CN118190826A - Wide-field photoacoustic microscopic imaging method and device - Google Patents
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Abstract
The invention discloses a wide-field photoacoustic microscopic imaging method and a device thereof, wherein the method comprises the following steps: the pulse light excites the sample to generate a wide-field photoacoustic wave which causes each particle on the surface of the sample to generate vibration displacement; detecting instantaneous vibration displacement of each particle on the surface of the sample under the action of photoacoustic waves at a certain moment by using wide-field interference light; detecting all vibration displacement amounts of all particles on the surface of a sample under the action of full-period photoacoustic waves by setting trigger delay time of excitation light and detection light; and reconstructing a wide-field photoacoustic image by carrying out maximum projection on all vibration displacement amounts of each particle on the surface of the sample. The wide-field photoacoustic microscopy imaging apparatus includes: the device comprises a pulse light excitation module, an excitation detection coupling module, a displacement detection module, a pumping detection module and a signal processing module. The method provided by the invention overcomes the limitation that the traditional photoacoustic imaging is difficult to realize high-resolution wide-field imaging, realizes non-contact, non-scanning and rapid acquisition of optical absorption information, and can be applied to the fields of biomedicine, material detection and the like.
Description
Technical Field
The invention belongs to the technical field of photoacoustic imaging, and particularly relates to a wide-field photoacoustic microscopic imaging method and an imaging device thereof.
Background
Photoacoustic imaging technology has imaging capabilities of high resolution and high optical contrast. Conventional photoacoustic imaging cannot achieve both high resolution and wide field imaging, and photoacoustic signals are obtained for imaging using limited bandwidth ultrasonic transducers for contact detection. Traditional photoacoustic imaging is divided into photoacoustic microscopic imaging and photoacoustic computed tomography imaging, and photoacoustic microscopy has the characteristic of high resolution, but the point-by-point scanning mode is contrary to the concept of wide field, so that the imaging speed of the technology is restricted; while photoacoustic computed tomography achieves wide-field fast scanning, its resolution is far lower than that of photoacoustic microscopy. The traditional photoacoustic imaging uses an ultrasonic transducer when collecting signals, and the ultrasonic transducer utilizes piezoelectric material acoustic resonance to detect photoacoustic signals, so that the ultrasonic transducer can only receive photoacoustic signals near resonance frequency, and photoacoustic signals of other frequencies of a sample are lost. By combining the photoacoustic effect with the sheet optical interference technology and adopting the pumping detection to realize the detection of the wide-field photoacoustic signal, the information of the substance absorber can be quickly, widely and contactlessly acquired and imaged, the defect that the past photoacoustic imaging cannot be achieved due to high speed and high resolution is overcome, and the photoacoustic imaging can be applied to more application scenes.
Disclosure of Invention
The invention mainly aims to overcome the defects and shortcomings of the prior art, and provides a wide-field photoacoustic microscopic imaging method and an imaging device thereof, wherein large-spot pulse laser is used as an excitation source, and the principle of photoacoustic effect is utilized, and the wide-field displacement detection technology and the pumping detection technology are combined, so that the surface vibration displacement of a sample caused by the photoacoustic effect is detected under the condition of no contact, and further photoacoustic signals of the sample are obtained through inversion, so that the rapid and high-resolution wide-field photoacoustic microscopic imaging is realized.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in a first aspect, the present invention provides a wide-field photoacoustic microscopy imaging method, the method comprising the steps of:
S1, pulse laser generated by a laser is subjected to beam expansion and collimation to form a large light spot to excite a sample to generate a wide-field photoacoustic wave, and each particle on the surface of the sample can be caused to generate vibration displacement by the photoacoustic wave;
s2, detecting light with wide field polarization emitted by the imaging light source is used as detection light, detection of optical paths of all particles on the surface of the sample is achieved through polarized interference light, and the optical paths are used for representing instantaneous vibration displacement of all particles on the surface of the sample under the action of photoacoustic waves at the moment;
S3, setting trigger delay time of the excitation light and the detection light, and changing the trigger delay time to measure for a plurality of times to obtain all vibration displacement amounts of each particle on the surface of the sample under the action of the full-period photoacoustic wave;
And S4, reconstructing a wide-field photoacoustic image by carrying out maximum projection on all vibration displacement amounts of each particle on the surface of the sample under the action of the full-period photoacoustic wave.
