CN110623641A - Self-adaptive second and third harmonic joint detection microscopic imaging method and device - Google Patents
Self-adaptive second and third harmonic joint detection microscopic imaging method and device Download PDFInfo
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Abstract
The invention relates to a self-adaptive second and third harmonic joint detection microscopic imaging method and a device, belonging to the technical field of optical microscopic imaging; according to the invention, a second harmonic signal detection module and a third harmonic signal detection module are simultaneously arranged on a detection path of a traditional harmonic microscopic device, separation and filtering of a second harmonic signal and a third harmonic signal are realized by using a dichroic mirror and an optical filter, and the second harmonic signal and the third harmonic signal are respectively detected by using independent photoelectric detectors; an adaptive aberration correcting device is introduced into the system and is used for correcting the aberration existing when the sample is subjected to large-depth detection. The device comprises a laser scanning system, a self-adaptive aberration correction system, a harmonic signal excitation system, a harmonic signal detection system, a wide-field imaging system and an image reconstruction and data processing system. The invention realizes the joint detection of second harmonic signal and third harmonic signal and the complementation of sample structure information, and keeps the imaging quality during the large-depth detection.
Description
Technical Field
The invention belongs to the field of optical microscopic measurement, and mainly relates to a self-adaptive second harmonic and third harmonic joint detection microscopic imaging method and device for three-dimensional microstructure imaging of a living biological sample.
Background
With the continuous development of scientific technology, high-resolution and high-penetration depth imaging of living biological samples has become an indispensable condition for the research of system biology. However, the resolution of conventional optical microscopy imaging is limited due to the existence of the optical diffraction limit. Fluorescence microscopy enables super-resolution imaging of biological samples. Among them, the multiphoton microscopic imaging technique is the best non-invasive fluorescence microscopic imaging method. The two-photon excitation microscopic process has actual energy conversion, and the response time is in the nanosecond order. And the generation process of the second harmonic and the third harmonic only has virtual energy conversion, and the response time is in the femtosecond magnitude. Therefore, high-sensitivity and high-speed response imaging can be realized by using the harmonic generation process for imaging. The harmonic microscopic imaging method is a three-dimensional optical imaging technology and has the imaging characteristic of nonlinear optical imaging. The excitation of the harmonic signal requires a high intensity laser pulse, so that the harmonic signal is only generated in the focal region. The nonlinear strong local effect reduces the background noise interference outside the focus during imaging, and has high signal-to-noise ratio. The harmonic signal is a nonlinear effect generated by the sample itself, and does not need an external fluorescent label, so that the activity of the biological sample is not influenced. Excitation of harmonic signals typically uses near-infrared excitation light, which can achieve very high detection depths. The generation of the second harmonic requires that the sample does not have inverted symmetry. The generation of the third harmonic is not required for the properties of the sample. The second harmonic and the third harmonic can reflect different structural information of the sample, and the complementary of the structural information of the sample can be realized by combined detection.
When living biological tissues are imaged at high depth, obvious aberration exists in the imaging process due to the nonuniformity of the optical characteristics of a sample and the mismatch of refractive indexes. The harmonic microscopic imaging process is extremely sensitive to aberrations as a high-order nonlinear optical process. The presence of aberrations can reduce the intensity of harmonic signals and the quality of the imaging. And the larger the probe depth, the larger the influence of the aberration. Some detailed information of great research interest may not be imaged due to the presence of aberrations. In order to achieve high-depth and high-quality imaging, aberrations caused by the biological sample must be corrected.
Therefore, one technical problem that needs to be urgently solved by those skilled in the art is: how to simultaneously carry out the combined detection of second harmonic and third harmonic on a sample and realize the fusion of second harmonic and third harmonic images and the information complementation through data processing. In addition, when large-depth detection is performed by harmonic microscopic imaging, aberration caused by a sample can be corrected.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a self-adaptive second harmonic and third harmonic joint detection microscopic imaging method and device. The invention simultaneously arranges a second harmonic detection module and a third harmonic detection module on a detection path in a harmonic microscopic imaging device, utilizes a dichroic mirror and an optical filter to realize the separation and filtering of a second harmonic signal and a third harmonic signal, and adopts independent photoelectric detectors to respectively detect the second harmonic signal and the third harmonic signal; an adaptive aberration correcting device is introduced into the system and used for correcting the aberration existing when the sample is detected at a large depth. The invention overcomes the defects of single detection mode and aberration in large-depth detection of the traditional harmonic microscopic imaging.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a self-adaptive second harmonic and third harmonic joint detection microscopic imaging device, which is characterized by comprising the following components: the system comprises a laser scanning system, a self-adaptive aberration correction system, a harmonic signal excitation system, a harmonic signal detection system, a wide-field imaging system and an image reconstruction and data processing system;
wherein: the laser scanning system comprises an ultrashort pulse laser light source, an optical switch, a light beam conversion unit and a light beam scanning element; the ultrashort pulse laser light source is used for providing excitation pulse light for generating harmonic signals, the optical switch is used for controlling the on-off of the excitation light, the light beam conversion unit is used for adjusting the size of the light beam of the excitation pulse light, and the light beam scanning element is used for scanning a sample by the excitation light.
