CN109917538B

CN109917538B - Light source modulation method of confocal scanning microscope

Info

Publication number: CN109917538B
Application number: CN201811525282.1A
Authority: CN
Inventors: 张金莲; 张�杰
Original assignee: Nanjing Zhibo Medical Devices Co ltd
Current assignee: Nanjing Boshi Medical Technology Co ltd
Priority date: 2018-11-30
Filing date: 2018-12-13
Publication date: 2020-04-03
Anticipated expiration: 2038-12-13
Also published as: CN109917538A

Abstract

The invention discloses a light source modulation method of a confocal scanning microscope, which comprises the following steps: modulating a light source by adopting a light source power modulator, and carrying out nonlinear modulation on the power of a transmitting end of the light source by the light source power modulator; simultaneously modulating the power of the output end of the light source in a forward scanning sampling window and a reverse scanning sampling window of the fast resonance mirror; or the power of the output end of the light source is modulated in the forward scanning sampling window; or the power of the output end of the light source is modulated in the reverse scanning sampling window. By adopting the invention, the purpose of minimizing the light radiation quantity on the surface of the sample can be realized.

Description

Light source modulation method of confocal scanning microscope

Technical Field

The invention relates to a confocal scanning optical imaging instrument technology, in particular to a confocal scanning microscope light source modulation method which can be used for carrying out two-dimensional scanning on biological samples (such as skin, eye ground and other biological tissues, which can be called as samples for short) to obtain high-definition two-dimensional images of the biological samples.

Background

Confocal scanning microscopy imaging optical systems typically use a fast resonant scanning mirror and a slow linear scanning mirror in orthogonal directions to perform a two-dimensional scan of a sample to acquire a two-dimensional image of the sample.

FIG. 1A shows a typical scanning mirror operation. As shown in fig. 1A, the fast resonant mirror scanning window 11 continuously scans the sample in the first direction in a sinusoidal manner, wherein any one sinusoidal scanning period can be decomposed into a forward scanning segment 111 and a reverse scanning segment 112. The amplitude of the sinusoid of the fast resonant mirror scanning window 11 directly determines the optical field of view size in the fast scan direction.

For each scanning cycle of the fast resonant mirror scanning window 11, the slow linear scanning mirror scanning window 12 is displaced by one step 121 in a direction orthogonal (90 °) to the fast resonant mirror in the second direction. The number of steps and the step size of the forward scan 121 of the slow linear scan mirror directly determine the size of the optical field of view in the slow scan direction.

After the slow linear scan mirror completes the forward scan 121, according to the system settings, then the reverse scan 122 is completed. The steps of reverse scan 122 are often larger than the steps of forward scan 121 so that the slow linear scan mirror can quickly return to the original position to start a new frame of image. In this case, the data acquisition module of the imaging system often only samples data from the forward scan 121.

The steps and number of steps of the forward scan 121 and the reverse scan 122 of the slow linear scan mirror may be equally large, in which case the data acquisition module of the imaging system samples all of the data from the forward slow scan 121 and the reverse scan 122.

FIG. 1B shows another exemplary scanning mirror mode of operation. As shown in fig. 1B, similar to the image interleaving mode of operation in a conventional television system. The fast resonant mirror scan window 11 continuously scans the sample in the first direction in a sinusoidal manner, wherein any one sinusoidal scan cycle can be decomposed into a forward scan 111 and a reverse scan 112. The odd lines of the image are from the forward scan 111 of the fast resonant mirror and the even lines of the image are from the reverse scan 112 of the fast resonant mirror. The amplitude of the fast resonant mirror scanning window 11 sinusoid directly determines the optical field of view size in the fast scan direction.

For each half-cycle of the fast resonant mirror scan window 11, the slow linear scan mirror scan window 12 is displaced by one step 121 in a direction orthogonal (90 °) to the fast resonant mirror in the second direction. The number of steps and the step size of the forward scan 121 of the slow linear scan mirror directly determine the size of the optical field of view in the slow scan direction.

The steps and number of steps of the slow linear mirror forward scan 121 and the reverse scan 122 can also be as large, and the data acquisition module of the imaging system samples all the data from the slow mirror forward scan 121 and the reverse scan 122.

