam splitter and the right beam splitter are both semi-transparent semi-reflecting mirrors, a first beam of light is transmitted through the right beam splitter and then incident to the deformable mirror for reflection, the light reflected back through the deformable mirror is incident to the right beam splitter for reflection to generate a first reflected light beam, a second beam of light is transmitted through the left beam splitter and then incident to the deformable mirror for reflection, the light reflected back through the deformable mirror is incident to the left beam splitter for reflection to generate a second reflected light beam, the second reflected light beam is incident to the right beam splitter for transmission and then is combined with the first reflected light beam, and two beams of light reflected back through the deformable mirror are respectively incident to the left beam splitter and the right beam.
The deformable mirror is mainly composed of a plurality of micro-mirror compact arrays with reflecting surface types capable of being adjusted in three dimensions, and a spatial light modulator can be particularly adopted.
The scanning module comprises a front scanning galvanometer, a front beam collimating lens, a rear scanning galvanometer, a front scanning module lens and a rear scanning module lens; the front scanning galvanometer, the front beam collimating lens, the rear scanning galvanometer, the front scanning module lens and the rear scanning module lens are sequentially arranged behind the beam combining module along a light path, and a beam emergent after being combined by the beam combining module is reflected by the front scanning galvanometer, the front beam collimating lens, the rear scanning galvanometer, the front scanning module lens and the rear scanning module lens and then is incident to the microscope objective;
the light intensity detection module comprises a condenser, a light filter, a collimation focusing lens, a photodiode and a lock-in amplifier and is designed into a transmission type system, the condenser, the light filter, the collimation focusing lens, the photodiode and the lock-in amplifier are sequentially arranged behind an experimental sample along a light path, and scattered light beams in the experimental sample sequentially pass through the condenser, the light filter and the collimation focusing lens and then enter the photodiode and the lock-in amplifier to be collected and amplified.
Secondly, a stimulated Raman scattering imaging method based on rapid and efficient adaptive optical compensation comprises the following steps: the method utilizes a self-adaptive compensation method to modulate incident light of the stimulated Raman scattering system, so that the resolution is improved, and the incident light is a light beam incident to an experimental sample; the method comprises the following steps:
1) dividing the deformable mirror into two areas in half, wherein the laser emits two beams with different wavelengths, and the two beams are respectively incident to the two areas partitioned by the deformable mirror through the optical path to be focused, and the two areas correspond to the two beams with different wavelengths; obtaining a focusing light spot at a focal plane of the microscope objective;
2) placing no experimental sample at the focal plane of the microscope objective, performing phase modulation on two regions of the deformable mirror, performing step 1), obtaining an ideal focusing light spot at the focal plane of the microscope objective, detecting light intensity, and recording the center of the ideal focusing light spot as a focusing center position OfObtaining the ideal focusing spot at the focusing center position OfThe signal light intensity value is used as a reference;
3) placing an experimental sample at the focal plane of the microscope objective, carrying out phase modulation on two areas of the deformable mirror, carrying out the step 1), obtaining a distorted focused light spot at the focal plane of the microscope objective, carrying out light intensity detection, recording the position O of the distorted focused light spot at the focusing center, and obtaining the position of the distorted focused light spotfThe signal light intensity value of (d);
4) carrying out phase modulation on two areas of the deformable mirror, carrying out different phase modulation on the two areas, carrying out light intensity detection in a phase modulation mode, carrying out step 1), obtaining a phase modulation focusing light spot at a focal plane of the microscope objective, analyzing and extracting the phase modulation focusing light spot to obtain a focusing center position O of each of two light beamsfThe signal intensity value is processed to obtain the compensation phase distribution of each light beam on the deformable mirror, namely the compensation phase value corresponding to each micromirror of each light beam in the deformable mirror;
5) respectively loading the compensation phases obtained by the two beams of light onto two subareas corresponding to the deformable mirror during the step 3), and performing the step 1) in an experimental sampleForming a final optical focusing compensation light spot with a focusing center position of OfWhere a stronger nonlinear signal is excited.
The focusing of the light beam in the step 2) is specifically: the laser emits two beams with different wavelengths, the two beams are respectively collimated and expanded, then are reflected on the deformable mirror, and are focused through the objective lens after being combined by the scanning module.
