Accurate view field distortionless scanning high-resolution microscopic imaging method and device based on position feedback

Abstract

The invention discloses an accurate view field distortionless scanning high-resolution microscopic imaging method and device based on position feedback. The piezoelectric deflection mirror system is used for replacing a traditional scanning system, a position feedback signal of the deflection mirror is utilized, clock signals of light source switching and signal detection are obtained, the light source is controlled to be turned on when the deflection mirror is located at a target view field and turned off when the deflection mirror is located at other positions, and therefore the view field is accurately controlled, and the scanning precision is improved. The photosensitive sample can be protected on the hardware level, so that the utilization rate of the sample is improved; position feedback signals of the deflection mirror under different voltage amplitudes and different scanning frequencies are processed, a linear section proportion which can be considered to be in distortionless scanning imaging in a scanning stroke is extracted, the variable is deduced, a more accurate fitting relational expression is provided, and the linear section proportion is calculated. A correct voltage amplitude capable of enabling a target view field to be in a linear section in a scanning stroke is applied to the deflection mirror, so that distortionless target view field scanning is realized. The method and the device have a wide application range.

Description

Technical Field

[0001] The present invention relates to the field of high-speed scanning microscopy imaging technology, and in particular, to a precise field-of-view distortion-free scanning high-resolution microscopy imaging method and device based on position feedback. Background Art

[0002] Ultra-high resolution microscopy imaging technology is widely used in the fields of biomedicine, materials science, and semiconductors. With the continuous development of related fields, the related research on ultra-high resolution microscopy imaging technology is also constantly advancing.

[0003] Ultra-high resolution microscopy imaging technology can generally be divided into two types: point scanning and wide-field imaging. Among them, point scanning imaging technology refers to a class of imaging methods that control the beam to scan on the sample surface, collect the reflected light or scattered light signals carrying the sample information, and reconstruct each pixel point to restore the sample image. Representative technologies include laser confocal and stimulated emission depletion imaging technologies, etc. In point scanning imaging technology, there are common problems such as the decrease in sample utilization rate and distortion in the imaging result due to the mismatch between the non-linear movement of the scanning system and the sequential acquisition module.

[0004] The method and device proposed by the present invention will achieve precise control of the scanning field of view from the hardware, solve the above-mentioned problem of the decrease in sample utilization rate, and establish the relationship between the target field of view and the scanning parameters by extracting the scanning position feedback signal, so as to achieve fast and accurate distortion-free imaging. This will provide a new idea for imaging technologies such as point scanning, open up a road for distortion-free imaging methods other than adding additional positioning devices, inputting complex voltage waveforms for correction, and correcting through post-processing algorithms, promote the further development of ultra-high resolution imaging technology, and thus drive the rapid development of related application fields. Summary of the Invention

[0005] The object of the present invention is to provide a method and device for accurate field-of-view distortion-free scanning high-resolution microscopic imaging based on position feedback. Aiming at the problem of random field of view in the common point-scanning super-resolution imaging technology, and thus the decrease in utilization rate caused by the arbitrary exposure of the sample under beam scanning, it is proposed to use a Tip-tilt mirror to replace the common galvanometer scanning system at the hardware level, and use the position feedback signal of the mirror to achieve precise control of the field of view and improve the utilization rate of the sample; aiming at the problem of imaging field-of-view distortion in the common point-scanning super-resolution imaging technology, a method for correcting imaging distortion based on the proportion of the linear segment of the scanning stroke is proposed. By processing the position feedback signals of the Tip-tilt mirror at different voltage amplitudes and different scanning frequencies, the proportion of the linear segment that can be considered as distortion-free scanning imaging in the scanning stroke of the mirror is extracted. On this basis, a new and more accurate fitting relationship is derived for the variable of the proportion of the linear segment of the Tip-tilt mirror. By applying the correct voltage amplitude to the mirror to make the target field of view in the linear segment of the scanning stroke, distortion-free scanning of the target field of view is achieved.