In the preferred technical scheme, in the step S1, the wide-field photoacoustic wave may cause each particle on the surface of the sample to generate vibration displacement, specifically:
Excitation light irradiates the sample excitation light sound wave, when the photoacoustic wave propagates to the sample surface, vibration displacement is generated on the sample surface, the size of the vibration displacement is determined by the sound pressure size of the photoacoustic wave, and the normalized displacement size and sound pressure have the following relation:
Wherein, Is/>Displacement of sample surface at moment,/>For density of medium,/>As the speed of sound in the medium,For/>Sound pressure of ultrasonic wave acting on the sample surface at the moment.
In the step S2, detection of the optical path of each particle on the surface of the sample is realized by the detection light through polarized interference light, and the optical path is used for representing the instantaneous vibration displacement of each particle on the surface of the sample under the action of the photoacoustic wave at the moment, specifically: the detection light is divided into two beams of light rays with mutually orthogonal polarization directions by the nomadic prism, the two beams of light rays respectively irradiate adjacent areas on the surface of a sample after passing through the objective lens and are reflected back to the nomadic prism to be combined, the two beams of light rays carry different optical paths through different positions on the surface of the sample, wide-field polarized light interference is carried out through the polarization analyzer after the beam combination, and the interference light intensity of each particle and the corresponding vibration displacement follow the following formula:
Wherein, For the surface/>Interference intensity at particles,/>To detect the light amplitude,/>Is the center wavelength of the probe light,/>Is the direction in which the incident light splits after passing through the nomads prism,/>Is the distance that the incident light separates after passing through the nomads prism,/>And/>Determined by nomads prism,/>Is the sample surface/>The surface vibration displacement of the particles can be detected by the Normasky prism, so that the instantaneous vibration displacement of each particle on the surface of the sample under the action of the photoacoustic wave can be detected.
In the step S3, the trigger delay time of the excitation light and the detection light is set, and the trigger delay time is changed to perform multiple measurements to obtain all vibration displacement amounts of each particle on the surface of the sample under the action of the full-period photoacoustic wave, which specifically comprises the following steps:
S1, triggering an imaging camera to expose the camera;
S2, triggering a laser, and triggering the laser to emit pulse light to excite a sample to generate a wide-field photoacoustic wave after the exposure time of the imaging camera is started;
s3, triggering an imaging light source, so that the imaging light source is triggered behind the laser and within the exposure time of the imaging camera;
and S4, measuring the trigger delay time of the excitation light and the detection light for a plurality of times to obtain all vibration displacement amounts of each particle on the surface of the sample under the action of the full-period photoacoustic wave.
In step S4, the wide-field photoacoustic image is reconstructed by maximum projection of all vibration displacement amounts of each particle on the surface of the sample under the action of the full-period photoacoustic wave, specifically:
the instantaneous vibration displacement of each particle on the surface of the sample at a certain moment is obtained through the nomads prism and is as follows after being normalized:
the instantaneous vibration displacement represents the light absorption intensity of different depths of the sample, and the wide-field photoacoustic image can be reconstructed by carrying out maximum projection on all vibration displacement amounts of each particle on the surface of the sample under the action of the full-period photoacoustic wave.
In a second aspect, the invention provides a wide-field photoacoustic microscopic imaging device, which comprises a pulse light excitation module, an excitation detection coupling module, a displacement detection microscopic module, a pumping detection module and a signal processing imaging module;
The pulse light excitation module comprises a pulse laser, an energy regulator I and a collimation beam expander I, wherein the pulse laser, the energy controller I and the collimation beam expander I are sequentially connected; the excitation detection coupling module comprises a dichroic mirror and a multi-wavelength objective lens, wherein the dichroic mirror is connected with the multi-wavelength objective lens, and the dichroic mirror is also connected with the first collimating beam expander;
The displacement detection module comprises an imaging light source, an energy controller II, a collimation beam expander II, a polarizer, a beam splitting prism, a Nomars prism, an analyzer and an eyepiece, wherein the imaging light source, the energy controller II, the collimation beam expander II, the polarizer, the beam splitting prism and the Nomars prism are sequentially connected, and the beam splitting prism is also sequentially connected with the analyzer and the eyepiece; the nomads prism is also connected with a dichroic mirror;
The pumping detection module comprises a singlechip and an imaging camera, wherein the singlechip is connected with the imaging camera and is also respectively connected with a laser and an imaging light source;
The signal processing module comprises a computer and a two-dimensional displacement platform, wherein the computer is connected with the single chip microcomputer, and the single chip microcomputer is connected with the two-dimensional displacement platform.