The light intensity detection in the steps 2), 3) and 4) is specifically as follows: the laser emits two beams with different wavelengths, one beam is modulated by the acousto-optic modulator, reflected by the deformable mirror and focused on an experimental sample through the objective lens, a distorted scattering light spot is generated in the experimental sample, the intensity of the two beams generates intensity change at the modulation frequency of the acousto-optic modulator, the scattered beam of the distorted scattering light spot utilizes the optical filter in the light intensity detection module to collect the intensity change information of one beam, the scattered beam is focused by the collimating focusing lens, collected by the photodiode, and the signal amplified by the phase-locked amplifier is used as a focusing center position OfThe signal intensity value of (a).
The acousto-optic modulator modulates the light intensity of one beam of light so that the two beams of light match the requirements of stimulated Raman scattering imaging.
The phase compensation in step 5) specifically means loading an additional compensation phase value on the basis of the original phase of the light beam when step 3) is performed.
And step 4) specifically, fast modulating the two regions of the deformable mirror respectively by using a self-adaptive algorithm so as to obtain a compensation phase value when the acquired signal light intensity is maximum.
The experimental sample is but not limited to living biological tissue, isolated biological tissue, agar block containing small balls and the like.
The stimulated Raman scattering of the invention utilizes two beams of light with different wavelengths to excite the required signal through the nonlinear effect, and the obtained scattering signal is very weak due to the scattering effect in the sample. The system is adaptively corrected by a deformable mirror to improve the intensity of a signal, and two beams of light with different wavelengths are respectively modulated by the deformable mirror through partitioning. By using a deformable mirror, different compensation phases are loaded to the deformable mirror, a light beam is focused and then scattered by a sample, a focus with specific intensity is still formed, and scattered light emitted from the focus is collected and received by a photodiode to be recorded and a signal is amplified.
According to the invention, the compensation phase value of the deformable mirror is adjusted through an optimization method, so that a focusing light spot with stronger central light intensity can be formed in the sample after the phase compensation is carried out on the light beam, and the nonlinear effect can be better excited. The invention starts from the stimulated Raman scattering principle and the phase compensation method, improves the quality of deep imaging inside the scattering medium, and provides a new technical processing mode for living deep unmarked high-resolution microscopic imaging.
The invention has the beneficial effects that:
the invention realizes the fast self-adaptive beam focusing compensation by using the deformable mirror, overcomes the problem of slow speed when the spatial light modulator is used for phase correction in the prior art by using the fast image refreshing speed of the deformable mirror, and improves the beam focusing speed.
Based on the principle of stimulated Raman scattering, the invention obtains the partition phase value matched with the scattering sample by combining the stimulated Raman scattering imaging technology with the adaptive optics technology, thereby obviously improving the light intensity of a focusing center, improving the utilization rate of incident light, effectively improving the collection rate of Raman signals, realizing unmarked imaging with higher resolution ratio while improving the adaptive optics focusing quality, and reducing the damage and toxicity to biological tissues.
Drawings
FIG. 1 is a schematic diagram of the system of the present invention;
FIG. 2 is a graph of Airy spots generated when a 300mm lens focuses 850nm light in an ideal case;
FIG. 3 is a graph showing the results of scattering spots generated after a scattering medium is placed at the f/2 position of a lens;
FIG. 4 is a graph of the results of random phase used to model a scattering medium;
FIG. 5 is a graph of the result of compensating phase values used to modulate scattered light;
fig. 6 is a diagram showing the result of the focus spot after adaptive modulation.
Detailed Description
The invention is further illustrated with reference to the following figures and examples.