[0006] The object of the present invention is achieved by the following technical solutions:

[0007] According to the first aspect of the present specification, a method for accurate field-of-view distortion-free scanning high-resolution microscopic imaging based on position feedback is provided, and the method includes:

[0008] In the confocal microscopy imaging optical path, a piezoelectric mirror system with a position feedback function is used as the scanning module, and an electro-optic modulation device that realizes the function of switching light through pulse width modulation is added at the light source;

[0009] Obtain the fast-axis scanning position feedback information, load the voltage waveforms amplitude by amplitude and frequency by frequency according to the maximum amplitude corresponding to the maximum field of view of the scanning module, record the input voltage waveform and the actual position feedback information each time, and obtain the proportion of the linear segment at different voltage amplitudes and different scanning frequencies;

[0010] Fit the data of the proportion of the linear segment, fit the amplitude-frequency response of the actual position feedback information of each input voltage waveform, obtain the damping factor at each amplitude and fit the relationship between the damping factor and the amplitude under non-linearity to obtain an accurate amplitude-frequency response relationship, and then obtain an accurate relationship of the proportion of the linear segment;

[0011] Associate the scanning parameters with the proportion of the linear segment, verify whether the relationship of the proportion of the linear segment can obtain the accurate target field of view, and obtain the accurate scanning waveform after passing the verification;

[0012] Real-time collect the position feedback information of the scanning module, judge whether the scanning position is within the target field of view, obtain the switching optical timing signal of the electro-optical modulation device and output it to the electro-optical modulation device to achieve accurate field-of-view imaging.

[0013] Further, based on the proportion of the linear segment of the scanning stroke, imaging distortion correction is realized. The linear segment in the scanning stroke refers to the scanning range where the slope error of the stroke curve and the target maximum slope is within the set adjustable error δ. The proportion of the linear segment is the ratio between the size of the linear segment and the field of view corresponding to the maximum amplitude of the input voltage. The calculation of the proportion of the linear segment is specifically as follows: Determine the adjustable error δ, take the first derivative of the input voltage waveform and the actual position waveform, calculate the linear segment threshold. The range of the actual position waveform above this threshold is the linear segment stroke, and then obtain the proportion of the linear segment.

[0014] Further, set the adjustable error δ = 25%. The input voltage waveform adopts a sine waveform. Since the input waveform has periodicity and the same nature for round trips, only the data within the range of 1 / 4 cycle needs to be extracted for calculating the proportion of the linear segment.

[0015] Further, the fitting of the proportion data of the linear segment is specifically as follows: Fit the amplitude-frequency response of the second-order oscillation link to the position feedback signal to obtain the curves of the natural frequency and the damping factor changing with different amplitudes; The damping factor is negatively correlated with the amplitude. Perform non-linear fitting on the damping factor to obtain the relationship between the damping factor and the amplitude. Use this relationship to replace the damping ratio in the original amplitude-frequency response relationship to obtain an accurate amplitude-frequency response relationship, and then obtain an accurate proportion of the linear segment.

[0016] Further, use the iterative trial method to obtain an accurate scanning waveform based on the proportion of the linear segment. Specifically: Determine the line frequency and the size of the target field of view according to the user input parameters. Set the initial amplitude as the maximum loadable amplitude of the scanning module, perform preprocessing, calculate the proportion of the linear segment and the actual field of view size. Compare whether the difference between the target field of view size and the actual field of view size is within the adjustable threshold m. If it is satisfied, it is regarded that the current amplitude and the proportion of the linear segment meet the requirements and output as the result. Otherwise, subtract the adjustable parameter n from the current amplitude downward, substitute the new amplitude into the above process and compare again whether it is close to the target field of view size. When the amplitude is reduced to 0, it means that the current system cannot meet the target field of view size.

[0017] Further, by applying the correct voltage amplitude to the yaw mirror to make the target field of view in the linear segment of the scanning stroke, distortion-free scanning of the target field of view is realized.

[0018] Furthermore, when it is determined that the scanning position enters the target field of view through the position feedback information collected in real time, the electro-optical modulation device that controls the light source switch is turned on. When it is determined that the scanning position leaves the target field of view, the electro-optical modulation device is turned off, so as to achieve no redundant exposure in the non-field-of-view range of the sample and improve the sample utilization rate.

[0019] According to the second aspect of this specification, a high-resolution microscopic imaging device with accurate field-of-view non-distortion scanning based on position feedback is provided. The device includes a light source, an electro-optical modulation device, a scanning module, an acquisition card, and a computer. The electro-optical modulation device realizes the function of switching light through pulse width modulation. The scanning module adopts a piezoelectric yaw mirror system with a position feedback function. The position feedback signal output port of the scanning module is connected to the input ports of the acquisition card and the electro-optical modulation device to realize the acquisition of position feedback information and the switching light control of the electro-optical modulation device. The acquisition card is connected to the computer to realize the acquisition of the fast-axis scanning position feedback information, the processing of obtaining the linear segment ratio at different voltage amplitudes and different scanning frequencies, the fitting of the linear segment ratio data, the acquisition of the accurate scanning waveform, and when collecting the position feedback information in real time, controlling the electro-optical modulation device to realize accurate field-of-view imaging.

