CN113777045B - Super-resolution functional photoacoustic imaging method based on single-particle multilateral localization tracking - Google Patents
Super-resolution functional photoacoustic imaging method based on single-particle multilateral localization tracking Download PDFInfo
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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- G01N29/00—Investigating or analysing materials by the use of ultrasonic, sonic or infrasonic waves; Visualisation of the interior of objects by transmitting ultrasonic or sonic waves through the object
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/1702—Systems in which incident light is modified in accordance with the properties of the material investigated with opto-acoustic detection, e.g. for gases or analysing solids
- G01N2021/1706—Systems in which incident light is modified in accordance with the properties of the material investigated with opto-acoustic detection, e.g. for gases or analysing solids in solids
Abstract
The invention provides a super-resolution functional photoacoustic imaging method based on single-particle multilateral localization tracking, which comprises the following steps of: calibrating the position of the detector, namely calibrating the position of the detector by adopting a trilateral positioning method; positioning a photoacoustic source, namely acquiring photoacoustic signals of particles by using a detector and fitting to obtain the distance between the particles and the detector and the distance resolution exceeding the acoustic diffraction limit; multilateral positioning of the particles, namely, obtaining the distance between the particles and the detectors by adopting at least three detectors, and carrying out multilateral positioning on the particles by utilizing a sound source positioning algorithm to obtain the three-dimensional space position of the particles; performing multi-particle iterative positioning calculation, namely performing positioning calculation on a plurality of particles by adopting an iterative algorithm to obtain the three-dimensional space position of each particle; and forming a super-resolution functional photoacoustic image, positioning at least one particle on a time sequence to obtain a corresponding three-dimensional space position, and generating the super-resolution functional photoacoustic image according to the three-dimensional space position and the change of the position of each particle along with time.
Description
Technical Field
The invention belongs to the field of super-resolution photoacoustic imaging, and particularly relates to a super-resolution functional photoacoustic imaging method based on single-particle multi-edge positioning and tracking.
Background
The photoacoustic tomography (PAT) is a novel noninvasive hybrid imaging technology based on photoacoustic effect, can realize functional imaging with high optical contrast and high resolution in deep biological tissues, and combines the advantages of high selection characteristic in optical imaging and high penetration depth in ultrasonic imaging due to the fact that the PAT process comprises optical excitation and ultrasonic detection. The principle is that short pulse laser energy is absorbed by a light absorber in the tissue, ultrasonic wave emission is generated by transient thermoelastic expansion, then ultrasonic waves are received on the surface of the tissue by a transducer array, and the spatial distribution of the light absorber in the tissue is reconstructed according to detected ultrasonic signals.
Although PAT has significant advantages in imaging depth, its spatial resolution is much lower than other optical imaging techniques. The resolution expression of PAT can be written as R C =0.88c/Δ f, where c is the speed of sound and Δ f is the bandwidth of the ultrasound transducer (approximately proportional to the center frequency of the ultrasound transducer). Thus, fundamentally, the resolution of PAT is limited not only by acoustic diffraction, but also by the wavelength of the photoacoustic signal in the tissue and the bandwidth of the ultrasound probe (when a 5MHz bandwidth is used, the resolution is typically around 300 μm).
In the prior art, the resolution of local particle positioning super-resolution photoacoustic imaging has no upper limit theoretically, but depends on the reconstruction accuracy of photoacoustic images in use. In order to obtain high-quality PAT images, local particle localization super-resolution photoacoustic imaging requires the use of a large number of ultrasound transducer arrays (e.g. >256 transducers) to collect signals from multiple angles simultaneously, and thus the requirements on the equipment are extremely high and expensive. The image reconstruction needs to use a complex inverse algorithm to perform accurate PAT image reconstruction, and then a local positioning algorithm with huge calculation amount is adopted to position a single liquid drop or particle, so that real-time, dynamic, high-speed and large-range super-resolution photoacoustic imaging is difficult to realize.
Disclosure of Invention
In order to solve the problems, the invention provides a super-resolution functional photoacoustic imaging method based on single-particle multilateral positioning tracking, which avoids the complex inverse algorithm and the requirement on signal intensity in photoacoustic image reconstruction, and adopts the following technical scheme:
the invention provides a super-resolution functional photoacoustic imaging method based on single-particle multilateral localization tracking, which is characterized by comprising the following steps of: calibrating the position of the detector, namely calibrating the position of the detector by adopting a trilateral positioning method; positioning a photoacoustic source, namely acquiring photoacoustic signals of particles by using a detector and fitting to obtain the distance between the particles and the detector and the distance resolution exceeding the acoustic diffraction limit; multilateral positioning of particles, namely, obtaining the distance between the particles and the detectors by adopting at least three detectors, and performing multilateral positioning on the particles by utilizing a sound source positioning algorithm to obtain the three-dimensional space position of the particles; performing multi-particle iterative positioning calculation, namely performing positioning calculation on a plurality of particles by adopting an iterative algorithm to obtain the three-dimensional space position of each particle; and forming a super-resolution functional photoacoustic image, positioning at least one particle on a time sequence to obtain a corresponding three-dimensional space position, and generating the super-resolution functional photoacoustic image according to the three-dimensional space position and the change of the position of each particle along with time.