As shown in fig. 1, the specific implementation includes a laser 1, half- wave plates 2 and 3, polarization beam splitters 4 and 5, an acousto-optic modulator 6, a beam expanding module, a reflecting mirror 11, a dichroic mirror 16, a phase delay module, a beam combining module, a deformable mirror 19, a scanning module, a microscope objective 26, an experimental sample 27 and a light intensity detecting module; the two half- wave plates 2 and 3 and the two polarization beam splitters 4 and 5 are arranged in front of the output end of the laser 1, the laser emits two beams with different wavelengths, the two beams are adjusted to the same polarization direction through the respective half- wave plates 2 and 3 and the polarization beam splitters 4 and 5, the first beam is emitted from the polarization beam splitters 4 and 5 and then sequentially enters the reflecting mirror 11 through the acousto-optic modulator 6 and the beam expanding module, the second beam is sequentially enters the dichroic mirror 16 through the beam expanding module and the phase delay module, the first beam is reflected by the reflecting mirror 11, then is transmitted by the dichroic mirror 16 and is separated from the second beam reflected by the dichroic mirror 16, then enters two areas of the deformable mirror 19 through the beam combining module, then is reflected to the beam combining module through the deformable mirror 19 to form a beam, and then enters the microscope objective lens 26 through the scanning module to be focused, the experimental sample 27 is located on the focal plane of the microscope objective lens 26, and is transmitted by the experimental sample 27 to generate a scattered light beam which is received by the light intensity detection module for detection, the experimental sample is stimulated to generate a nonlinear signal by stimulated Raman reflection excitation, and the nonlinear signal is received by the light intensity detection module for detection.
Each beam expansion module comprises a front beam expansion module lens 7/8 and a rear beam expansion module lens 9/10; the front beam expanding module lens 7/8 and the rear beam expanding module lens 9/10 are sequentially arranged behind the polarization beam splitter 4/5 along the optical axis, and two light beams emitted by the laser 1 are sequentially expanded to the same diameter after passing through the respective beam expanding modules.
The phase delay module comprises an emergent reflector 12, a front deflection reflector 13, a rear deflection reflector 14 and an incident reflector 15 which are sequentially arranged along a light path, wherein a second light beam which is not modulated by the acousto-optic modulator 6 enters the emergent reflector 12, and is reflected by the emergent reflector 12, the front deflection reflector 13, the rear deflection reflector 14 and the incident reflector 15 in sequence and then is emitted to a dichroic mirror 16; the distance between the front deflection mirror 13 and the exit mirror 12 and the distance between the rear deflection mirror 14 and the entrance mirror 15 can be adjusted, so that different retardation effects can be achieved.
Two beams emitted by the laser are pulse beams, and the pulses of the two beams are synchronized through different delay adjustment of the phase delay module.
The beam combining module comprises a left beam splitter 17 and a right beam splitter 18; the connecting line between the left beam splitter 17 and the right beam splitter 18 is placed in parallel to the reflecting surface of the deformable mirror 19, the left beam splitter 17 and the right beam splitter 18 are semi-transparent semi-reflective mirrors, a first beam of light is transmitted through the right beam splitter 18 and then enters the deformable mirror 19 to be reflected, the beam reflected by the deformable mirror 19 enters the right beam splitter 18 to be reflected to generate a first reflected beam, a second beam of light is transmitted through the left beam splitter 17 and then enters the deformable mirror 19 to be reflected, the second beam of light is reflected through the deformable mirror 19 and then enters the left beam splitter 17 to be reflected to generate a second reflected beam, the second reflected beam of light enters the right beam splitter 18 to be transmitted and then is combined with the first reflected beam, and two beams of light reflected by the deformable mirror 19 respectively enter the left beam splitter 17 and the right beam splitter 18 to be reflected to form a coaxial line.
The deformable mirror 19 is mainly composed of a plurality of micro-mirrors with three-dimensionally adjustable reflective surface, and a spatial light modulator can be used.
The scanning module comprises a front scanning galvanometer 20, a front beam collimating lens 21, a rear beam collimating lens 22, a rear scanning galvanometer 23, a front scanning module lens 24 and a rear scanning module lens 25; the front scanning galvanometer 20, the front beam collimating lens 21, the rear beam collimating lens 22, the rear scanning galvanometer 23, the front scanning module lens 24 and the rear scanning module lens 25 are sequentially arranged behind the beam combining module along a light path, and light beams emitted after being combined by the beam combining module are sequentially reflected by the front scanning galvanometer 20, the front beam collimating lens 21, the rear beam collimating lens 22, the rear scanning galvanometer 23, the front scanning module lens 24 and the rear scanning module lens 25 and then enter the microscope objective lens 26;
the light intensity detection module comprises a condenser 28, a filter 29, a collimation focusing lens 30, a photodiode 31 and a lock-in amplifier 32, and is designed into a transmission system, the condenser 28, the filter 29, the collimation focusing lens 30, the photodiode 31 and the lock-in amplifier 32 are sequentially arranged behind the experimental sample 27 along a light path, and scattered light beams in the experimental sample 27 sequentially pass through the condenser 28, the filter 29 and the collimation focusing lens 30 and then enter the photodiode 31 and the lock-in amplifier 32 to be collected and amplified.