The self-adaptive aberration correction system is arranged behind the laser scanning system and comprises a lens group, a wavefront sensor and a dynamic optical element; the lens group is used for connecting the light beam scanning element and the dynamic optical element, the wavefront sensor is used for wavefront sensing, and the dynamic optical element is used for modulating the wavefront of the light path so as to correct the aberration in the imaging process.
The harmonic signal excitation system is arranged behind the self-adaptive aberration correction system, and comprises a lens group and a microscope objective, wherein the lens group is connected with the dynamic optical element and the entrance pupil surface behind the microscope objective to form a 4f system for exciting the focusing of light beams and exciting harmonic signals.
The harmonic signal detection system comprises a three-dimensional micro-displacement platform, a harmonic signal detection objective lens, a dichroic mirror, an optical filter and a photoelectric detector; for the reflection type detection, the harmonic signal detection objective is a microscope objective; for transmission detection, a harmonic signal detection objective lens is arranged at the transmission end of a sample; the lens is used for focusing harmonic signals, the dichroic mirror is used for separating second harmonic signals and third harmonic signals, the optical filter is used for filtering stray light except the harmonic signals, and the photoelectric detector is used for collecting the harmonic signals.
The wide-field imaging system comprises an LED light source, a lens group and a wide-field imaging camera; the LED light source is used for wide field illumination, the lens group is used for light path adjustment, and the wide field imaging camera is used for imaging.
The image reconstruction and data processing system is connected with a scanning element in the laser scanning system, a dynamic optical element in the self-adaptive aberration correction system and a photoelectric detector and is used for carrying out global image processing on a scanning image acquired by the imaging device.
The centers of the optical surfaces of all the optical elements are coincident with the optical axis formed by the incident laser and the central beam of the harmonic signal, and all the lenses are perpendicular to the optical axis.
Further, the dynamic optical element is a deformable mirror or a spatial light modulator, or a combined system consisting of the deformable mirror and the spatial light modulator.
Further, the beam scanning element may be a scanning galvanometer or an acousto-optic deflector.
Furthermore, in the ultrashort pulse laser source and the beam transformation system, the ultrashort pulse laser source is a femtosecond pulse laser source.
Furthermore, the harmonic signal detection system adopts second and third harmonic synchronous detection; the dichroic mirror is used for separating second and third harmonic signals; the transmission wavelengths of the optical filter correspond to the wavelengths of the second harmonic signal and the third harmonic signal respectively; two independent photoelectric detectors respectively detect second harmonic wave signals and third harmonic wave signals, and sensitive wavelengths of the detectors respectively correspond to corresponding second harmonic wave wavelengths and third harmonic wave wavelengths.
A self-adaptive second and third harmonic joint detection microscopic imaging method is characterized by comprising the following steps:
(1) the laser scanning system generates a short pulse laser scanning beam;
(2) the self-adaptive aberration correction system realizes self-adaptive correction of aberration by modulating the wavefront of an optical path;
(3) the harmonic signal excitation system excites the sample to generate a harmonic signal;
(4) the harmonic signal detection system collects harmonic signals and performs second harmonic signal separation, third harmonic signal separation, filtering and synchronous detection;
(5) and the image reconstruction and data processing system carries out image reconstruction and data processing on the signals acquired by the photoelectric detector to reconstruct a final harmonic image.
The self-adaptive second and third harmonic joint detection microscopic imaging method is characterized in that the self-adaptive aberration correction can be performed by selecting a wavefront sensing mode or a wavefront-free sensing mode according to the existence of wavefront sensing.
The self-adaptive second and third harmonic joint detection microscopic imaging method is characterized in that harmonic signals can be detected in a reflection mode or a transmission mode.