The super-resolution functional photoacoustic imaging method based on single-particle multilateral localization tracking provided by the invention can also have the characteristics that the specific process of the detector position calibration is as follows: calibrating the position of the detector by using the light absorption particles with known positions on a two-dimensional plane, and exciting single light absorption particles on the surface of the glass slide by adjusting a focusing lens to control the diameter and the position of an illumination area; step T2, recording the position of the light absorption particles by using a fluorescence microscope; step T3, exciting and recording photoacoustic signals of the light absorption particles to the three ultrasonic detectors respectively; t4, moving the light absorption particles to a new position or shifting the excitation light beam to excite the light absorption particles on the surface of the slide at different positions by controlling the two-dimensional linear translation stage; and T5, repeating the steps T2-T4 until at least 3 groups of photoacoustic signals are obtained, and T6, substituting the groups of photoacoustic signals obtained in the step T5 into a trilateral positioning algorithm to reversely calculate to obtain the position of the detector in the three-dimensional space.
The super-resolution functional photoacoustic imaging method based on single-particle multilateral localization tracking provided by the invention can also have the characteristic that the light absorption particles can be gold nanoparticles, carbon black particles or pigment-containing particles.
The super-resolution functional photoacoustic imaging method based on single-particle multilateral positioning tracking provided by the invention can also have the characteristics that the specific process of photoacoustic source positioning is as follows: the method comprises the steps of collecting the intensity of photoacoustic signals sent by particles by at least three detectors, obtaining an intensity-time curve changing along with time, fitting the intensity-time curve by adopting a Gaussian fitting method or a maximum likelihood estimation method to obtain a fitting curve, and carrying out time positioning on the photoacoustic signals according to the fitting curve to obtain distance vectors from the particles to the detectors, wherein the distance resolution exceeding the acoustic diffraction limit can be obtained by fitting the photoacoustic signals.
The super-resolution functional photoacoustic imaging method based on single-particle multilateral positioning tracking provided by the invention can also have the characteristics that the specific process of particle multilateral positioning is as follows: the sound source positioning algorithm is a least square positioning method, and the positions of n detectors in the space are p i ＝(x i ，y i ，z i ) Wherein i ∈ (1,2,. Eta, n), n is a positive integer, the position of the particle is p = (x, y, z), and the distance from the detector is r i ，r i The calculation formula of (2) is as follows: r is a radical of hydrogen i 2 ＝(x i -x)+(y i -y)+(z i -z) 2 Obtaining the three-dimensional space position of the particle through a least square positioning algorithm, wherein the calculation formula of the position is as follows:
in the formula (I), the compound is shown in the specification,
and is
The super-resolution functional photoacoustic imaging method based on single-particle multilateral localization tracking provided by the invention can also have the characteristics that the sound source localization algorithm can be a Fourier domain reconstruction algorithm, a back projection algorithm, an iterative reconstruction algorithm or a deep learning reconstruction.
The super-resolution functional photoacoustic imaging method based on single-particle multilateral positioning tracking provided by the invention can also have the characteristics that the specific process of multi-particle iterative positioning calculation is as follows: obtaining distance vectors from a plurality of particles to each detector by utilizing light source positioning, randomly selecting the distance vector in one detector, carrying out particle multilateral positioning with the distance vectors between the detectors except the detector and obtaining a unique solution or a maximum probability solution of the three-dimensional space position of the particle, selecting the distance vector which is not used for positioning calculation to carry out particle multilateral positioning and obtaining the unique solution or the maximum probability solution of the three-dimensional space position of another particle until all the particles finish particle multilateral positioning.
The super-resolution functional photoacoustic imaging method based on single-particle multilateral positioning and tracking provided by the invention can also have the characteristics that the specific process of the functional super-resolution photoacoustic imaging is as follows: by utilizing the absorption spectrum characteristics of the particles and the acoustic characteristics of the constituent materials, different types of particles are distinguished in the positioning process, and the change of the physical and chemical characteristics of the particles along with time is resolved, so that a super-resolution functional photoacoustic image with multiple contrasts is obtained.
Action and Effect of the invention
According to the single-particle multilateral positioning and tracking-based super-resolution functional photoacoustic imaging method, the multilateral positioning method is introduced into single-particle positioning super-resolution photoacoustic imaging, the complex inverse algorithm and the requirement on signal intensity in photoacoustic image reconstruction are avoided, the dependence of the super-resolution photoacoustic imaging technology on software and hardware can be greatly reduced, and the new imaging mechanism is utilized to break through the spatial resolution limit in the prior art.