The embodiment of the invention and the implementation process thereof are as follows:
1) the deformable mirror 19 is divided into two areas, which correspond to two light beams with different wavelengths respectively;
2) the objective lens is not placed with a test sample at the focal plane, the light beam is focused by the deformable lens 19 after being partitioned, an ideal focusing light spot is obtained at the focal plane of the objective lens, as shown in fig. 2, and the focusing center position O of the ideal focusing light spot is recordedfAnd signal intensity value, the ideal focused spot signal intensity value in fig. 2 is 0.042139(a.u.) for reference;
3) placing the experimental sample at the focal plane of the objective lens, preloading the initial full 0 phase by using the partitioned deformable mirror 19, detecting the light intensity, and recording the focus center position O of the distorted focus light spot obtained after scatteringf' and signal intensity values, fig. 3, where the intensity signal value for the center position of the distorted focused spot is 0.00042439(a.u.), the scattering medium used for the simulation was placed at the lens focal length f/2 in the simulation, fig. 4;
4) the phase compensation is performed in a phase modulation manner for the two regions of the deformable mirror 19, and the adaptive algorithm used is as follows:
for each beam, the time-varying signal value of the focus center is obtained by first modulating half of the area in the corresponding segment of the deformable mirror at a different frequency. Fourier transform is carried out on the frequency domain data to obtain frequency domain data, phase values of different modulation partitions corresponding to corresponding frequencies in a frequency spectrum are obtained according to the modulated frequencies, and a focusing center O is obtainedf' maximum light intensityThe phase value needed is obtained, and the phase value of the half area is fixed to be unchanged; and adopting the same method to maximize the central light intensity in the other half area of the subarea to obtain the required compensation phase value, and superposing the compensation phase value and the compensation phase value to obtain the complete modulation phase of the corresponding subarea of the light beam. The same operation is carried out on the subarea for modulating the other light, the obtained compensation phase values corresponding to all the areas in the other subarea are processed, the 850nm light beam is simulated (the focusing focal length is 300mm) at this time, and the obtained phase loaded on the deformable mirror 19 is as shown in FIG. 5;
5) loading the obtained compensation phase value on a deformable mirror 19 for light intensity detection, and forming a final optical focusing compensation light spot in the experimental sample, as shown in fig. 6, wherein the modulated light intensity value is 0.027333(a.u.), and compared with the distorted focusing light spot, the final optical focusing compensation light spot can obtain a signal value enhanced by about 64.41 times at the focusing center position, so that the focusing center position is Of' stronger nonlinear signals are excited.
6) The obtained signal is collected by the photodiode 31 and amplified by the lock-in amplifier 32, and the signal value of the point is obtained.
7) Scanning the whole experimental sample by using a scanning module, and repeating the steps 3) to 6) for each scanning point to obtain a signal value of each point in the whole scanning area so as to form an image.
Compared with a traditional stimulated Raman scattering imaging (SRS) system, the stimulated Raman scattering imaging system with the rapid and efficient adaptive optical compensation can obtain better focus quality in samples with the same scattering degree, more effectively utilizes the optical power, reduces the loss of the excessive power to biological tissues, can obtain higher signal-to-noise ratio under the condition that the input light intensity is the same, and effectively improves the imaging speed and the resolution ratio.
Claims (10)
1. A stimulated Raman scattering imaging system based on rapid and efficient adaptive optical compensation is characterized in that: the device comprises a laser (1), half-wave plates (2 and 3), polarization beam splitters (4 and 5), an acousto-optic modulator (6), a beam expanding module, a reflecting mirror (11), a dichroic mirror (16), a phase delay module, a beam combining module, a deformable mirror (19), a scanning module, a microscope objective (26), an experimental sample (27) and a light intensity detection module; the two half-wave plates (2 and 3) and the two polarization beam splitters (4 and 5) are arranged in front of the output end of the laser (1), the laser emits two light beams, the two light beams are adjusted to be in the same polarization direction through the respective half-wave plates (2 and 3) and the polarization beam splitters (4 and 5), the first light beam is emitted from the polarization beam splitters (4 and 5) and then sequentially enters the reflecting mirror (11) through the acousto-optic modulator (6) and the beam expanding module, the second light beam is sequentially enters the dichroic mirror (16) through the beam expanding module and the phase delay module, the first light beam is reflected by the reflecting mirror (11), then is transmitted by the dichroic mirror (16), and then is emitted to two areas of the deformable mirror (19) through the beam combining module at intervals together with the second light beam reflected by the dichroic mirror (16), and then is reflected to the beam combining module through the deformable mirror (19) to form a light beam, the light beam is incident to the microscope objective (26) through the scanning module to be focused, the experimental sample (27) is positioned on the focal plane of the microscope objective (26), and the scattered light beam generated by the transmission of the experimental sample (27) is received by the light intensity detection module to be detected.