The fast resonant mirror scan window 11 of fig. 1A and 1B outputs a line-synchronous clock signal (H-Sync) one at a time per complete cycle of scanning by the corresponding electronic component. The slow linear scan mirror scan window 12 of fig. 1A and 1B outputs a field synchronous clock signal (V-Sync) in real time for each full cycle of scanning by the corresponding electronic component.

The horizontal synchronization clock signal (H-Sync) and the field synchronization clock signal (V-Sync) are commonly used as synchronization input signals for an imaging system data acquisition module, digitizing an image of an analog signal.

The motion trajectory of a fast resonant mirror can be generally expressed in terms of the spatio-temporal relationship of equation (1).

x(t)＝A－A·cos(ωt) (1)

Where A is the amplitude of the fast resonant mirror, ω is the angular velocity of the fast resonant mirror, t is a time variable, and x (t) is the scan spatial position of the fast resonant mirror.

The spatio-temporal relationship of equation (1) may be further expressed by the cosine curve 10 of FIG. 2A. The cosine curve 10 shows the complete period of a fast resonant mirror, including the forward scanning window 11 and the reverse scanning window 12.

At both sides of the scanning window, the moving speed of the fast resonant mirror is reduced and a mathematical inflection point appears, and due to the excessive stretching and distortion of the image, the data acquisition module of the image system often cuts off the data at both sides or abandons the sampling of the data at both sides, and only samples the part of the cosine curve in the middle which is relatively linear, as shown in the

regions

111 and 121 in fig. 2A. The 111 and 121 regions are often defined as image windows or sampling windows.

Mathematically differentiating equation (1) yields:

Δx(t)＝Aω·sin(ωt)Δt (2)

the inverse relationship between Δ x (t) and Δ t can be obtained from equation (2),

Δt(x)＝Δx/(Aω·sin(ωt)) (3)

assuming an imaging light source for illuminating the sampleOutput constant power P₀(in practice, the output power of the imaging light source is constant. the stability of the output power of the light source is an important index for measuring whether the performance of the light source is excellent), then the radiation received by the unit space size of the scanned sample is as follows:

ΔI(x)＝P₀Δt(x)＝P₀Δx/(Aω·sin(ωt)) (4)

rewriting equation (4) into differential form yields:

fig. 2B shows the normalized equation (5). The leftmost and rightmost 1% spatial positions are omitted from the figure and these two parts can be considered as sample-receiving pulsed light sources-receiving extremely high amounts of radiation in an extremely short time. However, even if only the scan space of 99% of the graph is considered, the amount of radiation received by both

sides

11, 12 of the sample is 7 times or more the middle 10 as shown in fig. 2B.

In the imaging of a living sample, such as a retina of a living animal, it is necessary to strictly control the dose of the imaging light source irradiating the sample. The imaging light source, especially visible light laser and ultraviolet band, is more toxic than infrared light, and strict control of laser dose can ensure that living body samples are always in a safe radiation range. Confocal scanning optical imaging instruments need to calculate the radiation load that a living sample can receive before application to ensure the safety of the living sample.

As described above, the light dose received in the two end regions of the sample shown by the forward scanning window 11 and the reverse scanning window 12 in one scanning cycle in fig. 2B is more than 7 times that received in the middle region 10 of the scanning cycle. The calculation of the phototoxicity requires consideration of the area of illumination that is least safe, i.e. the two end regions of 7 times the dose. The result of this problem is that, to obtain a sufficiently good image signal of the sample, more than 7 times the light dose must be received on both sides of the sample than in the middle of the sample. If a sufficiently good signal-to-noise ratio is ensured for the images on both sides of the sample with only 1/7 dose, then the necessary underexposure in the middle of the sample results in an image quality that does not meet the specified requirements. The non-linear scanning mechanism of the fast resonant mirror results in a non-linear result as shown in figure 2B.

Disclosure of Invention

In view of the above, the main objective of the present invention is to provide a method for modulating a light source of a confocal scanning microscope, which achieves the requirement that the light radiation amount reaches linear distribution on the surface of a sample by modulating the power of the light source in the fast scanning direction, and simultaneously turns off the light source in the scanning area where data is not sampled, so as to minimize the light radiation amount reaching the surface of the sample.