Drawings
FIG. 1 is a schematic structural diagram of a system for super-resolution functional photoacoustic imaging based on single-particle multilateral localization tracking according to an embodiment of the present invention;
FIG. 2 is a flow chart of a super-resolution functional photoacoustic imaging method based on single event multilateral localization tracking according to an embodiment of the present invention;
fig. 3 is a schematic diagram of time location of a photoacoustic signal according to an embodiment of the present invention, where fig. 3 (a) is a graph showing intensity time of the photoacoustic signal of a single particle obtained through computer simulation, fig. 3 (b) is a graph showing the photoacoustic signal after Hilbert transform, fig. 3 (c) is a partially enlarged view of an area where a dashed box in fig. 3 (b) is located, and the dashed line is a gaussian fitting result;
FIG. 4 is a schematic diagram of single particle multilateral localization according to an embodiment of the present invention;
fig. 5 is a numerical simulation of single-particle multilateral localization according to an embodiment of the present invention, in which fig. 5 (a) and 5 (b) are schematic diagrams of the spatial positions of a detector (black circle) and a particle (gray circle) when the particle position is p = (0,0) mm and p = (1.5,0.5) mm, respectively, and fig. 5 (c) and 5 (d) are hilbert transform and gaussian fit (dotted line) of photoacoustic signals captured by the detector when the particle position is p = (0,0) mm and p = (1.5,0.5) mm, respectively;
fig. 6 is a scatter distribution of the localization positions of the particles in a two-dimensional plane and histograms of the localization positions in the x and y directions of an embodiment of the present invention, where fig. 6 (a) is a scatter distribution diagram of the localization positions of the particles in the two-dimensional plane at the center of the field of view, p = (0,0) mm, fig. 6 (b) and fig. 6 (c) are histograms of the localization positions of the particles in the x and y directions, respectively, p = (0,0) mm, fig. 6 (d) is a scatter distribution diagram of the localization positions of the particles in the two-dimensional plane, respectively, p = (1.5,0.5) mm, fig. 6 (e) and fig. (f) are histograms of the localization positions of the particles in the x and y directions, respectively, p = (1.5,0.5);
fig. 7 is a lateral positioning accuracy distribution of particles and lateral and longitudinal positioning accuracies corresponding to the particles at different depths according to an embodiment of the present invention, where fig. 7 (a) is a lateral positioning accuracy distribution diagram (imaging depth is 3 mm) of the particles when three symmetrically distributed ultrasonic detectors are used, and fig. 7 (b) is a lateral and longitudinal positioning accuracy distribution diagram corresponding to the particles at the center of a field of view at different depths;
FIG. 8 is a schematic diagram of the number and spatial distribution of detectors that are possible with an embodiment of the present invention, where the detectors (black circles) are located 5mm from the center position of the field of view (gray circles);
fig. 9 is a schematic diagram of three-dimensional super-resolution photoacoustic localization imaging of scintillating light-absorbing particles in an embodiment of the present invention, wherein fig. 9 (a), 9 (c), and 9 (e) are the reconstructions of super-resolution images of a 3 × 3 array of light-absorbing particles with inter-particle spacings of 5 μm,7.5 μm, and 10 μm, respectively. FIGS. 9 (b), 9 (d) and 9 (f) are particle localization probability curves at the dotted line labeled cross-sections in FIGS. 9 (a), 9 (c) and 9 (e), respectively;
fig. 10 is a schematic diagram of three-dimensional super-resolution photoacoustic localization tracking on a flowing light-absorbing particle in an embodiment of the present invention, where fig. 10 (a) is a flow trajectory of a single light-absorbing particle, and fig. 10 (b) is a channel structure obtained by overlapping flow trajectories of a plurality of light-absorbing particles.
Detailed Description
The following description of the embodiments of the present invention will be made with reference to the accompanying drawings.
< example >
Fig. 1 is a schematic structural diagram of a system of super-resolution photoacoustic imaging based on single-particle multilateral localization tracking according to an embodiment of the present invention.
As shown in FIG. 1, the VGEN-G-20 pulse laser with tunable repetition rate and wavelength of 532nm is used as the excitation light source for the photoacoustic excitation in this embodiment.
In this embodiment, a three-dimensional spatial position of a particle is obtained by trilateral positioning using three probes. In other embodiments, multilateration may be employed using more than three detectors.
The light beam emitted by the laser is collimated by the lens group, reflected by the dichroic mirror through the focusing lens and then approximately focused on the back focal plane of the objective lens. The illumination range can be controlled by adjusting the position of the focusing lens. Meanwhile, the excitation light pulse is reflected by the beam sampling mirror and then captured by the high-speed photodiode, so that the excitation light pulse is used for correcting the excitation light intensity and serving as a trigger signal source of the system.
The sample is placed in a water tank, the position of the sample stage is controlled by a side branch light path of a two-dimensional nano linear translation stage realization system to realize fluorescence microscopic imaging, and the sample stage is composed of a 561nm long-pass color filter and a CCD camera and is used for observing and recording the sample. And a long working distance water lens with the numerical aperture of 0.3 is selected for testing, and the long working distance water lens is used for realizing microscopic imaging with the resolution ratio higher than 1 mu m through an objective lens.
Three needle-shaped ultrasonic transducers with non-collinear center frequencies of 20MHz and diameters of 0.5mm are adopted to acquire photoacoustic signals generated by particles. The acoustic signal is converted into an electric signal, amplified by a broadband low noise amplifier, collected by a data acquisition card at a sampling rate of 200MS/s and stored in a computer. In this embodiment, labVIEW language is used to write a control program to realize system automation control and signal acquisition.