[0020] Furthermore, the piezoelectric yaw mirror system adopts a Tip-Tilt piezoelectric yaw mirror system, which can operate quickly and stably at a high resonance frequency within 1KHz and has a position sensor that can feedback the scanning position information.

[0021] Furthermore, the electro-optical modulation device realizes the change of the polarization direction of the incident light by applying different voltages. Cooperating with the polarizer and analyzer placed before and after the electro-optical modulation device with perpendicular orientations, when the applied voltage is at the half-wave voltage, the switching of light is completely realized.

[0022] Compared with the prior art, the present invention has the following advantages and innovation points:

[0023] 1. Based on the position feedback signal, the present invention uses the electro-optical modulation device to control the light source to be turned on only when the scanning position of the scanning module is in the target field of view and turned off at other positions. This not only realizes the accurate control of the field of view, but also can protect photosensitive samples at the hardware level, reduce the unnecessary exposure times in the non-imaging area of the sample, and improve the sample utilization rate.

[0024] 2. The present invention proposes an imaging distortion correction method based on position feedback. By processing the position feedback signals of the scanning module at different voltage amplitudes and different scanning frequencies, the proportion of the linear segment that can be considered as non-distorted scanning imaging in the scanning stroke is extracted. On this basis, the proportion of the linear segment is deduced, and combined with the actual physical meaning, considering various factors of non-linear motion, a new and more accurate fitting relationship is proposed. By applying the correct voltage amplitude that enables the target field of view to be in the linear segment of the scanning stroke to the scanning module, distortion-free scanning of the target field of view is achieved.

[0025] 3. The present invention proposes an algorithm flow for the actual scanning waveform suitable for a point-scanning ultra-high-resolution microscopy imaging device. The user operates using common and easily understandable parameters. According to the proposed correspondence of scanning parameters, the line frequency and the offset are obtained, and the accurate amplitude is obtained based on the proportion of the linear segment using the iterative trial method proposed by the present invention, so as to obtain an accurate scanning waveform.

[0026] 4. Compared with other scanning field distortion correction methods, the method and device proposed by the present invention only need to be changed at the hardware level, and there are many choices for changes. It only requires that the scanning module has a position feedback function and a switching optical device with high pulse width adjustment. The algorithm flow has high feasibility, and the processing speed and accuracy are adjustable. Only the extraction work of the proportion of the linear segment in the early stage is required, and subsequent work such as scanning waveform calculation and acquisition imaging can be executed by the processor. Therefore, the solution of the present invention can be extended to any point-scanning ultra-high-resolution microscopy imaging system, and has the advantages of a wide application range, small overall changes in the system, protecting samples, improving sample utilization rate, and the accuracy and speed can be freely adjusted according to system requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0028] Figure 1 Frequency characteristics of a second-order oscillation link under different damping factors shown in an exemplary embodiment;

[0029] Figure 2 Normalized position feedback information curve shown in an exemplary embodiment;

[0030] Figure 3 Graph of natural frequency and damping factor at different amplitudes after fitting shown in an exemplary embodiment;

[0031] Figure 4Flow chart of the iterative trial value method shown for an exemplary embodiment;

[0032] Figure 5 Operation process shown for an exemplary embodiment;

[0033] Figure 6 Optical path diagram of the system device shown for an exemplary embodiment;

[0034] Figure 7 Process diagram of extracting the proportion of the linear segment shown for an exemplary embodiment. Detailed implementation manners

[0035] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention. It should be noted that, without conflict, the features in the following embodiments and implementation manners can be combined with each other.

[0036] The present invention provides a precise field-of-view non-distortion scanning high-resolution microscopic imaging method and device based on position feedback. Aiming at the problem of random field of view in the common point-scanning super-resolution imaging technology, and thus the utilization rate decrease caused by the arbitrary exposure of the sample under the beam scanning, it is proposed to use a Tip-tilt mirror to replace the common galvanometer scanning system at the hardware level, and use the position feedback signal of the mirror to achieve precise control of the field of view and improve the sample utilization rate; aiming at the problem of imaging field-of-view distortion in the common point-scanning super-resolution imaging technology, a method for correcting imaging distortion based on the proportion of the linear segment of the scanning stroke is proposed. By processing the position feedback signals of the Tip-tilt mirror at different voltage amplitudes and different scanning frequencies, the proportion of the linear segment that can be considered as non-distortion scanning imaging in the scanning stroke of the mirror is extracted. On this basis, a new and more accurate fitting relationship is deduced for the variable of the proportion of the linear segment of the Tip-tilt mirror, and by applying the correct voltage amplitude to the mirror to make the target field of view in the linear segment of the scanning stroke, the scanning of the target field of view without distortion is achieved.