In order to achieve the purpose, the technical scheme of the invention is realized as follows:

a light source modulation method of a confocal scanning microscope comprises the following steps:

a light source power modulator is adopted to modulate a light source, and the power of the transmitting end of the light source is modulated in a nonlinear way according to a sine curve or a cosine curve through the light source power modulator;

simultaneously modulating the power of the output end of the light source in a forward scanning sampling window and a reverse scanning sampling window of the fast resonance mirror; or the power of the output end of the light source is modulated in the forward scanning sampling window; or the power of the output end of the light source is modulated in the reverse scanning sampling window.

Wherein, the light source power modulator is an acousto-optic modulator AOM.

Carrying out nonlinear modulation on the transmitting end power of the light source through an acousto-optic modulator AOM to obtain the radiant quantity distribution of the light source in a sample space:

I′(x)＝ΔI(x)/Δx＝k·P₀/ω

wherein k is the attenuation coefficient of the AOM and is a constant; p₀Is the transmitting end power of the light source.

power modulation is carried out on the light source output end at a forward scanning image window and a reverse scanning image window of the fast resonance mirror at the same time; or the power of the light source output end is modulated in the forward direction scanning image window; or, the power of the output end of the light source is modulated in the reverse scanning image window.

Wherein, the light source power modulator is an acousto-optic modulator AOM.

I′(x)＝ΔI(x)/Δx＝k·P₀/ω

The light source modulation method of the confocal scanning microscope has the following beneficial effects:

the confocal scanning microscope light source modulation method is used for modulating the light source of a confocal scanning microscope imaging system which uses a fast resonance scanning reflector and a slow linear scanning reflector in the orthogonal direction, and two-dimensional scanning of a biological sample is carried out to obtain a two-dimensional image of the sample. The radiation quantity of a light source falling to a sample in the fast scanning direction is in nonlinear distribution due to the sine motion track of the fast resonance scanning reflector; the operation of the slow linear scan mirror results in the image acquisition module not acquiring all of the images from the slow scan. The modulation method realizes that the light source radiation quantity achieves linear distribution on the surface of the sample by modulating the power of the light source in the rapid scanning direction, and simultaneously reduces the light radiation quantity on the surface of the sample by closing the light source in a scanning area without sampling data, thereby achieving the purpose of minimizing the light radiation quantity of the sample.

Drawings

FIG. 1A is a schematic diagram of the operation of the SLO fast resonant scanning mirror and the slow linear scanning mirror in a non-interleaved mode (the slow mirror is linearly increased by one step per scanning cycle of the fast resonant mirror);

FIG. 1B is a schematic diagram of the operation of the SLO fast resonant scanning mirror and the slow linear scanning mirror in the interleaved mode (the slow mirror is linearly increased by one step for each half cycle of the fast resonant mirror scan);

FIG. 2A is a schematic diagram of the temporal-spatial relationship of the motion trajectory of the fast resonant scanning mirror (all shown in the diagram are normalized values);

FIG. 2B is a diagram showing a non-linear relationship between the amount of light radiation received per unit area of a sample and the position of a scanning space in the fast scanning direction;

FIG. 3A is a schematic diagram of the generation of a modulation signal on the electronic hardware to turn the light source on and off in real time based on the fast resonant scanning mirror window and the image sampling window;

FIG. 3B is a schematic diagram of the generation of a modulated signal on the electronic hardware to turn the light source on and off in real time (the light source is turned on only in the forward scanning position) based on the fast resonant scan mirror window and the image sampling window;

FIG. 3C is a schematic diagram of the generation of a modulation signal on the electronic hardware to turn the light source on and off in real time (the light source is turned on only in the reverse scan position) based on the fast resonant scan mirror window and the image sampling window;

FIG. 4A is a schematic diagram of the simultaneous modulation of the output power of the light source in the resonant scanning direction and the non-linear forward and backward scanning in the scanning window;

FIG. 4B is a schematic diagram of nonlinear forward scan modulation of the light source output power in the resonant scan direction at the scan window;