Fig. 2 is a flowchart of a super-resolution functional photoacoustic imaging method based on single-particle multilateral localization tracking according to an embodiment of the present invention.
As shown in fig. 2, the super-resolution functional photoacoustic imaging method based on single-particle multilateral localization tracking according to this embodiment includes the following steps:
and S1, calibrating the position of the detector by adopting a trilateral positioning method.
The position of the detector is calibrated by using light absorbing particles with known positions on a two-dimensional plane, and the specific process is as follows:
step T1, controlling the diameter and the position of an illumination area by adjusting a focusing lens, and exciting single light absorption particles on the surface of the glass slide;
step T2, recording the position of the light absorption particles by using a fluorescence microscope;
step T3, exciting and recording photoacoustic signals from the light absorption particles to the three ultrasonic detectors respectively;
t4, moving the light absorption particles to a new position or shifting the excitation light beam to excite the light absorption particles on the surface of the slide at different positions by controlling a two-dimensional nano linear translation stage;
step T5, repeating the steps T2-T4 until at least 3 groups of photoacoustic signals are obtained,
and step T6, substituting the multiple groups of photoacoustic signals obtained in the step T5 into a trilateral positioning algorithm to perform reverse calculation to obtain the position of the detector in the three-dimensional space.
In this embodiment, the light absorbing particles used are gold nanoparticles. In other embodiments, the light-absorbing particles may also be particles such as carbon black particles or pigment-containing particles.
And S2, acquiring photoacoustic signals of the particles by using the detector and fitting to obtain the distance between the particles and the detector and the distance resolution exceeding the acoustic diffraction limit. The specific process is as follows:
collecting the time-varying curve of the intensity of the photoacoustic signals emitted by the particles by using at least three detectors;
fitting the intensity-time curve by adopting a Gaussian fitting method or a maximum likelihood estimation method to obtain a fitting curve, performing time positioning on the photoacoustic signal according to the fitting curve, and obtaining the distance between the particle and the detector from the centroid position of Gaussian fitting as a distance vector. Wherein the fitting to the photoacoustic signal enables obtaining a distance resolution beyond the acoustic diffraction limit.
Fig. 3 is a schematic diagram of time positioning of a photoacoustic signal according to an embodiment of the present invention, where fig. 3 (a) is a graph showing intensity time of the photoacoustic signal of a single particle obtained through computer simulation, fig. 3 (b) is a graph showing the photoacoustic signal after undergoing hilbert transform, fig. 3 (c) is a partially enlarged view of an area where a dashed box in fig. 3 (b) is located, and the dashed line is a result of gaussian fitting.
As shown in fig. 3, fig. 3 (a) is a graph of the intensity of photoacoustic signals emitted from single particles detected by an ultrasonic transducer with a center frequency of 20MHz, which is obtained by computer simulation, as a function of time, the signal-to-noise ratio of 200MHz signal is 10dB, which is defined as the signal-to-noise ratio
Wherein P is signal And P noise The effective photoacoustic signal intensity and the noise intensity received by the ultrasonic transducer are respectively, and the noise is in random normal distribution. It can be seen from fig. 3 (b) and 3 (c) that the photoacoustic signal after the hilbert transform has a gaussian distribution.
The traditional photoacoustic imaging directly performs Hilbert transformation on signals, performs image reconstruction by using amplitude information of the signals, and the image resolution is generally determined by the full width at half maximum of the signals, so that a 20MHz detector can generally obtain 75 μm resolution.
The light gray curve in fig. 3 (c) is a fitted curve obtained by gaussian fitting of the photoacoustic signal, and the central accuracy (σ) of the gaussian fitting is calculated by introducing random noise in the numerical simulation t ) To obtain the precision of the location of the photoacoustic sourceDegree σ = v · σ t Where v is the speed of sound in the tissue. When the signal-to-noise ratio of the signal is 10dB, the positioning accuracy of the photoacoustic source obtained through calculation can be better than 1.7 mu m, and the resolution is improved by about 44 times compared with that of the traditional photoacoustic imaging. Compared with the single particle local area photoacoustic imaging in the prior art, the resolution is improved by about 7 times on the basis of 6 times.
And S3, collecting the intensity of photoacoustic signals emitted by the particles by adopting at least three detectors, and performing multilateral positioning on the particles by utilizing a sound source positioning algorithm to obtain the three-dimensional space positions of the particles.
FIG. 4 is a diagram of single particle multilateral localization according to an embodiment of the present invention.