2. The system of claim 1, wherein the system comprises: each of the beam expansion modules comprises a front beam expansion module lens (7/8) and a rear beam expansion module lens (9/10); the front beam expanding module lens (7/8) and the rear beam expanding module lens (9/10) are sequentially arranged behind the polarization beam splitter (4/5) along an optical axis, and two light beams emitted by the laser (1) are sequentially expanded to the same diameter after passing through the respective beam expanding modules.
3. The system of claim 1, wherein the system comprises: the phase delay module comprises an emergent reflector (12), a front deflection reflector (13), a rear deflection reflector (14) and an incident reflector (15) which are sequentially arranged along a light path, wherein a second light beam which is not modulated by the acousto-optic modulator (6) is incident to the emergent reflector (12), and is reflected by the emergent reflector (12), reflected by the front deflection reflector (13), reflected by the rear deflection reflector (14) and reflected by the incident reflector (15) in sequence and then is emitted to the dichroic mirror (16).
4. The system of claim 1, wherein the system comprises: the beam combining module comprises a left beam splitter (17) and a right beam splitter (18); the connecting line between the left beam splitter (17) and the right beam splitter (18) is placed in parallel to the reflecting surface of the deformable mirror (19), a first light beam transmits through the right beam splitter (18) and then enters the deformable mirror (19) to be reflected, a light beam reflected back through the deformable mirror (19) enters the right beam splitter (18) to be reflected to generate a first reflected light beam, a second light beam transmits through the left beam splitter (17) and then enters the deformable mirror (19) to be reflected, the second light beam enters the left beam splitter (17) to be reflected to generate a second reflected light beam, the second reflected light beam enters the right beam splitter (18) to be transmitted and then is combined with the first reflected light beam, and two light beams reflected back from the deformable mirror (19) respectively enter the left beam splitter (17) and the right beam splitter (18) to be reflected to form a coaxial line.
5. The system of claim 1, wherein the system comprises: the deformable mirror (19) is mainly composed of a plurality of micro-mirror compact arrays with three-dimensionally adjustable reflecting surface types.
6. The system of claim 1, wherein the system comprises: the scanning module comprises a front scanning galvanometer (20), a front beam collimating lens (21), a rear beam collimating lens (22), a rear scanning galvanometer (23), a front scanning module lens (24) and a rear scanning module lens (25); the front scanning galvanometer (20), the front beam collimating lens (21), the rear beam collimating lens (22), the rear scanning galvanometer (23), the front scanning module lens (24) and the rear scanning module lens (25) are sequentially arranged behind the beam combining module along a light path, and beams emitted after being combined by the beam combining module are sequentially reflected by the front scanning galvanometer (20), the front beam collimating lens (21), the rear beam collimating lens (22), the rear scanning galvanometer (23), the front scanning module lens (24) and the rear scanning module lens (25) and then enter the micro-objective (26); the light intensity detection module comprises a condenser (28), a light filter (29), a collimation focusing lens (30), a photodiode (31) and a phase-locked amplifier (32), wherein the condenser (28), the light filter (29), the collimation focusing lens (30), the photodiode (31) and the phase-locked amplifier (32) are sequentially arranged behind an experimental sample (27) along a light path, and scattered light beams in the experimental sample (27) sequentially pass through the condenser (28), the light filter (29) and the collimation focusing lens (30) and enter the photodiode (31) and the phase-locked amplifier (32) to be collected and amplified.