FIG. 4C is a schematic diagram of nonlinear inverse scan modulation of the light source output power in the resonant scan direction at the scan window;

FIG. 5A is a schematic diagram of the simultaneous modulation of the light source output power in the resonant scanning direction with non-linear forward and reverse scanning at the image window;

FIG. 5B is a schematic diagram of nonlinear forward scan modulation of the light source output power in the resonant scan direction at the image window;

FIG. 5C is a schematic diagram of the nonlinear inverse scan modulation of the light source output power in the resonant scan direction at the image window;

FIG. 6A is a schematic diagram of the simultaneous modulation of the output power of the light source in the resonant scanning direction and the non-linear forward scanning and reverse scanning in the image window (simultaneously performing the on-off adjustment of the light source);

FIG. 6B is a schematic diagram of nonlinear forward scanning modulation of the light source output power in the resonant scanning direction at the image window (while performing light source on-off debugging);

FIG. 6C is a schematic diagram of the nonlinear inverse scan modulation of the light source output power in the resonant scan direction at the image window (while performing the light source switch debugging);

FIG. 7 is a schematic diagram of light source modulation in the slow linear scan direction;

FIG. 8 is a schematic diagram of a signal control and processing device of a confocal scanning microscopy imaging system according to an embodiment of the invention;

FIG. 9 is a schematic diagram of the process of using the pixel clock signal p and the line synchronization signal h from the ADC or the dedicated PLL circuit to generate the light source modulation signal;

FIG. 10 is a schematic diagram of the generation process of the light source modulation signal in the slow linear scanning direction;

fig. 11 is a schematic diagram of an image obtained by integrating pixel values to obtain sinusoidal distortion removal.

Detailed Description

The following describes in detail the signal control and processing device of the confocal scanning microscopy imaging system and the method thereof with reference to the accompanying drawings and embodiments of the present invention.

[ example 1 ]

Referring to fig. 2A, a portion of the image window 111 and the image window 121, which is relatively linear between the fast resonant mirror scanning window 11 and the slow linear scanning mirror scanning window 12, is taken, and the image is not suitable for use because the sample image is severely stretched and distorted on both sides of the sample with a low fast resonant mirror speed.

In the above embodiment 1, in the case of using a Super Luminescent Diode (SLD) as an imaging light source, this is relatively easy to implement. Typical SLDs typically have a built-in opto-isolator with a bandwidth of 100kHz to 200kHz, while the resonant frequency of common fast resonant mirrors is typically between 4kHz and 16 kHz. The SLD thus has a bandwidth of 100kHz to 200kHz that is sufficiently fast to reflect the switching frequency from the fast resonant mirror, and it can be guaranteed that no sampling distortion occurs according to the Nyquist sampling theory.

Referring to fig. 3A, a modulation Signal 20 may be generated on electronic hardware, such as a Field Programmable Gate Array (FPGA), or a Digital Signal Processor (DSP), or other electronic components to turn the light source on or off in real time, based on the fast resonant scanning mirror window and the image sampling window.

The modulation signal 20 may be a transistor-transistor logic (TTL) signal, a Complementary Metal Oxide Semiconductor (CMOS) signal, or a Low Voltage Differential Signaling (LVDS) signal according to the light source input modulation parameters of the SLD. In this case, the data acquisition module obtains forward scan and reverse scan image data simultaneously.

Referring to FIG. 3B, there is shown a method for generating another modulation signal 20 to turn the light source on or off in real time, but only in the forward scanning position, based on the fast resonant scanning mirror window and the image sampling window, on electronic hardware such as a Field Programmable Gate Array (FPGA), Digital Signal Processor (DSP), or other electronic device. According to the light source input modulation parameter of the SLD, the modulation signal 20 may be a TTL signal, a CMOS signal, or an LVDS signal. In this case, the data acquisition module only obtains image data for the forward scan.