As shown in fig. 4, in the present embodiment, the sound source localization algorithm is a least square localization method. N detectors in space at p i ＝(x i ，y i ，z i ) Wherein i ∈ (1,2,. Eta, n), n is a positive integer, the position of the particle is p = (x, y, z), and the distance from the detector is r i ，r i The calculation formula of (2) is as follows:
r i 2 ＝(x i -x)+(y i -y)+(z i -z) 2
the matrix form of the above formula can be written as:
wherein
And is provided with
Thus, the position of the particle can be found by a linear least squares localization algorithm:
in the process of calculating the position of the particle, the known spatial coordinates of the detector and the distance between the detector and the particle are used, and it is obvious that the more the spatial coordinate points of the detector are, the higher the position accuracy of the particle is, and meanwhile, the more the distance is measured, the higher the position accuracy of the particle is. Theoretically, at least 4 non-coplanar detectors are needed to realize the positioning of the particles in the three-dimensional space, but since the detectors are generally always positioned on one side of the test object in the practical photoacoustic imaging application, the three-dimensional positioning of the single particles can be realized even if only 3 detectors are used.
In other embodiments, the sound source localization algorithm may also adopt any algorithm capable of realizing sound source localization, such as a fourier domain reconstruction algorithm, a back projection algorithm, an iterative reconstruction algorithm, or a deep learning reconstruction algorithm, to realize multilateral localization of particles.
Fig. 5 is a numerical simulation of single-particle multilateral localization according to an embodiment of the present invention, in which fig. 5 (a) and 5 (b) are schematic diagrams of the spatial positions of a detector (black circle) and a particle (gray circle) when the particle position is p = (0,0) mm and p = (1.5,0.5) mm, respectively, and fig. 5 (c) and 5 (d) are hilbert transform and gaussian fit (dotted line) of photoacoustic signals captured by the detector when the particle position is p = (0,0) mm and p = (1.5,0.5) mm, respectively.
As shown in fig. 5, the imaging resolution of single-particle multi-edge positioning super-resolution photoacoustic imaging when the minimum number of detectors (3) is used is taken as an example for short description. In fig. 5 (a), in order to obtain the most uniform particle positioning accuracy distribution, three ultrasonic detectors D1, D2 and D3 are symmetrically distributed in a regular triangle, the distance from the detector to the center of the field of view is set to be 5mm, and the particle is located at the center of the detector array. As can be seen from fig. 5 (b), since the distance from the particle to each detector is equal, the time when the detector receives the signal is also the same. In fig. 5 (c), the particle is not at the center of the detector array, so it can be seen in fig. 5 (d) that the time at which the detector receives the signal is also different. The distance from the particle to the detector can be further calculated through the signal receiving time, and the multilateral positioning of the particle is achieved.
Fig. 6 is a scatter distribution diagram of the localization positions of the particles in the two-dimensional plane and histograms of the localization positions in the x and y directions of the particles of the embodiment of the present invention, where fig. 6 (a) is a scatter distribution diagram of the localization positions of the particles in the two-dimensional plane at the center of the field of view, p = (0,0) mm, fig. 6 (b) and fig. 6 (c) are histograms of the localization positions of the particles in the x and y directions, respectively, p = (0,0) mm, fig. 6 (d) is a scatter distribution diagram of the localization positions of the particles in the two-dimensional plane, respectively, p = (1.5,0.5) mm, and fig. 6 (e) and fig. (f) are histograms of the localization positions of the particles in the x and y directions, respectively, p = (1.5,0.5).
As shown in fig. 6, the multilateration of the particles was numerically simulated at a signal-to-noise ratio of 10 dB. The same particle is excited 1000 times, random noise is added into the signal, and the photoacoustic signal is detected by three detectors through Gaussian fitting, so that the distance from the particle to the detectors is obtained. In fig. 6 (b) and 6 (c), the curves are gaussian-fit curves, and by calculating the standard deviation, it is possible to obtain the positioning accuracy of particles of p = (0,0) mm in the x and y directions of 1.67 μm and 1.62 μm, respectively. In fig. 6 (e) and fig. (f), the curves are gaussian-fit curves, and the positioning accuracy of particles with p = (1.5,0.5) mm can be 1.88 μm and 1.69 μm, respectively. Because the positioning precision is less than 2 μm, the requirements of the people on super-resolution imaging are met.
Fig. 7 is a transverse positioning accuracy distribution of particles and transverse and longitudinal positioning accuracies corresponding to the particles at different depths according to an embodiment of the present invention, where fig. 7 (a) is a transverse positioning accuracy distribution diagram (depth is 3 mm) of the particles when three symmetrically distributed ultrasonic detectors are used, and fig. 7 (b) is a transverse and longitudinal positioning accuracy distribution diagram corresponding to the particles at the center of a field of view at different depths.
As shown in FIG. 7 (a), the positioning accuracy of the region having a center diameter of 3mm is uniformly distributed, and the accuracy is about 2 μm. In practical application, the size of the imaging system is close to the size of a brain window of a common mouse, so that the requirement of cerebrovascular imaging can be met, and the imaging application requirement of the system is met. In the region close to the detector, the particle positioning accuracy is somewhat degraded, but also remains below 3 μm, differing from the central region by only 50%.
Fig. 8 is a diagram showing the number and spatial distribution of detectors that are possible in an embodiment of the present invention, where the detectors (black circles) are located 5mm from the center position of the field of view (gray circles).