As an optimal technical scheme, in the pulse light excitation module, a light beam emitted by a pulse laser sequentially passes through an energy controller and a collimation beam expander to obtain large-spot pulse light suitable for excitation of photoacoustic signals by a sample;
The energy controller consists of a polarizer, a half wave plate and a polarization beam splitter;
the collimating beam expander consists of a first lens, a diaphragm and a second lens.
In the excitation detection coupling module, excitation light and detection light are combined through a dichroic mirror, and focused through a multi-wavelength objective lens, so that excitation of wide-field photoacoustic waves and detection of instantaneous vibration displacement of each particle on the surface of a sample are performed on the sample.
In the preferred technical scheme, in the displacement detection module, light beams emitted by an imaging light source are adjusted to proper energy and light spot size through an energy controller and a beam expanding collimator, the light beams enter a polarizer to be changed into polarized light, the polarized light enters a nomads prism through a beam splitting prism and is divided into two light beams with mutually orthogonal polarization directions, the two light beams respectively irradiate adjacent areas on the surface of a sample after passing through an objective lens and are reflected back to the nomads prism to be combined, the two light beams carry different optical paths through different positions on the surface of the sample, interference intensity of each particle on the surface of the sample is subjected to wide-field imaging through an eyepiece after being combined.
In the pump detection module, the trigger time sequence of the output pulse laser, the imaging light source and the imaging camera is set through the singlechip, the trigger delay time between the pulse laser and the imaging camera is set to obtain the instantaneous vibration displacement of each particle on the surface of the sample at different moments, and the trigger delay time is changed to measure for multiple times to obtain all vibration displacement amounts of each particle on the surface of the sample under the action of the full-period photoacoustic wave.
In the signal processing module, an imaging camera collects interference intensity of each particle on the surface of a sample, then a computer restores the interference intensity of each particle on the surface of the sample to instantaneous vibration displacement, and finally maximum projection is carried out on all vibration displacement amounts of each particle on the surface of the sample under the action of full-period photoacoustic waves to reconstruct a wide-field photoacoustic image.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the invention provides a wide-field photoacoustic microscopic imaging method,
The method comprises the steps of detecting instantaneous vibration displacement of each particle on the surface of a sample by using a polarized light interference technology, detecting all vibration displacement of each particle on the surface of the sample under the action of a full-period photoacoustic wave by using a pumping detection technology, and reconstructing a wide-field photoacoustic image by carrying out maximum projection on all vibration displacement of each particle on the surface of the sample under the action of the full-period photoacoustic wave; the invention overcomes the limitation that the traditional photoacoustic imaging can not simultaneously realize high resolution and rapid imaging, provides photoacoustic wide-field microscopic imaging, provides a novel non-contact and rapid measurement and imaging strategy for the photoacoustic detection field, and is expected to play an important role in the fields of biomedical imaging, material structure detection and the like.
2. The invention has the capability of rapid detection, and the device for realizing the method has simple structure and convenient use, and can be widely applied to the fields of detection of internal structures of organisms and precision machinery, and the like.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
Fig. 1 is a flow chart of a wide-field photoacoustic microscopy imaging method according to embodiment 1 of the present invention;
fig. 2 (a) is a bright field image of a sample in a wide-field photoacoustic microscopy method according to embodiment 1 of the present invention;
Fig. 2 (b) is an imaging image of the sample surface in the wide-field photoacoustic microscopy imaging method according to embodiment 1 of the present invention;
Fig. 3 is an image of the surface of a tumor cell in a wide-field photoacoustic microscopy method according to embodiment 1 of the present invention.