The self-adaptive second and third harmonic joint detection microscopic imaging method is characterized in that the image reconstruction and data processing can independently form a second harmonic image or a third harmonic image and a second harmonic and third harmonic fusion image.
The invention has the beneficial effects that the second harmonic signal detection module and the third harmonic signal detection module are simultaneously arranged on the detection path in the traditional harmonic microscopic method, so that the joint detection of the second harmonic and the third harmonic and the complementation of the sample structure information are realized. In addition, an adaptive aberration correction module is introduced into the imaging system and used for correcting aberration caused by a sample during large-depth imaging, so that the imaging depth and the imaging quality of harmonic microscopic imaging are improved.
Drawings
Fig. 1 is a schematic diagram of an adaptive second harmonic and third harmonic joint detection microscopic imaging method and device in embodiment 1 of the present invention.
Fig. 2 is a schematic diagram of an adaptive second harmonic and third harmonic joint detection microscopic imaging method and apparatus in embodiment 2 of the present invention.
Wherein: 1-ultrashort pulse laser light source, 2-optical switch, 3-light beam conversion unit, 4-light beam scanning element, 5-first lens group, 6-dynamic optical element, 7-second lens group, 8-first dichroic mirror, 9-third lens group, 10-microobjective, 11-sample, 12-three-dimensional micro displacement stage, 13-harmonic signal detection objective, 14-first lens, 15-second dichroic mirror, 16-first optical filter, 17-third dichroic mirror, 18-second harmonic optical filter, 19-first pinhole, 20-second harmonic photoelectric detector, 21-second lens, 22-third harmonic optical filter, 23-second pinhole, 24-third harmonic photoelectric detector, 25-LED light source, 26-fourth lens group, 27-wide field imaging camera, 28-fourth dichroic mirror.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below.
The invention provides a self-adaptive second harmonic and third harmonic joint detection microscopic imaging method, which comprises the following steps:
(1) the laser scanning system generates a short pulse laser scanning beam;
(2) the self-adaptive aberration correction system realizes self-adaptive correction of aberration by modulating the wavefront of an optical path;
(3) the harmonic signal excitation system excites the sample to generate a harmonic signal;
(4) the harmonic signal detection system collects harmonic signals and performs second harmonic signal separation, third harmonic signal separation, filtering and synchronous detection;
(5) and the image reconstruction and data processing system carries out image reconstruction and data processing on the signals acquired by the photoelectric detector to reconstruct a final harmonic image.
The invention also provides a self-adaptive second harmonic and third harmonic joint detection microscopic imaging device according to the method.
Example 1:
fig. 1 shows a schematic diagram of an adaptive second-third harmonic joint detection microscopy imaging device according to this embodiment 1. The device comprises an ultrashort pulse laser light source 1, an optical switch 2, a light beam conversion unit 3, a light beam scanning element 4, a first lens group 5, a dynamic optical element 6, a second lens group 7, a first dichroic mirror 8, a third lens group 9, a microscope objective 10, a sample 11, a three-dimensional micro-displacement table 12, a harmonic signal detection objective 13, a first lens 14, a second dichroic mirror 15, a first optical filter 16, a third dichroic mirror 17, a second harmonic optical filter 18, a first pinhole 19, a second harmonic photoelectric detector 20, a second lens 21, a third harmonic optical filter 22, a second pinhole 23, a third harmonic photoelectric detector 24, an LED light source 25, a fourth lens group 26 and a wide field imaging camera 27. The connection relationship of the components is as follows: the centers of the optical surfaces of all the optical elements are superposed with an optical axis formed by the incident laser and the central beam of the harmonic signal, and all the lenses are perpendicular to the optical axis; the laser scanning system consists of an ultrashort pulse laser light source 1, an optical switch 2, a light beam conversion unit 3 and a light beam scanning element 4, and generates short pulse laser scanning light beams; the first lens group 5, the dynamic optical element 6 and the second lens group 7 form a self-adaptive aberration correction system which is arranged behind the laser scanning system to modulate the wavefront of the optical path to realize the self-adaptive correction of the aberration; the third lens group 9, the microscope objective 10, the sample 11 and the three-dimensional micro displacement table 12 form a harmonic signal excitation system which is arranged behind the self-adaptive aberration correction system and used for exciting the sample and generating a harmonic signal; a harmonic signal detection system is composed of a harmonic signal detection objective lens 13, a first lens 14, a second dichroic mirror 15, a first optical filter 16, a third dichroic mirror 17, a second harmonic optical filter 18, a first pinhole 19, a second harmonic photoelectric detector 20, a second lens 21, a third harmonic optical filter 22, a second pinhole 23 and a third harmonic photoelectric detector 24, and is used for collecting harmonic signals and performing second harmonic signal and third harmonic signal separation, filtering and synchronous detection; the first dichroic mirror 8, the LED light source 25, the fourth lens group 26, and the wide-field imaging camera 27 constitute a wide-field imaging system.