Referring to FIG. 3C, there is shown a method for generating another modulation signal 20 to turn the light source on or off in real time, but only in the reverse scan position, on electronic hardware, such as a Field Programmable Gate Array (FPGA), Digital Signal Processor (DSP), or other electronic device, based on the fast resonant scanning mirror window and the image sampling window. The modulation signal 20 may be a TTL signal, a CMOS signal, or an LVDS signal according to the input modulation parameter of the SLD. In this case, the data acquisition module only obtains image data for the inverse scan. The FPGA supports a user to randomly change the logic of an internal circuit according to the own needs, and customizes and generates digital signals required by the user.

As shown in fig. 3A, 3B, and 3C, the modulation signal 20 indicates that the high level light source is on and the low level light source is off. According to the parameters of the modulation signal input by the light source, the modulation signal 20 can be switched between a high level and a low level, so that the light source is turned off at the high level and turned on at the low level.

In this embodiment 1, the effect of reducing the amount of light radiation from the sample surface can be achieved by turning off the light source outside the image window.

[ example 2 ]

The method adopted in the embodiment is to use a power Modulator of the light source, and one typical application is to use an Acousto-optic Modulator (AOM) to modulate the light source. The modulation method is from equation (5) as the entry point. In equation (5), if the power P is applied to the emitting end of the light source₀After AOM, carrying out nonlinear modulation on the AOM to obtain:

where k is the attenuation coefficient of the AOM and is a constant. Substituting the formula (6) into the formula (5) to obtain the radiant quantity distribution of the light source in the sample space:

I′(x)＝ΔI(x)/Δx＝k·P₀/ω (7)

wherein: Δ i (x) is the amount of radiation received per unit spatial dimension of the scan sample, x is the scan spatial location; Δ x is the amount of position change; ω is the angular velocity of the fast resonant mirror.

That is, the distribution of the amount of radiation of the light source in the sample space becomes a constant through the power modulation of equation (6).

The formula (6) is further simplified to obtain,

referring to fig. 4A, there is shown

simultaneous power modulation

21, 22 of the light source output at the forward scanning (sampling) window 11 and the reverse scanning (sampling) window 12 of the fast resonant mirror.

Referring to fig. 4B, power modulation 21 of the light source output in the forward scanning (sampling) window 11 is shown.

Referring to fig. 4C, the power modulation 22 of the light source output at the inverse scanning (sampling) window 12 is shown.

Another modulation is to take into account the severe stretching distortion of the sample image outside the image window. As shown in fig. 5A,

simultaneous power modulation

211, 221 is performed on the light source output at the forward-scan image window 111 and the reverse-scan image window 121 of the fast resonant mirror.

As shown in fig. 5B, the light source output is power modulated 211 in the forward direction to scan the image window 111.

As shown in fig. 5C, the light source output is power modulated 221 in the reverse scan image window 121.

The embodiment adopts the modulation mode to modulate the light source, and can also achieve the effect of reducing the light radiation quantity on the surface of the biological sample.

[ example 3 ]

The method adopted in this embodiment is to combine the methods of

embodiments

1 and 2, and to turn off the SLD light source outside the image window, and at the same time, superimpose a power modulator, such as an AOM, to perform power modulation on the output end of the light source.

As shown in fig. 6A,

simultaneous power modulation

211, 221 and light source on-

off modulation

311, 321 are performed on the light source by AOM at the forward scan image window 111 and the reverse scan image window 121 of the fast resonant mirror.

As shown in fig. 6B, simultaneous power modulation 211 and light source on-off modulation 311 is performed on the light source by AOM in the forward scanning image window 111 of the fast resonant mirror.

As shown in fig. 6C, the light source is simultaneously power modulated 221 and light source on-off modulated 321 by AOM at the reverse scan image window 121 of the fast resonant mirror.

The three embodiments described above can be implemented separately.

[ example 4 ]

The method employed in this embodiment is to implement light source modulation in the slow scan direction.

In the slow scan mechanism shown in fig. 1A and 1B, the image acquisition module is used to acquire the most linear portion of the slow mirror forward scan 121, and the most linear portion is retained as the final image data.