Fig. 4 is a schematic structural diagram of a wide-field photoacoustic microimaging apparatus according to embodiment 2 of the present invention;
1-1 is a pulse laser, 1-2 is an energy controller I, 1-3 is a collimation beam expander I, 2-1 is a dichroic mirror, 2-2 is a multi-wavelength objective lens, 3-1 is an imaging light source, 3-2 is an energy controller II, 3-3 is a collimation beam expander II, 3-4 is a polarizer, 3-5 is a beam splitting prism, 3-6 is a nomads prism, 3-7 is an analyzer, 3-8 is an eyepiece, 4-1 is a singlechip, 4-2 is an imaging camera, 5-1 is a computer, and 5-2 is a two-dimensional displacement platform.
Detailed Description
In order to enable those skilled in the art to better understand the present application, the following description will make clear and complete descriptions of the technical solutions according to the embodiments of the present application with reference to the accompanying drawings. It will be apparent that the described embodiments are only some, but not all, embodiments of the application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
Reference in the specification to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment may be included in at least one embodiment of the application. The appearances of such phrases in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments. Those of skill in the art will explicitly and implicitly appreciate that the described embodiments of the application may be combined with other embodiments.
Example 1
As shown in fig. 1, the present embodiment provides a wide-field photoacoustic microscopy imaging method, and referring to fig. 1, the present invention includes the steps of:
S1, exciting a sample by large-spot pulse light to generate a wide-field photoacoustic wave;
Further, the step S1 specifically includes:
S11, generating energy suitable for absorbing a sample to generate a wide-field photoacoustic signal by pulse laser emitted by a pulse laser through energy control;
The light emitted by the pulse laser is pulse laser with the wavelength of 266 nm and the pulse width of 10 ns, the pulse laser is suitable for exciting absorption of a tumor sample, the laser energy is regulated and controlled after being combined by a half wave plate and a polarization beam splitter prism, the polarization state of the half wave plate is changed, and the incident light can only pass through the polarization beam splitter prism to determine the linear polarized light component, so that the energy control can be realized by changing the polarization state before passing; the sample is a tumor sample having strong absorption at wavelength 266 nm.
S12, the beam subjected to energy control is subjected to beam expansion collimation to obtain a large light spot size suitable for wide-field imaging, so that the maximum light spot area capable of exciting a sample photoacoustic signal is achieved.
The beam expansion collimation is realized by adopting two lens combinations, and can be realized by adopting two convex lens combinations, or can be formed by adopting a concave lens and a convex lens combination.
S2, detecting instantaneous vibration displacement information of each particle on the surface of a sample under the action of photoacoustic waves at a certain moment by using wide-field polarized interference light, wherein the instantaneous vibration displacement information is specifically as follows:
S21, using an imaging light source as detection light, dividing the detection light into two beams of light rays with mutually orthogonal polarization directions by a Nomaski prism, respectively irradiating the two beams of light rays to adjacent areas on the surface of a sample after passing through an objective lens, reflecting the two beams of light rays back to the Nomaski prism to combine beams, carrying different optical paths by passing through different positions on the surface of the sample, carrying out wide-field polarized light interference by an analyzer after combining the beams, and leading the interference light intensity of each particle and the corresponding vibration displacement to follow the following formula:
Wherein, For the surface/>Interference intensity at particles,/>To detect the light amplitude,/>Is the center wavelength of the probe light,/>Is the direction in which the incident light splits after passing through the nomads prism,/>Is the distance that the incident light separates after passing through the nomads prism,/>And/>Determined by nomads prism,/>Is the sample surface/>The surface vibration displacement of the particles can be detected by the Normasky prism, so that the instantaneous vibration displacement of each particle on the surface of the sample under the action of the photoacoustic wave can be detected.
The imaging light source is super-continuum spectrum laser, the output wavelength is 400 nm to 800 nm, the pulse width is 100 ps, and external triggering can be performed.
As shown in fig. 2 (a) and 2 (b), a hematoxylin eosin stained mouse skin section was used as a verification experiment for the detection of the sample height by the wide-field polarized interference light. FIG. 2 (a) is an image of a sample under a bright field microscope, and has been subjected to gray value processing; fig. 2 (b) is an image of a sample using the wide-field polarized interference light in this step, and has been subjected to gray value processing. As can be seen from a comparison of fig. 2 (a) and fig. 2 (b), the wide-field polarized interference light energy converts the surface level difference of the sample into a light intensity difference, and the light intensity value changes in the case where the height changes, represent the change in the surface level difference.