[0037] The technical solutions adopted by the present invention for precisely controlling the field of view are as follows: The confocal imaging technology is an important basis for super-resolution imaging technology. Therefore, the present invention is an improvement on the basis of the traditional confocal imaging optical path. In the traditional confocal imaging optical path, the system responsible for driving the light spot to scan on the sample surface is called the scanning module. Common scanning modules include devices such as prism scanning, Nipkow disk scanning, galvanometer scanners, resonant scanners, and combinations thereof. The latter two are currently widely used. Among them, the galvanometer scanner has the advantages of simple mechanical structure, small moment of inertia, being able to change the frequency within its controllable frequency range and having linear control, and a large scanning field of view, but it also has the disadvantages of slow scanning speed and large jitter. The advantages of the resonant scanner are fast speed, long service life, and small required power. Its disadvantages are that the scanning frequency is fixed, the scanning field of view is small, and there is scanning distortion. The present invention makes improvements on the scanning module and proposes to use a Tip-Tilt piezoelectric mirror system to replace the common scanning system. This device can operate quickly and stably at a high resonance frequency within 1KHz, and it has a position sensor that can feedback the scanning position information, which provides a hardware basis for achieving a highly linear field of view scanning. By collecting and obtaining the position feedback information during the light spot scanning, when the scanning position enters the corresponding field of view range, the electro-optical modulator (EOM) that controls the laser switch will be turned on, and when the scanning position leaves the corresponding field of view range, the EOM will be turned off, so as to achieve no extra exposure in the non-field of view range of the sample, thereby improving the sample utilization rate.

[0038] The technical solutions adopted by the present invention for achieving non-distorted imaging results are as follows: In a point-scanning super-resolution microscopy system, in order to achieve two-dimensional scanning, the scanning module is usually divided into two parts, which respectively implement the scanning tasks of the horizontal and vertical axes. One axis is called the fast axis because its waveform has a relatively high change frequency, and the other axis is called the slow axis, which needs to wait for the end of the fast axis task before changing to the next state. The present invention mainly focuses on the fast axis task and solves its common field of view distortion problem. The piezoelectric mirror system has a position and angular velocity negative feedback link, and it is similar to the common galvanometer scanner system in terms of amplitude-frequency response characteristics. For simplicity, it can be regarded as a second-order response linear time-invariant system, and its transfer function can be expressed by the following formula:

[0039]

[0040] where K D (scanner damping constant) is the damping constant of the mirror, K θ is the transducer position constant, K is the system gain, J is the moment of inertia, D is the system's inherent damping coefficient, K Sis the stiffness constant; θ(S) is the Laplace transform function of the deflection angle θ(t) of the deflecting mirror, V m (S) is the Laplace transform function of the input control voltage V m (t), C is the voltage amplification factor, and S is the complex frequency variable in the Laplace transform. As Figure 1 shown, Equation (1) indicates that when the frequency is not very high, the deflection angle of the deflecting mirror is proportional to the input control voltage and there is no phase lag phenomenon. The deflecting mirror system has good dynamic characteristics. At this time, signals of any waveform can be input and the mirror can move normally. As the frequency increases, the ratio between the two changes in two ways. One is that the ratio is no longer a real number but becomes an imaginary number, which causes the phase between the deflection angle and the input control voltage to be no longer synchronized. The other change is that the absolute value of the ratio gradually becomes smaller, which is equivalent to the proportional coefficient between the deflection angle and the input control voltage gradually becoming smaller, resulting in the actual deflection angle of the deflecting mirror being smaller than expected. In actual motion, when a reciprocating motion waveform is applied to the deflecting mirror, when the motion approaches the edge, the speed will drop sharply and change the direction of motion, so there is a large acceleration. Therefore, in the actual acquisition process, the scanning beam in the edge field of view does not move at a constant speed, that is, there are non-ideal distortion regions in the front and back parts of the entire scanning field of view, and the middle can be regarded as a target uniform field of view with linear motion.

[0041] More specifically, in mathematical terms, the linear segment in the scanning stroke defined by the present invention refers to the scanning range where the curve slope of the stroke has an error within δ from the maximum slope of the target, that is, the magnitude of the first derivative is above (1 - δ) of the maximum derivative value, expressed as: P'(t) ≥ (1 - δ)max{P'(t)}, where P(t) is the position feedback information changing with time, P'(t) is the first derivative of the position feedback information, which can be understood as the speed feedback information, and δ is the set adjustable error. The smaller the value, the stricter the definition of the linear ratio, and the larger the value, the looser the requirement for the linear ratio. For example, in the specific implementation process of the present invention, it is set to 25%, that is, the regions where the speed fluctuates around 25% above and below the maximum speed required by the target scanning field of view during the actual scanning process are regarded as the acceptable linear segments. The ratio between the size of the linear segment and the field of view corresponding to the maximum amplitude of the input voltage is defined as the linear segment ratio.