In this embodiment, the detection mode of the harmonic signal is transmission detection;
the aberration correction system adopts a wavefront-free sensing mode to correct aberration, and the dynamic optical element adopts a spatial light modulator;
the light beam scanning element is a scanning galvanometer;
the wide field imaging system employs backlighting.
Example 2:
fig. 2 shows a schematic diagram of an adaptive second-third harmonic joint detection microscopy imaging device in this embodiment 2. The device comprises an ultrashort pulse laser light source 1, an optical switch 2, a light beam conversion unit 3, a light beam scanning element 4, a first lens group 5, a dynamic optical element 6, a second lens group 7, a first dichroic mirror 8, a third lens group 9, a microscope objective 10, a sample 11, a three-dimensional micro-displacement table 12, a first lens 14, a first optical filter 16, a third dichroic mirror 17, a second harmonic optical filter 18, a first pinhole 19, a second harmonic photoelectric detector 20, a second lens 21, a third harmonic optical filter 22, a second pinhole 23, a third harmonic photoelectric detector 24, an LED light source 25, a fourth lens group 26, a wide field imaging camera 27 and a fourth dichroic mirror 28. The connection relationship of the components is as follows: the centers of the optical surfaces of all the optical elements are superposed with an optical axis formed by the incident laser and the central beam of the harmonic signal, and all the lenses are perpendicular to the optical axis; the laser scanning system consists of an ultrashort pulse laser light source 1, an optical switch 2, a light beam conversion unit 3 and a light beam scanning element 4, and generates short pulse laser scanning light beams; the first lens group 5, the dynamic optical element 6 and the second lens group 7 form a self-adaptive aberration correction system which is arranged behind the laser scanning system to modulate the wavefront of the optical path to realize the self-adaptive correction of the aberration; the third lens group 9, the microscope objective 10, the sample 11 and the three-dimensional micro displacement table 12 form a harmonic signal excitation system which is arranged behind the self-adaptive aberration correction system and used for exciting the sample and generating a harmonic signal; a harmonic signal detection system is composed of a microscope objective 10, a first lens 14, a first optical filter 16, a third dichroic mirror 17, a second harmonic optical filter 18, a first pinhole 19, a second harmonic photoelectric detector 20, a second lens 21, a third harmonic optical filter 22, a second pinhole 23, a third harmonic photoelectric detector 24 and a fourth dichroic mirror 28, harmonic signals are collected, and second harmonic and third harmonic signal separation, filtering and synchronous detection are carried out; the first dichroic mirror 8, the LED light source 25, the fourth lens group 26, and the wide-field imaging camera 27 constitute a wide-field imaging system.
In this embodiment, the detection mode of the harmonic signal is a reflective detection;
the microscope objective is a focusing objective of exciting light and a detection objective of harmonic signals;
the aberration correction system adopts a wavefront-free sensing mode to correct aberration, and the dynamic optical element adopts a deformable reflector;
the light beam scanning element is a scanning galvanometer;
the wide field imaging system employs backlighting.
The adaptive confocal line scanning harmonic microscopic imaging method and device proposed by the present invention are described in detail above, and the principle and the implementation of the present invention are explained in the present document by applying specific examples, and the description of the above examples is only used to help understanding the method and the core idea of the present invention; meanwhile, for a person skilled in the art, according to the idea of the present invention, there are changes in the specific embodiments and the application scope, and these changes should be covered by the protection scope of the appended claims. In view of the above, the present disclosure should not be construed as limiting the invention.