As shown in fig. 3, wide-field polarized interference light imaging was performed using human colorectal cancer cell line HCT116 to verify that the detection system resolution was able to resolve tumor cells, as shown in the figure, the wide-field polarized interference light was able to identify the number and morphology of tumor cells.
As shown in fig. 4, wide-field polarized interference light imaging was performed using human colorectal cancer cell line HCT116 to verify that the detection system resolution was able to resolve tumor cells, as shown in the figure, the wide-field polarized interference light was able to identify the number and morphology of tumor cells.
S3, obtaining complete vibration displacement signals of all particles on the surface of the sample through pumping detection measurement, wherein the complete vibration displacement signals are specifically as follows:
The vibration displacement of all particles on the surface of the sample at a certain moment can be detected through the nomads prism, but the photoacoustic imaging also needs to obtain all vibration displacement signals of each particle on the surface of the sample under the action of full-period photoacoustic waves; the propagation of the photoacoustic signal in the sample is an ultra-fast process, when the photoacoustic signal is detected by using an ultrasonic transducer, the required time resolution is about 10 ns, and because the resolution is difficult to reach by a common camera, the photoacoustic signal is detected by adopting a detection mode of pumping detection, the pulse laser, an imaging light source used for detecting light and a triggering time sequence of an imaging camera for recording the interference intensity of each point are output by using a singlechip, and the imaging camera is firstly subjected to time sequence triggering to expose the camera; triggering the pulse laser, and transmitting pulse light to excite the sample to generate a wide-field photoacoustic wave after the exposure time of the imaging camera is started; finally, triggering the imaging light source, enabling the imaging light source to trigger after the pulse laser and in the exposure time of the imaging camera, setting the trigger delay time between the pulse laser and the imaging camera to obtain instantaneous vibration displacement of each particle on the surface of the sample under the effect of photoacoustic waves at different moments, and changing the trigger delay time to measure for multiple times to obtain all vibration displacement amounts of each particle on the surface of the sample under the effect of full-period photoacoustic waves; the initial value of the time delay of the pulse laser and the detection light is 10 ns, and the time delay of the laser and the detection light is controlled by a singlechip to be increased by 10 ns each time.
S4, reconstructing a light absorption image of the sample through the vibration displacement signal, wherein the light absorption image is specifically:
the instantaneous vibration displacement of each particle on the surface of the sample at a certain moment is obtained through the nomads prism and is as follows after being normalized:
the instantaneous vibration displacement represents the light absorption intensity of different depths of the sample, and the wide-field photoacoustic image can be reconstructed by carrying out maximum projection on all vibration displacement amounts of each particle on the surface of the sample under the action of the full-period photoacoustic wave.
Example 2
As shown in fig. 4, the present embodiment provides a wide-field photoacoustic microimaging apparatus, which includes a pulse light excitation module for providing excitation light, an excitation detection coupling module for adapting light acoustic wave excitation and wide-field polarized interference light, a displacement detection module for detecting a height difference of a sample surface, a pump detection module for detecting all vibration displacements generated by full-period light acoustic waves on the sample surface, and a signal processing module for image reconstruction.
The pulse light excitation module comprises a pulse laser 1-1, an energy controller 1-2 and a collimation beam expander 1-3, wherein the pulse laser 1-1, the energy controller 1-2 and the collimation beam expander 1-3 are sequentially connected.
The excitation detection coupling module comprises a 2-1 dichroic mirror and a multi-wavelength object mirror 2-2, wherein the dichroic mirror 2-1 is connected with the multi-wavelength object mirror 2-2, and the dichroic mirror 2-1 is also connected with a beam expansion collimator I1-3.
The displacement detection module comprises an imaging light source 3-1, an energy controller II 3-2, a collimating beam expander II 3-3, a polarizer 3-4, a beam splitting prism 3-5, a nomads prism 3-6, an analyzer 3-7 and an eyepiece 3-8, wherein the imaging light source 3-1, the energy controller II 3-2, the collimating beam expander II 3-3, the polarizer 3-4, the beam splitting prism 3-5 and the nomads prism 3-6 are sequentially connected, and the beam splitting prism 3-5 is also connected with the analyzer 3-4 and the eyepiece 3-8; the nomads prism 3-6 is also connected to a dichroic mirror 2-1.