Referring to fig. 7, a data truncated portion 13 is scanned in the forward direction and a data truncated portion 14 is scanned in the reverse direction using a slow scan. Since the forward scan data is intercepted 13 and the entire reverse scan data intercepted 14 is not captured, this part of the light source is turned off. The modulation signal in the slow scanning direction is in the interval 15 of fig. 7, and the modulation signal may directly control the light source (e.g., a built-in optoelectronic isolation device), may modulate the power of the output end of the light source by the AOM, or may be implemented in both modulation modes.

The modulation method of embodiment 4 described above may be used alone, or may be used in combination with any one of the modulation methods of

embodiments

1, 2, and 3 described above.

Furthermore, in order to accurately generate the modulation signal in the modulation method described in the above embodiment and finally convert the modulation signal into an image signal, the present invention further utilizes a synchronization signal of the data acquisition module. Fig. 8 shows typical input and output signals of the data acquisition module.

Fig. 8 is a schematic diagram of a signal control and processing device of the confocal scanning microscopy imaging system according to the embodiment of the invention.

As shown in fig. 8, the signal input and output process of the components such as the data acquisition module in the signal control and processing device of the confocal scanning microscopy imaging system is shown. The signal control and processing device of the confocal scanning microscopic imaging system mainly comprises a fast resonant mirror module 80, a field synchronizing signal and slow mirror scanning signal generator 81, a slow scanning mirror module 86 and a data acquisition and digital-to-analog conversion module 87.

Wherein the fast resonant mirror module 80 is independently operated by internal oscillator excitation. In operation, the fast resonant mirror module 80, on the one hand, excites the fast resonant mirror into sinusoidal motion and, on the other hand, generates a row-sync pulse signal (H-sync) 82. The line synchronization pulse signal (H-sync)82 is input to the field synchronization signal and slow mirror scanning signal generator 81, and generates a field synchronization signal (V-sync)84 and a slow mirror scanning signal 85. The field synchronization pulse signal 84 is input to the data acquisition and digital-to-analog conversion module 87, and the slow mirror scanning signal 85 is input to the slow scanning mirror module 86 to drive the slow scanning mirror to make linear motion in a direction orthogonal to the fast scanning mirror. The horizontal synchronization pulse signal (H-sync)82 is also input to the data acquisition and digital-to-analog conversion module 87 to generate a digitized horizontal synchronization pulse signal H.

The field synchronizing signal and slow mirror scanning signal generator 81 is in the form of a digital circuit, and may be formed by a circuit including an FPGA, a DSP, or other electronic components. The field sync signal (V-sync)84 is a digital signal and the slow mirror scan signal 85 is an analog signal that needs to be converted to a digital signal by an analog-to-digital converter DAC. In this embodiment, the analog-to-digital converter may be implemented by a DAC5672 analog-to-digital converter of Texas Instrument. The phases of the field sync pulse signal (V-sync)84 and the slow mirror scan signal 85 are phase-locked by the line sync pulse signal (H-sync) 82.

The slow speed mirror scans an analog signal 85, which is used to directly drive the slow speed mirror, or drive the slow speed mirror to work after the signal is amplified by the power amplifier.

As shown in fig. 8, after the multi-channel SLO (multi-channel image Data) video signal 83, the line synchronization pulse signal 82 and the field synchronization pulse signal 84 are input to the Data acquisition and digital-to-analog conversion module 87, the three signals are digitized through digital-to-analog conversion, and a set of digitized signals 88 is output, including a digitized image signal (SLO-Data), a line synchronization pulse signal h, a digitized field synchronization pulse signal v and a pixel clock signal p.

The pixel clock signal p may be generated by programming a Phase-locked Loop (PLL) built in a digital-to-analog converter ADC of the data acquisition and digital-to-analog conversion module 87. In this embodiment, the digital-to-Analog converter ADC employs an AD9984a chip of Analog Device Inc. The pixel clock signal p is derived from the row synchronization pulse signal (H-sync)82 and can be generally determined by the following mathematical relationship:

f_p＝N×f_H(9)

wherein f is_pIs the frequency of the pixel clock signal, f_HIs the frequency of the line sync pulse, and N is an integer used to determine how many pixel clocks need to be generated in a line sync pulse. The size of N is determined by the user through programming.