In practical applications, we can increase the imaging resolution and accuracy by increasing the number of detectors, as shown in fig. 8. As shown in fig. 8 (a-d), in the present embodiment, the distance from the detector to the center position of the field of view is set to be fixed 5mm, and then all the detectors are symmetrically distributed. Fig. 8 (e) shows the relationship between imaging resolution and the number of detectors by using numerical calculation. In other embodiments, the distance setting from the detector to the center of the field of view needs to be adjusted according to imaging requirements, and the distribution of the detectors does not need to be symmetrical all the time.
And S4, positioning and calculating the plurality of particles by adopting an iterative algorithm to obtain the three-dimensional space position of each particle.
In photoacoustic single particle multilateration, multiple particles will be excited by a single light pulse to generate ultrasound, and since the speed of light in tissue is much greater than the speed of sound, all particles can be considered to emit signals at the same time. When the ultrasound probes receive their signals, their time-series correspondence is not known, and the signals may also overlap in time-series. Therefore, the iterative algorithm is adopted to carry out positioning calculation, and the simultaneous positioning of multiple particles is realized.
Obtaining distance vectors between a plurality of particles and each detector by utilizing the positioning of the light source,
randomly selecting a distance vector in one detector, carrying out particle multilateral positioning process with distance vectors between other detectors except the detector and obtaining a unique solution or a most probable solution of the three-dimensional space position of the particle,
and selecting the distance vectors which are not used for positioning calculation to perform particle multilateral positioning and obtaining a unique solution or a solution with the maximum probability of the three-dimensional space position of another particle until all the particles finish the particle multilateral positioning.
And S5, positioning at least one particle on a time sequence to obtain a corresponding three-dimensional space position, and generating a super-resolution photoacoustic image according to the three-dimensional space position and the change of the positions of the particles.
By utilizing the absorption spectrum characteristics of the particles and the acoustic characteristics of the constituent materials, different types of particles are distinguished in the positioning process, and the change of the physical and chemical characteristics of the particles along with time is distinguished, so that a super-resolution functional photoacoustic image with multiple contrasts is obtained.
In the embodiment of the invention, the scintillation light absorption particles and the flow light absorption particles are respectively subjected to three-dimensional super-resolution photoacoustic positioning imaging and tracking.
Fig. 9 is a schematic diagram of three-dimensional super-resolution photoacoustic localization imaging of scintillating light-absorbing particles in an embodiment of the present invention, wherein fig. 9 (a), 9 (c), and 9 (e) are the reconstructions of super-resolution images of a 3 × 3 array of light-absorbing particles with inter-particle spacings of 5 μm,7.5 μm, and 10 μm, respectively. Fig. 9 (b), 9 (d) and 9 (f) are particle localization probability curves at the broken line-marked cross-sections in fig. 9 (a), 9 (c) and 9 (e), respectively.
As shown in fig. 9, the embodiment of the present invention performs three-dimensional super-resolution photoacoustic localization imaging on the scintillating light absorbing particles. Certain light-absorbing particles will flicker continuously under external excitation, i.e. the emission intensity of their photo acoustic signals will change significantly over time. This property can be used to distinguish and localize individual particles in a collection of multiple particles. As can be seen from fig. 9 (b), 9 (d) and 9 (f), light-absorbing particles showing a pitch as small as 5 μm can be clearly resolved.
Fig. 10 is a schematic diagram of three-dimensional super-resolution photoacoustic localization tracking of a flowing light-absorbing particle in an embodiment of the present invention, where fig. 10 (a) is a flow trajectory of a single light-absorbing particle, and fig. 10 (b) is a channel structure obtained by overlapping flow trajectories of a plurality of light-absorbing particles.
As shown in fig. 10, the embodiment of the present invention performs three-dimensional super-resolution photoacoustic localization tracking on the flowing light-absorbing particles. The signal sampling rate is determined by the repetition rate of the excitation laser, typically 1kHz to 100kHz. The method comprises the steps of performing three-dimensional positioning on light absorption particles flowing in a microfluidic chip or a blood vessel by using a positioning algorithm, connecting continuous light absorption particles in two adjacent positioning steps by using line segments, and obtaining a flow velocity vector for a single particle by using a difference method. 10 (a) is the flow trajectory of the individual light absorbing particles. As shown in fig. 10 (b), a channel structure can be obtained by overlapping the flow trajectories of a plurality of light-absorbing particles. Also, the fluid dynamics analysis can be obtained by calculating the flow trajectory and flow velocity of the particles.
By utilizing the absorption spectrum characteristics of the light absorption particles and the acoustic characteristics of the constituent materials, the super-resolution functional photoacoustic imaging method for single-particle multilateral positioning tracking can be used for distinguishing different types of particles and the change of the physical and chemical characteristics of the particles along with time in the positioning process, and obtaining a super-resolution photoacoustic image with multiple contrasts and functionality.