The pumping detection module comprises a singlechip 4-1 and an imaging camera 4-2, wherein the singlechip 4-1 is connected with the imaging camera 4-2, and the singlechip 4-1 is also connected with a pulse laser 1-1 and an imaging light source 3-1 respectively;
The signal processing imaging module comprises a computer 5-1 and a two-dimensional displacement platform 5-2, wherein the computer 5-1 is connected with a single chip microcomputer 4-1, and the single chip microcomputer 4-1 is connected with the two-dimensional displacement platform 5-2.
Furthermore, the pulse light excitation module is connected with the displacement detection module through the excitation detection coupling module, and the signal processing module is connected with the pulse light excitation module and the displacement detection module.
Furthermore, the pulse light excitation module is specifically composed of a pulse laser, an energy controller I and a collimation beam expander I, and pulse laser which is suitable for an imaging sample absorption wave band and can excite a photoacoustic signal can be obtained through the pulse light excitation module.
In this embodiment, the laser is a solid-state pulse laser with a wavelength of 266, 266 nm and a pulse width of 10, 10 ns.
In this embodiment, the first lens is a convex lens, and the focal length is 5 mm; the second lens is a convex lens, and the focal length is 10 mm.
In this embodiment, a pulse laser and a pulse light excitation module are used to obtain a large-spot pulse laser suitable for excitation of a sample wide-field photoacoustic signal, where the method specifically includes:
The laser emitted by the pulse laser firstly passes through the half wave plate to obtain linearly polarized light in any direction, and the linearly polarized light passes through the polarization beam splitter prism and then only passes through light components with vertical or horizontal polarization directions, and the components passing through the polarization beam splitter prism are controlled by rotating the half wave plate so as to realize energy control. The light beam is collimated and expanded by the first lens, the diaphragm and the second lens, and the expansion ratio is determined by the focal lengths of the first lens and the second lens, and in this embodiment, the expansion ratio is 2 times.
The beam after beam expansion is irradiated to the sample through the dichroic mirror in the excitation detection coupling module to generate a wide-field photoacoustic wave. The photoacoustic wave, when propagating to the sample surface, causes the sample surface to expand and displace.
The displacement detection module and the pumping detection module are used for detecting the surface displacement of the sample, and the detection module is specifically as follows:
The imaging light source obtains a detection light beam suitable for imaging through the energy controller II and the collimation beam expander II, the detection light beam becomes polarized light after passing through the polarizer, the polarized light enters the Nomason prism through the beam splitting prism and is divided into two beams of light rays with mutually orthogonal polarization directions, the two beams of light rays respectively irradiate the adjacent area of the sample surface after passing through the objective lens and are reflected back to the Nomason prism for beam combination, the two beams of light rays carry different optical paths through different positions of the sample surface, the beams are mutually interfered after being combined, and the interference light intensity of each particle on the sample surface and the corresponding vibration displacement follow the following formula:
Wherein, For the surface/>Interference intensity at particles,/>To detect the light amplitude,/>Is the center wavelength of the probe light,/>Is the direction in which the incident light splits after passing through the nomads prism,/>Is the distance that the incident light separates after passing through the nomads prism,/>And/>Determined by nomads prism,/>Is the sample surface/>Surface vibration displacement at the particles.
In the pumping detection module, a singlechip outputs trigger time sequences of a pulse laser, an imaging light source and an imaging camera, and the imaging camera is firstly subjected to time sequence triggering to expose the camera; triggering the pulse laser, and transmitting pulse light to excite a sample photoacoustic signal after the exposure time of the imaging camera is started; and finally triggering the imaging light source, so that the imaging light source is triggered after the laser and within the exposure time of the imaging camera, and an ultrafast photoacoustic signal is detected.
In this embodiment, the imaging light source adopts supercontinuum laser, the output wavelength is 400 nm to 800 nm, the pulse width is 100 ps, and external triggering can be performed.
In the signal processing module, an imaging camera collects interference intensity of each particle on the surface of a sample, a computer is used for reducing the interference intensity of each particle on the surface of the sample into instantaneous vibration displacement, and finally maximum projection is carried out on all vibration displacement amounts of each particle on the surface of the sample under the action of full-period photoacoustic waves to reconstruct a wide-field photoacoustic image.