7. A stimulated Raman scattering imaging method based on rapid and efficient adaptive optical compensation applied to the system of any one of claims 1 to 6, characterized in that: the method utilizes a self-adaptive compensation method to modulate incident light of a stimulated Raman scattering system, and comprises the following steps:
1) the deformable mirror (19) is divided into two areas in half, the laser emits two beams with different wavelengths, the two beams are respectively incident to the two areas of the deformable mirror (19) through the optical path to be focused, and a focused light spot is obtained at the focal plane of the microscope objective (26);
2) an experimental sample (27) is not placed at the focal plane of the microscope objective (26), the two areas of the deformable mirror (19) are not subjected to phase modulation, the step 1) is carried out, an ideal focusing light spot is obtained at the focal plane of the microscope objective (26) and is subjected to light intensity detection, and the center of the ideal focusing light spot is recorded as a focusing center position OfObtaining the ideal focusing spot at the focusing center position OfThe signal light intensity value of (d);
3) placing an experimental sample (27) at the focal plane of a microscope objective (26), carrying out phase modulation on two areas of a deformable mirror (19), carrying out the step 1), obtaining a distorted focused light spot at the focal plane of the microscope objective (26), carrying out light intensity detection, recording the position O of the distorted focused light spot at the focusing center, and obtaining the position of the distorted focused light spotfThe signal light intensity value of (d);
4) two regions of the deformable mirror (19) are phase-modulated and are not phase-modulatedThe same phase modulation is carried out, step 1) is carried out, phase modulation focusing light spots are obtained at the focal plane of the microscope objective (26), and each light beam is obtained at the focusing center position O by analyzing and extracting the phase modulation focusing light spotsfThe signal intensity value of the light source is processed to obtain the compensation phase distribution of each light beam on the deformable mirror (19);
5) respectively loading the compensation phases obtained by the two light beams on two subareas corresponding to the deformable mirror (19) during the step 3) to perform the step 1), forming a final optical focusing compensation spot in the experimental sample (27), wherein the focusing center position is OfWhere a stronger nonlinear signal is excited.
8. The method of claim 1, wherein the method comprises: the light intensity detection in the steps 2), 3) and 4) is specifically as follows:
the laser emits two beams with different wavelengths, wherein one beam is modulated by an acousto-optic modulator (6), is focused on an experimental sample (27) through an objective lens after being reflected and combined on a deformable mirror (19), a distortion scattering light spot is generated in the experimental sample (27), the intensities of the two beams generate intensity change at the modulation frequency of the acousto-optic modulator (6), the scattering light beam of the distortion scattering light spot acquires the intensity change information of one beam of light by using an optical filter (29), is focused by a collimation focusing lens (30), is collected by a photodiode (32) and uses a lock-in amplifier (32) to amplify the signal as a focusing center position OfThe signal intensity value of (a).
9. The method of claim 1, wherein the method comprises: the phase compensation in step 5) specifically means loading an additional compensation phase value on the basis of the original phase of the light beam when step 3) is performed.
10. The method of claim 1, wherein the method comprises: the experimental sample (27) is, but not limited to, a living biological tissue, an isolated biological tissue, an agar block containing a pellet, and the like.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010125026.4A CN111202499B (en) | 2020-02-27 | 2020-02-27 | Stimulated Raman scattering imaging system and method with rapid and efficient self-adaptive optical compensation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010125026.4A CN111202499B (en) | 2020-02-27 | 2020-02-27 | Stimulated Raman scattering imaging system and method with rapid and efficient self-adaptive optical compensation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111202499A true CN111202499A (en) | 2020-05-29 |