In the embodiment, the imaging capability of the single-particle multilateral positioning super-resolution photoacoustic imaging system is verified by combining a fluorescence microscope. Gold nanoparticles (10 nm) were sparsely dispersed on the surface of the slide by a nano spin coating process. The gold nanoparticles have autofluorescence under 532nm laser irradiation, and the distribution of the gold nanoparticles on the space can be observed through a side-branch optical path fluorescence microscope. In the implementation, a 10xNA0.3 water mirror is adopted, under the condition, the resolution of the fluorescence microscope is about 1 micron, is superior to the theoretical resolution of a single-particle multi-edge positioning photoacoustic microscopy system, and can be used for recording accurate position information of gold nanoparticles as a reference standard.
After the spatial position calibration of the detector is completed, positioning single gold nanoparticles and multiple gold nanoparticles is carried out, and the specific process is as follows:
step A1, positioning single and multiple gold nanoparticles, moving the gold nanoparticles, exciting and collecting photoacoustic signals to perform positioning, and recording 1000 positions at each position. And calculating the standard variance of the positioning position to obtain three-dimensional positioning precision, and obtaining a spatial distribution map of the positioning precision.
And A2, enlarging an illumination area and simultaneously exciting a plurality of gold nanoparticles. And calculating the three-dimensional positioning precision of each gold nanoparticle. And (4) recording the position of each gold nanoparticle by using a fluorescence microscope, comparing the position with a multilateral positioning result, and analyzing errors.
The microfluidic chip is manufactured by Polydimethylsiloxane (PDMS) to simulate a blood vessel micro-channel structure and is used for single-particle multi-edge positioning super-resolution photoacoustic imaging to realize particle tracking, wherein the whole thickness of the chip is designed to be 200 mu m. The pipe diameter of the micro-channel is designed to be less than 40 mu m, and the pipe diameter of the minimum micro-channel is 6-8 mu m, so that the micro-channel is used for simulating a capillary structure. Since the sound velocity in PDMS is about 1100m/s, which is slightly slower than that in water, compensation is needed in the image reconstruction process. And injecting the blood mixed with the gold nanoparticles into the microfluidic chip, and controlling the flow rate by using an injection pump. The focusing lens was adjusted to produce an illumination field of approximately 3mm in diameter through a 10-fold water mirror. And exciting and collecting photoacoustic signals generated by the flowing gold nanoparticles, wherein the sampling rate range is 1kHz-100kHz. And performing multilateral positioning on the gold nanoparticles by using a positioning algorithm, connecting continuous gold nanoparticles in two adjacent positioning steps by using a line segment, reconstructing the flow track of a single gold nanoparticle, and obtaining a flow velocity vector for the single particle by using a difference method. The microchannel structure can be obtained by overlapping the flow trajectories of a plurality of gold nanoparticles. Also, the fluid dynamics analysis can be obtained by calculating the flow trajectory of the particles.
In the process, a fluorescence microscope is adopted to record the flow of the gold nanoparticles in the microfluidic chip as a reference image, the acquisition integration time of the fluorescence image is 20ms, and the sampling frame rate is 50 frames/s. Since the sampling rates of photoacoustic signal imaging and fluorescence imaging are different, we need to synchronize them first at the time of data processing. And finally, comparing the reconstructed super-resolution photoacoustic image with the fluorescence image to verify the accuracy of the single-particle multilateral positioning photoacoustic imaging.
Examples effects and effects
According to the super-resolution functional photoacoustic imaging method based on single-particle multilateral positioning tracking, the multilateral positioning method is introduced into single-particle positioning super-resolution photoacoustic imaging, the complex inverse algorithm and the requirement on signal intensity in photoacoustic image reconstruction are avoided, the dependence of a super-resolution photoacoustic imaging technology on software and hardware can be greatly reduced, and a new imaging mechanism is utilized to break through the resolution limit of the prior art.
The super-resolution functional photoacoustic imaging method based on single-particle multilateral localization tracking further breaks through the existing resolution limit, and the three-dimensional photoacoustic imaging technology with wide field, sampling rate of more than 1kHz and resolution superior to 3 microns is realized. The limit resolution of the new method is improved by about 7 times compared with the existing super-resolution local particle photoacoustic imaging, and is improved by about 44 times compared with the traditional photoacoustic imaging. In addition, the reconstruction of the image by an inverse algorithm is not needed any more, so that the imaging speed of the new method is improved by more than 50 times compared with that of the existing super-resolution local particle photoacoustic imaging.
The super-resolution functional photoacoustic imaging method based on single-particle multilateral positioning tracking has the characteristic of functional imaging, can be used for distinguishing different types of particles in the positioning process by utilizing the spectrum and ultrasonic characteristics of the particles, and realizes cross-scale high-resolution photoacoustic imaging with multiple contrast and multifunctional characteristics.
The super-resolution functional photoacoustic imaging method based on single-particle multilateral localization tracking can provide imaging parameters complementary to other optical imaging modes, and obtain important physiological information. In the past, due to resolution mismatch, photoacoustic imaging was difficult to integrate perfectly with other optical imaging techniques. The single-particle multilateral positioning photoacoustic imaging technology has the possibility of providing spatial resolution matched with other optical imaging modes, and the combination of photoacoustic imaging and other optical imaging technologies is facilitated.
The above-described embodiments are merely illustrative of specific embodiments of the present invention, and the present invention is not limited to the description of the above-described embodiments.
Claims (8)
1. A super-resolution functional photoacoustic imaging method based on single-particle multilateral localization tracking is characterized by comprising the following steps:
calibrating the position of the detector, namely calibrating the position of the detector by adopting a trilateral positioning method;
positioning a photoacoustic source, namely acquiring a photoacoustic signal of a particle by using a detector and fitting to obtain the distance between the particle and the detector and the distance resolution exceeding the acoustic diffraction limit;
multilateral positioning of particles, namely, obtaining the distance between the particles and the detectors by adopting at least three detectors, and performing multilateral positioning on the particles by utilizing a sound source positioning algorithm to obtain the three-dimensional space positions of the particles;
performing multi-particle iterative positioning calculation, wherein an iterative algorithm is adopted to perform positioning calculation on a plurality of particles to obtain the three-dimensional space position of each particle;
and forming a super-resolution functional photoacoustic image, positioning at least one particle on a time sequence to obtain the corresponding three-dimensional space position, and generating the super-resolution functional photoacoustic image according to the three-dimensional space position and the change of the position of each particle along with time.
2. The single-particle multilateral localization tracking-based super-resolution functional photoacoustic imaging method according to claim 1, wherein the specific process of the detector position calibration is as follows:
the position of the detector is calibrated with light absorbing particles of known position in a two-dimensional plane,
step T1, controlling the diameter and the position of an illumination area by adjusting a focusing lens, and exciting a single light absorption particle on the surface of the glass slide;
step T2, recording the position of the light absorption particles by using a fluorescence microscope;
step T3, exciting and recording the photoacoustic signals from the light absorption particles to three ultrasonic detectors respectively;
step T4, moving the light absorption particles to a new position or shifting an excitation beam to excite the light absorption particles on the surface of the slide at different positions by controlling a two-dimensional linear translation stage;
step T5, repeating the steps T2-T4 until at least 3 groups of the photoacoustic signals are obtained,
and T6, substituting the multiple groups of photoacoustic signals obtained in the step T5 into a trilateral positioning algorithm to perform reverse calculation to obtain the position of the detector in the three-dimensional space.
3. The super-resolution functional photoacoustic imaging method based on single-particle multilateral localization tracking according to claim 2, characterized in that:
wherein the light absorbing particles may be gold nanoparticles, carbon black particles or pigment-containing particles.
4. The single event multilateral localization tracking-based super-resolution functional photoacoustic imaging method according to claim 1, wherein the specific process of photoacoustic source localization is as follows:
acquiring the intensity of the photoacoustic signals emitted by the particles with at least three of the detectors and obtaining an intensity-time curve over time,
fitting the intensity-time curve by adopting a Gaussian fitting method or a maximum likelihood estimation method to obtain a fitting curve, performing time positioning on the photoacoustic signal according to the fitting curve to obtain a distance vector from the particle to the detector,
wherein the fitting of the photoacoustic signal enables obtaining a distance resolution beyond the acoustic diffraction limit.
5. The single-particle multi-edge localization tracking-based super-resolution functional photoacoustic imaging method according to claim 1, wherein the specific process of particle multi-edge localization is as follows:
the sound source positioning algorithm is a least square positioning method, and the positions of n detectors in the space are p i ＝(x i ,y i ,z i ) Wherein i e (1,2,. Once, n), n is a positive integer, the position of the particle is p = (x, y, z), and the distance from the detector is r i ，r i The calculation formula of (2) is as follows:
r i 2 ＝(x i -x)+(y i -y)+(z i -z) 2
obtaining the three-dimensional space position of the particle through a least square positioning algorithm, wherein the calculation formula of the position is as follows:
in the formula (I), the compound is shown in the specification,
and is
6. The super-resolution functional photoacoustic imaging method based on single-particle multilateral localization tracking according to claim 1, characterized in that:
the sound source positioning algorithm can be a Fourier domain reconstruction algorithm, a back projection algorithm, an iterative reconstruction algorithm or a deep learning reconstruction.
7. The single-particle multilateral localization tracking-based super-resolution functional photoacoustic imaging method according to claim 1, wherein the specific process of the multi-particle iterative localization calculation is as follows:
obtaining distance vectors between a plurality of the particles to the respective detectors by using the photoacoustic sound source localization,
randomly selecting the distance vector in one detector, carrying out the multilateral particle positioning process with the distance vectors between the detectors except the detector, and obtaining a unique solution or a most probable solution of the three-dimensional space position of the particle,
and selecting the distance vectors which are not used for positioning calculation to perform the particle multilateral positioning and obtain a unique solution or a probability maximum solution of the three-dimensional space position of another particle until all the particles finish the particle multilateral positioning.
8. The single-particle multilateral localization tracking-based super-resolution functional photoacoustic imaging method according to claim 1, wherein the specific process of the functional super-resolution photoacoustic imaging is as follows:
and distinguishing different types of particles and the change of the physical and chemical characteristics of the particles along with time in the positioning process by utilizing the absorption spectrum characteristics of the particles and the acoustic characteristics of the constituent materials to obtain the super-resolution functional photoacoustic image with multiple contrasts.
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