Those skilled in the art will understand that all or part of the procedures in the methods of the foregoing embodiments may be arbitrarily combined, and for brevity, all of the possible combinations of the features of the foregoing embodiments are not described, however, as long as there is no contradiction between the combinations of the features, they should be considered as the scope of the disclosure. The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (11)
1. A wide-field photoacoustic microscopy imaging method, comprising the steps of:
S1, pulse laser generated by a laser is subjected to beam expansion and collimation to form a large light spot to excite a sample to generate a wide-field photoacoustic wave, and each particle on the surface of the sample can be caused to generate vibration displacement by the photoacoustic wave;
s2, detecting light with wide field polarization emitted by the imaging light source is used as detection light, detection of optical paths of all particles on the surface of the sample is achieved through polarized interference light, and the optical paths are used for representing instantaneous vibration displacement of all particles on the surface of the sample under the action of photoacoustic waves at the moment;
S3, setting trigger delay time of the excitation light and the detection light, and changing the trigger delay time to measure for a plurality of times to obtain all vibration displacement amounts of each particle on the surface of the sample under the action of the full-period photoacoustic wave;
And S4, reconstructing a wide-field photoacoustic image by carrying out maximum projection on all vibration displacement amounts of each particle on the surface of the sample under the action of the full-period photoacoustic wave.
2. The method of claim 1, wherein in the step S1, the wide-field photoacoustic wave causes each particle on the sample surface to generate a vibration displacement, specifically:
Excitation light irradiates the sample excitation light sound wave, when the photoacoustic wave propagates to the sample surface, vibration displacement is generated on the sample surface, the size of the vibration displacement is determined by the sound pressure size of the photoacoustic wave, and the normalized displacement size and sound pressure have the following relation:
；
Wherein, Is/>Displacement of sample surface at moment,/>For density of medium,/>Is the sound velocity in the medium,/>For/>Sound pressure of ultrasonic wave acting on the sample surface at the moment.
3. The method of claim 1, wherein in the step S2, the detection light realizes the detection of the optical path length of each particle on the sample surface by polarized interference light, and the optical path length is used to characterize the instantaneous vibration displacement of each particle on the sample surface under the action of the photoacoustic wave, specifically: the detection light is divided into two beams of light rays with mutually orthogonal polarization directions by the nomadic prism, the two beams of light rays respectively irradiate adjacent areas on the surface of a sample after passing through the objective lens and are reflected back to the nomadic prism to be combined, the two beams of light rays carry different optical paths through different positions on the surface of the sample, wide-field polarized light interference is carried out through the polarization analyzer after the beam combination, and the interference light intensity of each particle and the corresponding vibration displacement follow the following formula:；
Wherein, For the surface/>Interference intensity at particles,/>To detect the light amplitude,/>Is the center wavelength of the probe light,/>Is the direction in which the incident light splits after passing through the nomads prism,/>Is the distance that the incident light separates after passing through the nomads prism,/>And/>Determined by nomads prism,/>Is the sample surface/>The surface vibration displacement of the particles can be detected by the Normasky prism, so that the instantaneous vibration displacement of each particle on the surface of the sample under the action of the photoacoustic wave can be detected.
4. The method of claim 1, wherein in the step S3, a trigger delay time of the excitation light and the probe light is set, and the trigger delay time is changed to perform multiple measurements to obtain all vibration displacement amounts of each particle on the surface of the sample under the action of the full-period photoacoustic wave, and the specific steps are as follows:
S1, triggering an imaging camera to expose the camera;
S2, triggering a laser, and triggering the laser to emit pulse light to excite a sample to generate a wide-field photoacoustic wave after the exposure time of the imaging camera is started;
s3, triggering an imaging light source, so that the imaging light source is triggered behind the laser and within the exposure time of the imaging camera;
and S4, measuring the trigger delay time of the excitation light and the detection light for a plurality of times to obtain all vibration displacement amounts of each particle on the surface of the sample under the action of the full-period photoacoustic wave.