[0042] The derivation of the linear segment ratio needs to consider the amplitude-frequency response characteristics of the scanning module. First, the transfer function of the second-order oscillation link mentioned above is sorted out to obtain its amplitude-frequency characteristics:

[0043]

[0044] where A(f) is the amplitude-frequency response relationship, f is the frequency of the input voltage waveform applied to the deflecting mirror, a is the natural frequency, b is the damping ratio, and the curve is asFigure 1 As shown in the amplitude-frequency response. The actual scanning stroke is the product of the input voltage waveform and this amplitude-frequency response. In the present invention, a sine waveform commonly used in the scanning module is selected as the input. The sine waveform is commonly used as the input for the scanning waveform due to its advantages of smoothness, stability, and relatively small impact force at the edges, which can protect the scanning system. Let f(t) and g(t) represent the input voltage waveform and the actual scanning stroke respectively, then there is

[0045] f(t) = A0sin(2πft) (3)

[0046] g(t) = A(f)·A0sin(2πft) (4)

[0047] where A0 is the maximum amplitude of the input voltage waveform.

[0048] According to the above definition of the linear segment, there is

[0049]

[0050] q = (1 - δ) 2 (7)

[0051] where q represents a constant related to the set adjustable error δ. Equation (6) shows that the proportion Ratio of the linear segment of the scanning module is related to the amplitude-frequency response of the module itself. The overall trend is similar to the amplitude-frequency response curve. Ratio is related to frequency. Different waveform frequencies will result in different Ratios. When a certain frequency is reached and its corresponding amplitude-frequency response decays to (1 - δ), there is no linear segment in the scanning field of view. It should be noted that the above derivation is based on the scanning module being a linear time-invariant system, which means that its amplitude-frequency response curve is determined, that is, its natural frequency and damping ratio are both constants. However, when actually collecting and analyzing the position feedback information of the scanning module as Figure 2 shown, it can be found that there are differences in the amplitude-frequency responses of different input amplitudes. Specifically, the amplitude-frequency response curves of different amplitudes are each similar to the second-order oscillation response curve as Figure 1 shown, all showing a trend of changing with frequency. However, this trend is not the same among different amplitudes. Therefore, the present invention further derives the formula for the proportion of the linear segment of the scanning module.

[0052] The present invention takes into account that the amplitude-frequency response curve of each amplitude conforms to the amplitude-frequency response characteristics of the above second-order oscillation link. Combining with the actual physical meaning, it is analyzed that during actual operation, there are multiple factors in the scanning module such as materials, structures, electricity, and thermal effects, which lead to changes in the natural frequency or damping factor. The amplitude-frequency responses of the data for each amplitude are fitted to obtain the natural frequency and damping factor data for each amplitude, as Figure 3As shown in (a) and (b) therein. It can be seen that the natural frequency fluctuates around 200 Hz (factory value) (200 ± 8 Hz), which can be regarded as not changing with the amplitude; the damping factor is negatively correlated with the amplitude and has an important influence on the amplitude-frequency response. Considering that the damping coefficient in the nonlinear second-order system is related to the above-mentioned many factors and will show characteristics related to the amplitude, an empirical model is set to fit the damping factor,

[0053] b(A) = b0 + αA n (8)

[0054] where b(A) refers to the damping factor that changes with the amplitude A of the input waveform, b0 refers to the initial value of the damping constant, and α and n are both constants.

[0055] The relationship between the damping factor and the amplitude change is obtained. Combining with Equation (2), the actual and accurate amplitude-frequency response relationship A(f) is obtained, and then the accurate ratio Ratio of the linear segment is obtained, laying a foundation for the follow-up.

[0056]

[0057] When the user actually operates the super-high-resolution microscopic device, the parameters that the user pays attention to are the scanning range X Range, Y Range, the set pixel size Pixel Size, and the dwell time Dwell Time of the pixel points. These parameters construct three important parameters of the line frequency f, voltage amplitude A, and voltage offset offset that the system needs to output to the scanning module. The specific relationship is as follows:

[0058]

[0059] where k is the mapping relationship ratio between the actual field of view and the input voltage, and XR1 and XR2 are the starting and ending positions of the fast-axis scanning range XRange.