| CN111202499B CN111202499B (en) | 2023-05-23 |
Family
ID=70781117
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010125026.4A Active CN111202499B (en) | 2020-02-27 | 2020-02-27 | Stimulated Raman scattering imaging system and method with rapid and efficient self-adaptive optical compensation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111202499B (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112044870A (en) * | 2020-07-15 | 2020-12-08 | 中国科学院西安光学精密机械研究所 | Laser cleaning method and device based on coaxial ranging and real-time adjustable focal length |
| CN112378858A (en) * | 2020-11-11 | 2021-02-19 | 上海交通大学 | Chiral detection system |
| CN112539705A (en) * | 2020-07-14 | 2021-03-23 | 深圳中科飞测科技股份有限公司 | Light-emitting device and focusing method and detection equipment thereof |
| CN112684463A (en) * | 2020-12-21 | 2021-04-20 | 武汉光目科技有限公司 | Area array sweep frequency measuring device and method |
| CN113324540A (en) * | 2021-06-03 | 2021-08-31 | 合肥工业大学 | Spliced mirror pose testing system and method based on scanning galvanometer |
| CN113655026A (en) * | 2021-08-05 | 2021-11-16 | 中国科学院苏州生物医学工程技术研究所 | Large-visual-field high-flux two-photon microscope with elliptic hemispherical curved surface |
| CN113812928A (en) * | 2021-11-22 | 2021-12-21 | 北京航空航天大学 | Multimode imaging device based on Raman spectrum and optical coherence tomography |
| CN114975689A (en) * | 2022-06-02 | 2022-08-30 | 深圳市韵腾激光科技有限公司 | Coaxial laser processing temperature control system of N type Topcon battery |
| CN117870574A (en) * | 2024-03-13 | 2024-04-12 | 广东普洛宇飞生物科技有限公司 | Laser optical device capable of correcting in real time and correction method thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104359892A (en) * | 2014-11-20 | 2015-02-18 | 福建师范大学 | Different modal molecular vibration spectrum detection and imaging device and method |
| US20160103307A1 (en) * | 2014-10-13 | 2016-04-14 | Robert David Frankel | Method and system for stokes interference stimulated fluorescent scattering for in-vivo imaging |
| CN107132646A (en) * | 2017-05-09 | 2017-09-05 | 浙江大学 | Based on the enhanced quick efficient self-adapted optical imagery compensation method of interference and system |
| CN107219617A (en) * | 2017-05-09 | 2017-09-29 | 浙江大学 | Fast accurate optical focus Enhancement Method and system based on DMD |
| JP2018008040A (en) * | 2016-07-05 | 2018-01-18 | キヤノン株式会社 | Wavefront control apparatus, wavefront control method, information acquiring apparatus, program, and storage medium |
| CN107991270A (en) * | 2017-12-28 | 2018-05-04 | 中国科学院苏州生物医学工程技术研究所 | Blood species discrimination system based on coherent antistockes Raman spectroscopy |
| US20190360937A1 (en) * | 2016-12-19 | 2019-11-28 | Chien-Sheng Liao | Stimulated raman spectroscopic microscope by resonant delay-line tuning |
-
2020
- 2020-02-27 CN CN202010125026.4A patent/CN111202499B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160103307A1 (en) * | 2014-10-13 | 2016-04-14 | Robert David Frankel | Method and system for stokes interference stimulated fluorescent scattering for in-vivo imaging |
| CN104359892A (en) * | 2014-11-20 | 2015-02-18 | 福建师范大学 | Different modal molecular vibration spectrum detection and imaging device and method |
| JP2018008040A (en) * | 2016-07-05 | 2018-01-18 | キヤノン株式会社 | Wavefront control apparatus, wavefront control method, information acquiring apparatus, program, and storage medium |
| US20190360937A1 (en) * | 2016-12-19 | 2019-11-28 | Chien-Sheng Liao | Stimulated raman spectroscopic microscope by resonant delay-line tuning |
| CN107132646A (en) * | 2017-05-09 | 2017-09-05 | 浙江大学 | Based on the enhanced quick efficient self-adapted optical imagery compensation method of interference and system |
| CN107219617A (en) * | 2017-05-09 | 2017-09-29 | 浙江大学 | Fast accurate optical focus Enhancement Method and system based on DMD |
| CN107991270A (en) * | 2017-12-28 | 2018-05-04 | 中国科学院苏州生物医学工程技术研究所 | Blood species discrimination system based on coherent antistockes Raman spectroscopy |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112539705A (en) * | 2020-07-14 | 2021-03-23 | 深圳中科飞测科技股份有限公司 | Light-emitting device and focusing method and detection equipment thereof |
| CN112044870A (en) * | 2020-07-15 | 2020-12-08 | 中国科学院西安光学精密机械研究所 | Laser cleaning method and device based on coaxial ranging and real-time adjustable focal length |