5. The method of claim 1, wherein in step S4, the wide-field photoacoustic image is reconstructed by maximum projection of all vibration displacement amounts of particles on the surface of the sample under the action of the full-period photoacoustic wave, specifically:
the instantaneous vibration displacement of each particle on the surface of the sample at a certain moment is obtained through the nomads prism and is as follows after being normalized:
；
the instantaneous vibration displacement represents the light absorption intensity of different depths of the sample, and the wide-field photoacoustic image can be reconstructed by carrying out maximum projection on all vibration displacement amounts of each particle on the surface of the sample under the action of the full-period photoacoustic wave.
6. The wide-field photoacoustic microscopic imaging device is characterized by comprising a pulse light excitation module, an excitation detection coupling module, a displacement detection module, a pumping detection module and a signal processing module;
The pulse light excitation module comprises a pulse laser, an energy regulator I and a collimation beam expander I, wherein the pulse laser, the energy controller I and the collimation beam expander I are sequentially connected; the excitation detection coupling module comprises a dichroic mirror and a multi-wavelength objective lens, wherein the dichroic mirror is connected with the multi-wavelength objective lens, and the dichroic mirror is also connected with the first collimating beam expander;
The displacement detection module comprises an imaging light source, an energy controller II, a collimation beam expander II, a polarizer, a beam splitting prism, a Nomars prism, an analyzer and an eyepiece, wherein the imaging light source, the energy controller II, the collimation beam expander II, the polarizer, the beam splitting prism and the Nomars prism are sequentially connected, and the beam splitting prism is also sequentially connected with the analyzer and the eyepiece; the nomads prism is also connected with a dichroic mirror;
The pumping detection module comprises a singlechip and an imaging camera, wherein the singlechip is connected with the imaging camera and is also respectively connected with a laser and an imaging light source;
The signal processing module comprises a computer and a two-dimensional displacement platform, wherein the computer is connected with the single chip microcomputer, and the single chip microcomputer is connected with the two-dimensional displacement platform.
7. The wide-field photoacoustic microscopic imaging apparatus of claim 6, wherein in the pulse light excitation module, the light beam emitted by the pulse laser sequentially passes through the first energy controller and the first collimation beam expander to obtain large-spot pulse light suitable for photoacoustic signal excitation of the sample;
the first energy controller consists of a polarizer, a half wave plate and a polarization beam splitter;
The first collimating and beam expander consists of a first lens, a diaphragm and a second lens.
8. The device of claim 6, wherein in the excitation detection coupling module, excitation light and detection light are combined by a dichroic mirror and focused by a multi-wavelength objective lens, so as to excite a wide-field photoacoustic wave and detect instantaneous vibration displacement of each particle on the surface of the sample.
9. The wide-field photoacoustic microscopic imaging apparatus of claim 6, wherein in the displacement detection module, the light beam emitted by the imaging light source is adjusted to have proper energy and light spot size by the energy controller II and the beam expanding collimator II, and enters the polarizer to become polarized light, the polarized light enters the nomads prism by the beam splitting prism and is split into two beams of light beams with mutually orthogonal polarization directions, the two beams of light beams respectively irradiate the adjacent area of the sample surface after passing through the objective lens and are reflected back to the nomads prism for beam combination, the two beams of light beams carry different optical paths after passing through different positions of the sample surface, the beams mutually interfere after beam combination, and the interference intensity of each particle on the sample surface is imaged in a wide field by the eyepiece.
10. The wide-field photoacoustic microscopic imaging apparatus of claim 6, wherein in the pump detection module, the trigger time sequences of the output pulse laser, the imaging light source and the imaging camera are set through the single chip microcomputer, the trigger delay time between the pulse laser and the imaging camera is set to obtain the instantaneous vibration displacement of each particle on the surface of the sample at different moments, and the trigger delay time is changed to obtain all the vibration displacement of each particle on the surface of the sample under the action of the full-period photoacoustic wave through multiple measurements.
11. The device of claim 6, wherein in the signal processing module, the imaging camera collects the interference intensity of each particle on the surface of the sample, the computer restores the interference intensity of each particle on the surface of the sample to instantaneous vibration displacement, and finally the maximum projection is performed on all the vibration displacement amounts of each particle on the surface of the sample under the action of the full-period photoacoustic wave to reconstruct the wide-field photoacoustic image.
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