[0060] The above three important parameters together form the fast-axis input voltage waveform of the scanning module,

[0061] f(t) = A * sin(2πft) + offset (13)

[0062] According to the more accurate and practical Ratio expression (9) above, it can be found that the value of Ratio is related to the amplitude A. Therefore, after determining f, Ratio cannot be directly determined, and further the amplitude parameter A of the input waveform cannot be determined. Other methods need to be adopted to solve it.

[0063] The present invention proposes a method for obtaining the input voltage amplitude based on the iterative trial number method. The specific process is as Figure 4As shown, first determine the value of the line frequency f and the target field of view size (Wanted Field of View, WFOV) according to the user input parameters. Set an initial amplitude for the overall process. This value is often the maximum loadable amplitude A0 of the scanning module. Perform preprocessing and calculate according to equations (2) and (9) to obtain the Ratio value and the actual field of view size (Real Field of View, RFOV) at this time.

[0064] RFOV = A * Ratio (14)

[0065] Calculate and compare whether the difference between the target field of view size and the actual field of view size is within the adjustable threshold m. If it is satisfied, consider the current amplitude A and the Ratio at this time to meet the requirements and output them as the result. Otherwise, subtract the adjustable parameter n from the current amplitude, substitute the new amplitude into the above process, and compare again whether it is close to the target field of view size. Note that when the amplitude is reduced to 0, this process should be exited, indicating that the current system cannot meet the target field of view size. The above-mentioned parameters m and n can be adjusted according to the requirements of the device system to achieve a controllable scanning waveform acquisition speed and accuracy.

[0066] As Figure 5 shown, the system of the present invention operates as follows: After preparing the above devices, first connect the acquisition card to each device and the computer to ensure that the position feedback signal output port of the scanning module can obtain the position feedback signal from the acquisition card and the communication of other devices; then load the waveform amplitude by amplitude and frequency according to the maximum loadable amplitude corresponding to the maximum field of view of the scanning module used, record and save each input and the actual feedback signal; then according to the above linear segment extraction method, first fit the amplitude-frequency response of the actual feedback signal of each waveform, obtain the damping factor at each amplitude and fit to obtain the expression of the damping factor b(A) under non-linearity, so as to obtain the accurate linear segment ratio relationship of the scanning module; then establish the relationship between the scanning parameters and the linear segment ratio, and verify whether this relationship can obtain the accurate target field of view; next, run the overall system, read the position feedback signal of the scanning module in real time, complete the judgment of whether the scanning position enters the target scanning field of view position in the computer, and obtain the switching optical timing signal of the EOM and output it to the EOM.

[0067] Figure 6This is an optical path diagram realized by the device of the present invention, which is an improvement based on the traditional confocal microscopy imaging device. The method proposed by the present invention is applicable to replacing the scanning module with a scanning system having a position feedback function, and adding a device capable of realizing the switching light function by high pulse width modulation at the light source. In this embodiment, the traditional galvanometer scanning system on the scanning module is replaced by a Tip-Tilt piezoelectric deflection mirror system, and an electro-optic modulation device EOM capable of high pulse width adjustment is added at the light source. The EOM can change the polarization direction of the incident light by loading different voltages, and cooperate with the polarizer and analyzer placed vertically before and after the EOM. When the loaded voltage is at the half-wave voltage V π , the switching of light can be completely realized. After the overall device is ready, connect the device to the acquisition card and the computer. In addition to the necessary connections of the point-scanning super-high-resolution microscopy imaging system, the present invention additionally needs to connect the position feedback information port of the Tip-Tilt device and the input port of the EOM to the acquisition card to obtain the position feedback information and control the switching light of the EOM.

[0068] In this embodiment, then obtain the fast-axis scanning position feedback information. By loading the amplitudes and different-frequency sine scanning waveforms that decrease successively from the maximum loadable voltage amplitude of the scanning system, collect and save the input waveforms and position feedback information. The position feedback curve is as Figure 2 shown.

[0069] After obtaining the input and actual position feedback information, perform the processing to obtain the proportion of the linear segment. According to the definition of the linear segment, it is necessary to first determine the value of the adjustable error δ, which is set to 25% in this embodiment. Since the input waveform has periodicity and the same nature of going back and forth, only extract the data within the range of 1 / 4 cycle, take the first derivative of the input and actual waveforms within this range, calculate the threshold that can be considered as the linear segment, and the range of the actual position waveform above this threshold is the linear segment, and then obtain the proportion of the linear segment. Repeat the above operations to obtain the proportion of the linear segment at different amplitudes and different frequencies. The specific operation process of obtaining the proportion of the linear segment is as Figure 7 shown, Figure 7 in (a) is the input waveform and position feedback information, Figure 7 in (b) and (c) are the data of a certain 1 / 4 cycle and the corresponding first derivative image respectively, and the calculated linear segment threshold is displayed in the first derivative image. Extract the data within the threshold and display it in Figure 7 in (d), which can intuitively display the linear segment travel in the actual position, Figure 7 in (e) is the overall signal relationship diagram, which includes the processing schematic diagram of the signal output to the EOM. When the scanning position enters the linear segment, that is, the target field of view, turn on the light, and turn off the light at other times.

[0070] Next, perform fitting on the linear segment ratio data. First, perform amplitude-frequency response fitting on the position feedback signal for a second-order oscillation link to obtain curves of the natural frequency and damping factor varying with different amplitudes as shown in Figure 3 . The natural frequency fluctuates around 200 Hz (factory value) (200 ± 8 Hz) and can be regarded as not changing with the amplitude; the damping factor is negatively correlated with the amplitude and has an important influence on the amplitude-frequency response. Therefore, perform non-linear fitting on the damping factor to obtain the relationship between the damping factor and the amplitude, obtain the actual and accurate amplitude-frequency response relationship, and then obtain the accurate linear segment ratio.

[0071] After that, after the user inputs common parameters, the iterative trial method proposed in the present invention can be used to obtain an accurate scanning waveform. As shown in Figure 4 , first determine the value of the line frequency and the target field of view size WFOV according to the user input parameters. Set an initial amplitude in the overall process, which is often the maximum loadable amplitude of the scanning module. After preprocessing, obtain the Ratio value and the actual field of view size RFOV at this time. Calculate and compare whether the difference between the target field of view size and the actual field of view size is within a settable adjustment threshold m. If it is satisfied, it is considered that the current amplitude A and the Ratio at this time meet the requirements and are output as the result. Otherwise, subtract a settable adjustment parameter n from the current amplitude, substitute the new amplitude into the above process, and compare again whether it is close to the target field of view. Note that when the amplitude is reduced to 0, this process should be exited, indicating that the current system cannot meet the target field of view size. The above-mentioned m and n parameters can be adjusted according to the requirements of the device system to achieve a controllable scanning waveform acquisition speed and accuracy. In this embodiment, m and n set in the iterative trial method are 0.001 and 0.0001 respectively, which can be adjusted according to needs. By comparing the linear segment ratios obtained by actual extraction and the algorithm, it can be found that the algorithm has a high acquisition effect. Then, combine the calculated offset to obtain the scanning waveform.

[0072] Finally, when the system is running, collect the position feedback signal in real time, determine whether the scanning position is within the target field of view, and then obtain the switching optical signal of the EOM to achieve accurate field of view imaging.

[0073] The above is only the preferred embodiment of the present invention. Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible changes and modifications to the technical solution of the present invention, or modify it into an equivalent embodiment with equivalent changes, without departing from the scope of the technical solution of the present invention. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention all fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for high-resolution microscopic imaging with accurate field of view and distortion-free scanning based on position feedback, characterized in that: The method comprises: In the confocal microscopy imaging optical path, a piezoelectric deflection mirror system with position feedback function is used as a scanning module, and an electro-optical modulation device that realizes the switching light function through pulse width modulation is added to the light source; The fast-axis scanning position feedback information is obtained, and the voltage waveform is loaded by amplitude and frequency according to the maximum loadable amplitude corresponding to the maximum field of view of the scanning module. The input voltage waveform and the actual position feedback information are recorded each time, and the linear segment proportion under different voltage amplitudes and different scanning frequencies is obtained; Fit the linear segment proportion data, fit the amplitude-frequency response of the actual position feedback information of each input voltage waveform, obtain the damping factor under each amplitude, and fit the relationship between the damping factor and the amplitude under nonlinearity to obtain an accurate amplitude-frequency response relationship, and then obtain an accurate linear segment proportion relationship; The scanning parameters are associated with the linear segment ratio to verify whether the linear segment ratio relationship can obtain the accurate target field of view. After the verification, the accurate scanning waveform is obtained; The position feedback information of the scanning module is collected in real time to determine whether the scanning position is within the target field of view, and the switching light timing signal of the electro-optical modulation device is obtained and output to the electro-optical modulation device to achieve precise field of view imaging.

2. The method for accurate field of view distortion-free scanning high-resolution microscopic imaging based on position feedback according to claim 1, characterized in that: Imaging distortion correction is achieved based on the proportion of linear segments in the scanning stroke. The linear segment in the scanning stroke refers to a scanning range in which the error between the slope of the curve of the stroke and the target maximum slope is within a set adjustable error δ. The linear segment proportion is the ratio of the linear segment size to the field of view corresponding to the maximum amplitude of the input voltage. The calculation of the linear segment proportion is specifically as follows: determining the adjustable error δ, taking the first-order derivative of the input voltage waveform and the actual position waveform, and calculating the linear segment threshold. The range of the actual position waveform above the threshold is the linear segment stroke, and then obtaining the linear segment proportion.

3. The method for accurate field of view distortion-free scanning high-resolution microscopic imaging based on position feedback according to claim 1, characterized in that: The adjustable error δ is set to 25%, and the input voltage waveform adopts a sinusoidal waveform. Since the input waveform has periodicity and the same properties of round trip, only the data within the 1 / 4 cycle range needs to be extracted to calculate the linear segment ratio.

4. The method for accurate field of view distortion-free scanning high-resolution microscopic imaging based on position feedback according to claim 1, characterized in that: The fitting of the linear segment proportion data is specifically as follows: fitting the amplitude-frequency response of the second-order oscillation link of the position feedback signal to obtain the curve of the natural frequency and the damping factor changing with different amplitudes; the damping factor is negatively correlated with the amplitude, and the damping factor is nonlinearly fitted to obtain a relationship between the damping factor and the amplitude, and the relationship is used to replace the damping ratio in the original amplitude-frequency response relationship to obtain an accurate amplitude-frequency response relationship, and then obtain an accurate linear segment proportion.

5. The method for accurate field of view distortion-free scanning high-resolution microscopic imaging based on position feedback according to claim 1, characterized in that: An iterative trial method is used to obtain an accurate scanning waveform based on the linear segment ratio. Specifically, the line frequency and target field of view size are determined according to the user input parameters, the initial amplitude is set to the maximum loadable amplitude of the scanning module, and preprocessing is performed to calculate the linear segment ratio and the actual field of view size. The difference between the target field of view size and the actual field of view size is compared to see if it is within the settable adjustable threshold m. If so, the current amplitude and linear segment ratio are considered to meet the requirements and are output as the result. Otherwise, the current amplitude is subtracted from the settable adjustable parameter n, and the new amplitude is substituted into the above process to compare again whether it is close to the target field of view size. When the amplitude is reduced to 0, it means that the current system cannot meet the target field of view size.

6. The method for accurate field of view distortion-free scanning high-resolution microscopic imaging based on position feedback according to claim 1, characterized in that: By applying the correct voltage amplitude to the deflection mirror so that the target field of view is in the linear segment of the scanning stroke, distortion-free scanning of the target field of view is achieved.

7. The method for accurate field of view distortion-free scanning high-resolution microscopic imaging based on position feedback according to claim 1, characterized in that: When the scanning position is judged to enter the target field of view through the position feedback information collected in real time, the electro-optical modulation device that controls the light source switch is turned on. When it is judged that the scanning position leaves the target field of view, the electro-optical modulation device is turned off to achieve no redundant exposure in the non-field of view range of the sample and improve the sample utilization rate.

8. A high-resolution microscopic imaging device with accurate field of view and distortion-free scanning realized by the method according to any one of claims 1 to 7, characterized in that: The device includes a light source, an electro-optical modulation device, a scanning module, an acquisition card and a computer; the electro-optical modulation device realizes the switching light function through pulse width modulation; the scanning module adopts a piezoelectric deflection mirror system with a position feedback function; the position feedback signal output port of the scanning module is connected to the acquisition card and the input port of the electro-optical modulation device to realize the acquisition of position feedback information and the switching light control of the electro-optical modulation device; the acquisition card is connected to the computer to realize the acquisition of fast-axis scanning position feedback information, the processing of linear segment proportions under different voltage amplitudes and different scanning frequencies, linear segment proportion data fitting, accurate scanning waveform acquisition, and when collecting position feedback information in real time, control the electro-optical modulation device to achieve precise field of view imaging.

9. The high-resolution microscopic imaging device with accurate field of view and distortion-free scanning based on position feedback according to claim 8, characterized in that: The piezoelectric deflection mirror system adopts a Tip-Tilt piezoelectric deflection mirror system, which can operate quickly and stably at a high resonant frequency within the 1KHz range, and has a position sensor that can feedback scanning position information.

10. The precise field of view distortion-free scanning high-resolution microscopic imaging device based on position feedback according to claim 8, characterized in that: The electro-optical modulation device achieves the change of the polarization direction of the incident light by applying different voltages, and cooperates with the vertically oriented polarizers and analyzers placed before and after the electro-optical modulation device to fully realize the switching of light when the applied voltage is at a half-wave voltage.

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