Claims (9)
1. An adaptive second harmonic and third harmonic joint detection microscopic imaging device, which is characterized by comprising: the system comprises a laser scanning system, a self-adaptive aberration correction system, a harmonic signal excitation system, a harmonic signal detection system, a wide-field imaging system and an image reconstruction and data processing system;
wherein: the laser scanning system comprises an ultrashort pulse laser light source, an optical switch, a light beam conversion unit and a light beam scanning element; the ultra-short pulse laser light source is used for providing excitation pulse light for generating harmonic signals, the optical switch is used for switching control of the excitation light, the light beam conversion unit is used for adjusting the size of the light beam of the excitation pulse light, and the light beam scanning element is used for scanning a sample by the excitation light;
the self-adaptive aberration correction system is arranged behind the laser scanning system and comprises a lens group, a wavefront sensor and a dynamic optical element; the lens group is used for connecting the light beam scanning element and the dynamic optical element, the wavefront sensor is used for wavefront sensing, and the dynamic optical element is used for modulating the wavefront of the light path so as to correct the aberration in the imaging process;
the harmonic signal excitation system is arranged behind the self-adaptive aberration correction system, comprises a lens group and a microscope objective, is connected with the dynamic optical element and the entrance pupil surface behind the microscope objective by the lens group, and forms a 4f system for exciting the focusing of light beams and the excitation of harmonic signals;
the harmonic signal detection system comprises a three-dimensional micro-displacement platform, a harmonic signal detection objective lens, a dichroic mirror, an optical filter and a photoelectric detector; for the reflection type detection, the harmonic signal detection objective is a microscope objective; for transmission detection, a harmonic signal detection objective lens is arranged at the transmission end of a sample; the lens is used for focusing harmonic signals, the dichroic mirror is used for separating second harmonic signals and third harmonic signals, the optical filter is used for filtering stray light except the harmonic signals, and the photoelectric detector is used for collecting the harmonic signals;
the wide-field imaging system comprises an LED light source, a lens group and a wide-field imaging camera; the LED light source is used for wide-field illumination, the lens group is used for light path adjustment, and the wide-field imaging camera is used for imaging;
the image reconstruction and data processing system is connected with a scanning element in the laser scanning system, a dynamic optical element in the self-adaptive aberration correction system and a photoelectric detector and is used for carrying out global image processing on a scanning image acquired by an imaging device;
the centers of the optical surfaces of all the optical elements are coincident with the optical axis formed by the incident laser and the central beam of the harmonic signal, and all the lenses are perpendicular to the optical axis.
2. The adaptive second harmonic and third harmonic joint detection microscopy imaging device according to claim 1, wherein the dynamic optical element is a deformable mirror or a spatial light modulator, or a combined system of the deformable mirror and the spatial light modulator.
3. An adaptive second-third harmonic joint detection microscopy imaging device as defined in claim 1, wherein the beam scanning element is a scanning galvanometer or an acousto-optic deflector.
4. The adaptive second and third harmonic joint detection microscopy imaging device according to claim 1, wherein in the ultrashort pulse laser source and the beam transformation system, the ultrashort pulse laser source is selected from a femtosecond pulse laser source.
5. The adaptive second and third harmonic joint detection microscopic imaging device according to claim 1, wherein the harmonic signal detection system adopts second and third harmonic synchronous detection; the dichroic mirror is used for separating second and third harmonic signals; the transmission wavelengths of the optical filter correspond to the wavelengths of the second harmonic signal and the third harmonic signal respectively; two independent photoelectric detectors respectively detect second harmonic wave signals and third harmonic wave signals, and sensitive wavelengths of the detectors respectively correspond to corresponding second harmonic wave wavelengths and third harmonic wave wavelengths.
6. A self-adaptive second and third harmonic joint detection microscopic imaging method is characterized by comprising the following steps:
(1) the laser scanning system generates a short pulse laser scanning beam;
(2) the self-adaptive aberration correction system realizes self-adaptive correction of aberration by modulating the wavefront of an optical path;
(3) the harmonic signal excitation system excites the sample to generate a harmonic signal;
(4) the harmonic signal detection system collects harmonic signals and performs second harmonic signal separation, third harmonic signal separation, filtering and synchronous detection;
(5) and the image reconstruction and data processing system carries out image reconstruction and data processing on the signals acquired by the photoelectric detector to reconstruct a final harmonic image.
7. The adaptive second harmonic joint detection microscopy imaging method as claimed in claim 6, wherein the adaptive aberration correction is performed according to the presence or absence of wavefront sensing, and optionally by a wavefront sensing method or a wavefront-free sensing method.
8. The adaptive second and third harmonic joint detection microscopy imaging method of claim 6, wherein the harmonic signals are detected in both a reflection mode and a transmission mode.
9. The adaptive second and third harmonic joint detection microscopy imaging method of claim 6, wherein the image reconstruction and data processing are performed separately to form a second harmonic image or a third harmonic image and a second and third harmonic fused image.
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Application publication date: 20191231 |