| CN112378858A (en) * | 2020-11-11 | 2021-02-19 | 上海交通大学 | Chiral detection system |
| CN112378858B (en) * | 2020-11-11 | 2021-08-20 | 上海交通大学 | Chiral detection system |
| CN112684463A (en) * | 2020-12-21 | 2021-04-20 | 武汉光目科技有限公司 | Area array sweep frequency measuring device and method |
| CN112684463B (en) * | 2020-12-21 | 2024-03-26 | 武汉光目科技有限公司 | Area array sweep frequency measuring device and method |
| CN113324540B (en) * | 2021-06-03 | 2023-07-07 | 合肥工业大学 | System and method for testing pose of spliced mirror based on scanning galvanometer |
| CN113324540A (en) * | 2021-06-03 | 2021-08-31 | 合肥工业大学 | Spliced mirror pose testing system and method based on scanning galvanometer |
| CN113655026A (en) * | 2021-08-05 | 2021-11-16 | 中国科学院苏州生物医学工程技术研究所 | Large-visual-field high-flux two-photon microscope with elliptic hemispherical curved surface |
| CN113655026B (en) * | 2021-08-05 | 2024-01-23 | 中国科学院苏州生物医学工程技术研究所 | Elliptic hemispherical surface large-view high-flux two-photon microscope |
| CN113812928A (en) * | 2021-11-22 | 2021-12-21 | 北京航空航天大学 | Multimode imaging device based on Raman spectrum and optical coherence tomography |
| CN114975689A (en) * | 2022-06-02 | 2022-08-30 | 深圳市韵腾激光科技有限公司 | Coaxial laser processing temperature control system of N type Topcon battery |
| CN114975689B (en) * | 2022-06-02 | 2024-06-04 | 深圳市韵腾激光科技有限公司 | Coaxial laser processing temperature control system of N-type Topcon battery |
| CN117870574A (en) * | 2024-03-13 | 2024-04-12 | 广东普洛宇飞生物科技有限公司 | Laser optical device capable of correcting in real time and correction method thereof |
| CN117870574B (en) * | 2024-03-13 | 2024-05-14 | 广东普洛宇飞生物科技有限公司 | Laser optical device capable of correcting in real time and correction method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111202499B (en) | 2023-05-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111202499B (en) | Stimulated Raman scattering imaging system and method with rapid and efficient self-adaptive optical compensation | |
| JP5649828B2 (en) | Laser microscope equipment | |
| CN107941763B (en) | Coaxial three-dimensional stimulated radiation loss super-resolution microscopic imaging method and device | |
| US8817255B2 (en) | Apparatus and method for irradiating a scattering medium | |
| CN110836876B (en) | Super-resolution microscopy method and system based on saturated pumping-stimulated radiation detection | |
| CN212489863U (en) | Stimulated Raman scattering imaging system with rapid and efficient adaptive optical compensation | |
| US8097864B2 (en) | System, method and computer-accessible medium for providing wide-field superresolution microscopy | |
| de Aguiar et al. | Enhanced nonlinear imaging through scattering media using transmission-matrix-based wave-front shaping | |
| CN111521596B (en) | Fluorescence differential super-resolution imaging method and imaging system | |
| JP2003227796A (en) | Method and arrangement for grasping sample by depth decomposition | |
| CN108303421B (en) | Three-dimensional high-speed wide-field tomography method and device | |
| CN114895450B (en) | Super-resolution microscopic imaging system and method based on second harmonic | |
| CN104359892A (en) | Different modal molecular vibration spectrum detection and imaging device and method | |
| CN107478630B (en) | Device and method for improving single-molecule optical imaging contrast | |
| CN110146473B (en) | Axial super-resolution two-photon fluorescence microscopy device and method | |
| CN113251916B (en) | Femtosecond interference scattering microscopic imaging system and measuring method | |
| WO2023029471A1 (en) | Multi-modal nonlinear microscopic imaging system | |
| CN111122535B (en) | High-spectrum rapid imaging measurement system for molecular vibration mode | |
| CN111537478B (en) | Super-resolution optical microscopic imaging system based on frequency division multiplexing | |
| NL2017449B1 (en) | Simultaneous Hyperspectral Coherent Raman Microscope | |
| CN106404723B (en) | Second harmonic high-resolution imaging method and system | |
| CN115078326A (en) | Stimulated Raman microscopic imaging device combined with optical tweezers | |
| CN111579486B (en) | Super-resolution imaging method and imaging system based on low-power stimulated emission loss | |
| CN111678898B (en) | Time-resolved optical biological detection equipment based on broadband stimulated radiation and detection imaging method thereof | |
| US11808702B2 (en) | Hybrid multi-photon microscopy |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |