CN111110198A - Photoacoustic wavefront shaping microscopic imaging method for biological tissue - Google Patents
Photoacoustic wavefront shaping microscopic imaging method for biological tissue Download PDFInfo
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- CN111110198A CN111110198A CN202010024092.2A CN202010024092A CN111110198A CN 111110198 A CN111110198 A CN 111110198A CN 202010024092 A CN202010024092 A CN 202010024092A CN 111110198 A CN111110198 A CN 111110198A
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- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/0093—Detecting, measuring or recording by applying one single type of energy and measuring its conversion into another type of energy
- A61B5/0095—Detecting, measuring or recording by applying one single type of energy and measuring its conversion into another type of energy by applying light and detecting acoustic waves, i.e. photoacoustic measurements
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/72—Signal processing specially adapted for physiological signals or for diagnostic purposes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/72—Signal processing specially adapted for physiological signals or for diagnostic purposes
- A61B5/7203—Signal processing specially adapted for physiological signals or for diagnostic purposes for noise prevention, reduction or removal
Abstract
The invention provides a photoacoustic wavefront plastic microscopic imaging method for biological tissues, which comprises the following steps: acquiring an original photoacoustic signal; wavelet denoising and relevant detection are combined, and photoacoustic signals are extracted; using an optimization algorithm to control iterative optimization wave front shaping; after the optimization is completed, the optimal mask is loaded to the light modulation device, so that the photoacoustic signal is effectively enhanced; and scanning imaging. The invention combines wavelet de-noising and related detection to remove white noise and avoid clutter interference, and can effectively extract photoacoustic signals under the condition that incident light is seriously scattered and excited photoacoustic signals are weak; the invention combines an optimization algorithm with the optical modulation device, and performs iterative optimization wave front shaping by taking the normalized photoacoustic signal as feedback, thereby effectively enhancing the strength of the photoacoustic signal, obtaining a high-contrast image and improving the depth of photoacoustic imaging.
Description
Technical Field
The invention belongs to the field of photoacoustic imaging, and particularly relates to a photoacoustic wavefront shaping microscopic imaging method for biological tissues.
Background
Biomedical imaging has guiding effects on disease diagnosis, disease treatment, drug delivery and the like. Light travels along a straight line in a transparent medium. Biological tissue is typically a strongly scattering tissue in which light propagates and undergoes multiple scattering, rapidly becoming scattered light. Conventional high-resolution optical imaging methods such as confocal microscopy, optical coherence tomography or multiphoton imaging all use ballistic light to image, the depth of imaging in biological tissue does not exceed a transmission mean free path, usually about several microns, and are only suitable for imaging superficial biological tissue. The photoacoustic imaging method based on the photoacoustic effect has the advantages of both light absorption contrast and ultrasonic transmission. The photoacoustic effect refers to the absorption of energy from incident light by tissue, resulting in thermal expansion and thus the generation of an ultrasound signal. This signal is a photoacoustic signal whose intensity is proportional to the intensity of the incident light and the optical absorption coefficient of the tissue at that location. In the photoacoustic imaging method, a tissue is irradiated with laser light and photoacoustic signals are collected, and a light absorption contrast image of the tissue is restored. The influence of the scattering on the ultrasound is 2-3 orders of magnitude weaker than that of the scattering on the light, so that the depth of the biological tissue imaging is effectively improved by the photoacoustic imaging method. However, with further increase of the imaging depth, the scattering of incident light becomes more severe, and a situation that the photoacoustic signal cannot be effectively excited begins to occur, and the signal is weak and submerged in noise, so that the signal extraction is difficult, and the imaging quality is reduced or even the imaging cannot be performed. By merely increasing the light pulse energy to enhance the photoacoustic signal intensity, damage to the tissue surface is easily caused. There is a need for a method that can overcome the severe scattering of incident light, effectively excite photoacoustic signals, and further improve the photoacoustic imaging depth of biological tissues.
Disclosure of Invention
The photoacoustic imaging method aims to solve the problems that in photoacoustic imaging, incident light is seriously scattered along with the increase of imaging depth, a photoacoustic signal cannot be effectively excited, the signal is weak and is submerged in noise, the image quality of the photoacoustic imaging is reduced, even the photoacoustic imaging cannot be carried out, and the photoacoustic imaging depth is limited.
The invention provides a photoacoustic wavefront plastic microscopic imaging method for biological tissues, which comprises the following steps:
step (1): acquiring an original photoacoustic signal;
step (2): wavelet denoising and relevant detection are combined, and photoacoustic signals are extracted;
and (3): using an optimization algorithm to control iterative optimization wave front shaping;
and (4): after the optimization is completed, the optimal mask is loaded to the light modulation device, so that the photoacoustic signal is effectively enhanced;
and (5): and (5) scanning and imaging.
The method comprises the following steps that (1) a pulse laser source is used for emitting optical pulses with a certain repetition frequency; generating a random mask, loading the random mask on an optical modulation device, and performing wavefront modulation on an incident light pulse; comprising projecting modulated light pulses onto a biological tissue sample; comprises collecting photoacoustic signals using an ultrasonic detection device; including amplifying the photoacoustic signal using a signal amplifier.
Selecting a wavelet basis function similar to the waveform of the photoacoustic signal, determining the level, the threshold type and the threshold processing mode of the photoacoustic signal wavelet decomposition, and performing wavelet denoising on the photoacoustic signal wavelet basis function to remove white noise; the method comprises the following steps of pre-selecting a photoacoustic signal template which is not interfered by clutter, and according to a formula:
calculating a normalized correlation coefficient between the acquired photoacoustic signal and the photoacoustic signal template, wherein,γ xy is a normalized correlation coefficient of the correlation data,Mis the length of the signal or signals,x(m)is a template signalmThe value of the point is such that,y(m)is the collected photoacoustic signalmThe value of the point is such that,
is the average of the template signal and is,
is the mean of the collected photoacoustic signals; judging whether the collected photoacoustic signals are interfered by clutter or not according to the correlation coefficient so as to eliminate the interfered signals.
Wherein, the step (3) comprises the step of separating a part of the light pulse emitted by the pulse laser source for detecting the energy of the light pulse; dividing the photoacoustic signal by corresponding optical pulse energy to obtain a normalized photoacoustic signal; taking a peak value of a normalized photoacoustic signal as a feedback signal for iterative optimization wave front shaping; the method comprises the steps of using an optimization algorithm to control an iterative optimization process, and retrieving the optimal wave front distribution of incident light and the corresponding optimal mask.
Loading the optimal mask obtained by iterative optimization on the optical modulation device, modulating the wavefront of incident light, realizing light focusing at a target position in the biological tissue, and effectively exciting a photoacoustic signal; comprising collecting and amplifying photoacoustic signals; extracting photoacoustic signals by using a method combining wavelet denoising and related detection; including calculating and storing normalized photoacoustic signals.
Wherein, the step (5) comprises moving the sample for scanning; optimizing focusing and storing the normalized photoacoustic signal extracted after optimization is completed; including recovering an image from the photoacoustic signals at each point.
The invention has the beneficial effects that: the invention combines wavelet de-noising and related detection to remove white noise and avoid clutter interference, and can effectively extract photoacoustic signals under the conditions that incident light is seriously scattered and excited photoacoustic signals are weak; the invention combines an optimization algorithm with the optical modulation device, and performs iterative optimization wave front shaping by taking the normalized photoacoustic signal as feedback, thereby effectively enhancing the strength of the photoacoustic signal, obtaining a high-contrast image and improving the depth of photoacoustic imaging.
Drawings
The invention is further illustrated with reference to the following figures and examples.
FIG. 1 is a flow chart of a photoacoustic wavefront shaping microscopic imaging method for biological tissues according to the present invention;
FIG. 2 is a block diagram of the system components of the method of the present invention in example 1 and example 2;
FIG. 3 is a schematic structural diagram of the method of the present invention in example 1;
fig. 4 is a schematic structural diagram of the method of the present invention in embodiment 2.
In the figure, 1, a light source module, 2, an optical pulse energy monitoring module, 3, an optical wavefront modulation module, 4, a sample and displacement platform module, 5, a control module, 6, a signal acquisition and processing module, 7, a pulse laser source, 8, a beam splitter, 9, an attenuator, 10, a photoelectric detector, 11, a data acquisition card, 12, a beam expansion collimator, 13, a first spatial filter, 14, a reflector, 15, an optical modulation device, 16, a microscope objective, 17, a biological tissue sample, 18, a displacement platform, 19, an ultrasonic detection device, 20, a small signal amplifier, 21, a computer, 22, a first convex lens, 23, a second spatial filter and 24, a second convex lens.
Detailed Description
The invention is further illustrated with reference to the following figures and examples.
The invention relates to a photoacoustic wavefront shaping microscopic imaging method for biological tissues, which comprises the steps of firstly acquiring original photoacoustic signals as shown in figure 1; then, combining wavelet denoising with related detection to extract photoacoustic signals; then, using an optimization algorithm to control iterative optimization wavefront shaping; after the optimization is completed, the optimal mask is loaded to the light modulation device, so that the photoacoustic signal can be effectively enhanced; and finally scanning and imaging.
[ example 1 ]
The embodiment provides a biological tissue photoacoustic wavefront shaping microscopic imaging system applying the biological tissue photoacoustic wavefront shaping microscopic imaging method provided by the invention. The system comprises a light source module 1, a light pulse energy monitoring module 2, a light wavefront modulation module 3, a sample and displacement platform module 4, a control module 5 and a signal acquisition and processing module 6.
The embodiment realizes the basic flow of the photoacoustic wavefront shaping microscopic imaging of the biological tissue: the light source module 1 emits pulsed light, the pulsed light is split, a part of the pulsed light is turned to the optical pulse energy monitoring module 2, and the optical pulse energy change is monitored through the photoelectric detector 10, so that the photoacoustic signal is conveniently normalized, and the influence caused by the fluctuation of the optical pulse energy is eliminated. The rest light continues to propagate along the original light path, in the light wave front modulation module 3, the light modulation device modulates the wave front, the modulated light is emitted to a biological tissue sample 17 soaked in the solution through a microscope objective lens 16, an ultrasonic detection device 18 collects photoacoustic signals excited by the pulsed light, the signals are amplified by a small signal amplifier 20 and collected by a data acquisition card 11, and the signals are extracted through wavelet denoising and related detection methods. And finally, normalizing by combining the optical pulse energy data detected by the optical pulse energy monitoring module. The normalized photoacoustic signal is used as a feedback signal for the iterative optimization process. The optimization algorithm selected in the present embodiment is a genetic algorithm. The iterative optimization process is controlled by using a genetic algorithm, the optimal mask is retrieved and loaded on the light modulation device, wave front shaping light focusing at a certain depth inside the biological tissue is realized, the light intensity of a target point is effectively enhanced, the photoacoustic signal is enhanced, and the signal-to-noise ratio of the photoacoustic signal is improved. The sample is moved by the displacement platform 18 for scanning imaging.
The structural schematic diagram of the biological tissue photoacoustic wavefront shaping microscopic imaging system of the present embodiment is shown in fig. 3. The light source in the light source module 1 employed in the present embodiment is a pulse laser with a wavelength of 532nm and a repetition frequency of 10 kHz. The light source module 1 outputs an optical pulse having a repetition frequency of 10kHz, and is split into two beams of light by a 1:9 beam splitter 8. The 10% light is turned to the light pulse energy monitoring module 2, hit on the attenuation sheet 9 at first, hit on the photodetector 10 after attenuating sheet 9 again, the photodetector 10 is connected to the data acquisition card 11. 90% of the light continues to propagate along the original optical path and enters the optical wavefront modulation module 3. The beam diameter is adjusted by the beam expanding collimator 12, and the spot size is adjusted by the first spatial filter 13 to be equivalent to the size of the light modulation device 15. And then reflected by the mirror 14 onto the light modulation device 15. In the embodiment, a phase type light modulation device is selected, and a Spatial Light Modulator (SLM) modulates the wavefront phase of the phase type light modulation device. The modulated light pulses are focused by a microscope objective 16 onto a biological tissue sample 17. The sample of this example is mouse brain tissue, immersed in a solution, placed on a two-dimensional moving platform 18. The signal acquisition and processing module 6 acquires photoacoustic signal data in addition to the optical pulse energy data. The ultrasonic signal excited by the light pulse is detected by an ultrasonic detection device 19, which is a focused water immersion ultrasonic probe with a center frequency of 20MHz and a focal region size of 20 μm. The photoacoustic signal collected by the ultrasonic detection device 19 is amplified by the small signal amplifier 20 and sent to the data acquisition card 11. In this embodiment, an oscilloscope integrated with a data acquisition card is used for data acquisition and display.
The computer 21 reads the photoacoustic signal and the optical pulse energy data from the oscilloscope and extracts the photoacoustic signal using a method combining wavelet de-noising and correlation detection. In the embodiment, a coif5 wavelet basis function with a waveform close to that of the photoacoustic signal is selected, 8-layer wavelet decomposition is performed on the original signal, a square root threshold is selected, and the threshold type is a soft threshold. The white noise is effectively removed by wavelet denoising, then the normalized correlation coefficient is calculated by the white noise and a preselected photoacoustic signal waveform without the clutter, and the photoacoustic signal waveform with the normalized correlation coefficient lower than 0.7 is regarded as being seriously interfered by the clutter and is removed. Through wavelet denoising and relevant detection, the photoacoustic signal is effectively extracted. The ratio of the denoised photoacoustic signal peak value to the optical pulse energy is the normalized photoacoustic signal intensity, and is used as a feedback signal for iteratively optimizing photoacoustic wavefront shaping. A genetic algorithm is used to control an iteratively optimized wavefront shaping process. In this embodiment, first, 20 random phase masks are generated and sequentially loaded onto the SLM, and then the 20 masks are sorted according to the normalized photoacoustic signal intensity, the higher the sorting is, the more easily the masks are selected, the masks are used for generating the next generation of 20 masks through cross variation, and after 80 iterations are completed, the mask with the strongest photoacoustic signal is taken as the optimal mask. And loading the optimal mask on the SLM, forming a focusing point at the target position in the biological tissue by the modulated light, and exciting a stronger photoacoustic signal. And moving the biological tissue sample through a displacement platform, optimizing and focusing point by point, storing the normalized photoacoustic signal extracted after the optimization is completed, and finally recovering the light absorption contrast image of the biological tissue sample according to the photoacoustic signal of each point.
[ example 2 ]
The embodiment provides a biological tissue photoacoustic wavefront shaping microscopic imaging system applying the biological tissue photoacoustic wavefront shaping microscopic imaging method provided by the invention. The system composition block diagram is shown in fig. 2. The schematic diagram of the system structure is shown in fig. 4.
The present embodiment is different from embodiment 1 in that the light modulation device employed in the present embodiment is an amplitude type light modulation device, a Digital Micromirror Device (DMD).
In this embodiment, the DMD is used to implement common modulation of the amplitude and phase of the incident light in an off-axis configuration, and the iterative optimization of the photoacoustic wavefront shaping is performed: the DMD is divided into independent super pixel blocks, each super pixel block is composed of n multiplied by n sub-pixels, and 1 micro mirror on the DMD serves as 1 sub-pixel; the light reflected from the DMD surface passes through a 4f system consisting of a first convex lens 22, a second spatial filter 23 and a second convex lens 24 before being focused on the biological tissue sample by the microscope objective lens 16, wherein the second spatial filter 23 is selected to be at the +1 st or-1 st order diffracted light position, and only the +1 st or-1 st order diffracted light is allowed to pass; the light reaching the spatial filter from different sub-pixel positions in the same super-pixel block has different phases, and the super-pixel blocks with different phases can be obtained by superposition of different sub-pixel combinations, so that phase modulation of incident light by using the DMD is realized.
According to the specific embodiment, the invention provides a photoacoustic wavefront shaping microscopic imaging method for biological tissues. Wavelet denoising and related detection are combined firstly for removing white noise and avoiding clutter interference, and photoacoustic signals are effectively extracted. And then modulating the wave front of incident light, taking the normalized photoacoustic signal as feedback, and carrying out iterative optimization based on a genetic algorithm to find the optimal wave front, effectively enhance the strength of the photoacoustic signal, obtain a high-contrast image, and further improve the depth of photoacoustic imaging. It should be noted that the form of pulsed laser source and spatial light modulator required is not limiting to this patent; the adopted optimization algorithm can be any one of a sequence optimization algorithm, a block optimization algorithm, a genetic algorithm, a particle swarm algorithm, a multi-frequency parallel optimization algorithm and the like.
Claims (6)
1. The invention provides a photoacoustic wavefront plastic microscopic imaging method for biological tissues, which is characterized by comprising the following steps of:
step (1): acquiring an original photoacoustic signal;
step (2): wavelet denoising and relevant detection are combined, and photoacoustic signals are extracted;
and (3): using an optimization algorithm to control iterative optimization wave front shaping;
and (4): after the optimization is completed, the optimal mask is loaded to the light modulation device, so that the photoacoustic signal is effectively enhanced;
and (5): and (5) scanning and imaging.
2. A photoacoustic wavefront shaping microscopic imaging method for biological tissue according to claim 1, wherein said step (1): acquiring an original photoacoustic signal, including emitting an optical pulse with a certain repetition frequency by using a pulse laser source; generating a random mask, loading the random mask on an optical modulation device, and performing wavefront modulation on an incident light pulse; comprising projecting modulated light pulses onto a biological tissue sample; comprises collecting photoacoustic signals using an ultrasonic detection device; including amplifying the photoacoustic signal using a signal amplifier.
3. A photoacoustic wavefront shaping microscopic imaging method for biological tissue according to claim 1, wherein said step (2): combining wavelet denoising and related detection, extracting photoacoustic signals, selecting wavelet basis functions similar to waveforms of the photoacoustic signals, determining levels, threshold types and threshold processing modes of wavelet decomposition of the photoacoustic signals, and performing wavelet denoising on the photoacoustic signals to remove white noise; the method comprises the following steps of pre-selecting a photoacoustic signal template which is not interfered by clutter, and according to a formula:
calculating a normalized correlation coefficient between the acquired photoacoustic signal and the photoacoustic signal template, wherein,γ xy is a normalized correlation coefficient of the correlation data,Mis the length of the signal or signals,x(m)is a template signalmThe value of the point is such that,y(m)is the collected photoacoustic signalmThe value of the point is such that,
is the average of the template signal and is,
is the mean of the collected photoacoustic signals; judging whether the collected photoacoustic signals are interfered by clutter or not according to the correlation coefficient so as to eliminate the interfered signals.
4. A photoacoustic wavefront shaping microscopic imaging method for biological tissue according to claim 1, wherein said step (3): using an optimization algorithm to control iterative optimization wavefront shaping, wherein the iterative optimization wavefront shaping comprises the step of dividing a part of the optical pulse emitted by the pulse laser source for detecting the energy of the optical pulse; dividing the photoacoustic signal by corresponding optical pulse energy to obtain a normalized photoacoustic signal; taking a peak value of a normalized photoacoustic signal as a feedback signal for iterative optimization wave front shaping; the method comprises the steps of using an optimization algorithm to control an iterative optimization process, and retrieving the optimal wave front distribution of incident light and the corresponding optimal mask.
5. A biological tissue photoacoustic wavefront shaping microscopic imaging method according to claim 1, wherein said step (4): after the optimization is completed, the optimal mask is loaded on the optical modulation device, the photoacoustic signal is effectively enhanced, the optimal mask obtained by iterative optimization is loaded on the optical modulation device, the wave front of incident light is modulated, the light focusing at the target position in the biological tissue is realized, and the photoacoustic signal is effectively excited; comprising collecting and amplifying photoacoustic signals; extracting photoacoustic signals by using a method combining wavelet denoising and related detection; including calculating and storing normalized photoacoustic signals.
6. A biological tissue photoacoustic wavefront shaping microscopic imaging method according to claim 1, wherein said step (5): scanning imaging, including moving a sample for scanning; optimizing focusing and storing the normalized photoacoustic signal extracted after optimization is completed; including recovering an image from the photoacoustic signals at each point.
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