As shown in fig. 8, the pixel clock signal p may also be generated by programming a dedicated phase-locked loop circuit. In this embodiment, the pll circuit may employ a 501A pll chip of an IDT. The pixel clock signal p and the line synchronization pulse signal h also satisfy the condition of equation (9).

In this embodiment, the pixel clock signal p generated by the dedicated phase-locked loop circuit is used as an external pixel clock for an ADC (e.g., an ADS58C48 digital-to-analog conversion chip of texas instrument) for digitizing the analog image signal from the SLO.

The field sync signal (V-sync)14 and the pixel clock signal p as shown in fig. 8 are both obtained by phase-locking the row sync signal (H-sync) 82.

The pixel clock p and the digitized line synchronization signal h described above are used to phase-lock the light source modulation signals of the modulation methods described in embodiments 1 to 3 described above.

In addition, in the embodiment of the confocal imaging system of the present invention, the optical system is an 8kHz fast resonant scanning mirror model CRS8k from cambridge technology Inc.

The fast resonant mirror module 80 shown in FIG. 8 depicts the mechanical and electrical arrangement of the resonant scanning mirror, producing a line sync signal of 8kHz clock, i.e., the resonant clock described above, as well as the imaging system line sync signal (H-sync) 82. The clock signal is simultaneously transmitted to the field sync and slow mirror scan signal generators of the field sync and slow mirror scan signal generator 81 and the data acquisition and digital to analog conversion module 87.

In this embodiment, the field sync signal and slow mirror scan signal generator 81 may employ Xilinx FPGA chip, model ML507(Virtex-5) or SP605(Spartan-6), and generates the field sync signal (V-sync)84 (refer to FIGS. 8 and 10) and the slow mirror scan signal 85 (refer to FIG. 10) from the line sync signal (H-sync) 82. The field sync pulse signal 84 can be immediately sent to the data acquisition and digital-to-analog conversion module 87.

In this embodiment, the slow scan signal 85 generated by the FPGA chip via the custom circuit is also a digital signal that is converted to an analog signal via a digital-to-analog conversion chip DAC5672 of Texas Instruments for controlling the mechanical motion of the slow scan mirror. The analog signal obtained after digital-to-analog conversion, i.e., the slow mirror scanning signal 85, is directly or after being amplified by the AD8421 chip and transmitted to the slow mirror scanning module 86. One of the two axes of motion of a set of two-dimensional scan mirrors 6220H from cambridge technology Inc is applied to the slow scan mirror module 86.

In the present invention, the multichannel SLO video signal 83 comes from the SLO system. In the embodiment, a Hamamatsu Avalanche Photodiode (APD) with the model number of C10508-01 is adopted as a photodetector, and an optical signal returned by the biological sample is received. The multichannel SLO video signal 83 output by the APD is directly transmitted to the data acquisition and digital-to-analog conversion module 87. The SLO system may support receiving, by one or more APDs, one or more optical signals returned from a biological sample.

The data acquisition and digital-to-Analog conversion module 87 adopts an Analog Device Analog-to-digital conversion chip with the model number of AD9984 a; the multichannel analog signals transmitted by the APDs can be converted into corresponding digital signals in a one-to-one manner according to a line synchronization signal (H-sync)82 and a field synchronization signal (V-sync)84 provided by a system, and simultaneously, a pixel clock signal p, a digitized line synchronization signal H and a digitized field synchronization signal V of the system are output. Wherein, the horizontal synchronizing signal H and the horizontal synchronizing signal (H-sync)82 at the input end are synchronous phase-locked, and the field synchronizing signal V and the field synchronizing signal (V-sync)14 at the input end are phase-locked.

Wherein the FPGA chip is further programmed to generate the light source modulation signal of fig. 9 and 10, which is a digital signal. The FPGA chip may also be programmed to transmit digitized image signals of the set of digitized signals 88 from ML507 or SP605 to a Host (Host PC) via PCIe for image display, image processing, and data recording.

Signal 10 in fig. 9 shows a scatter plot of the complete sinusoidal motion trajectory of a fast resonant mirror, where any one point corresponds to the position of one pixel clock. Signal 11 of FIG. 9 shows the row sync signal (H-sync) output of the fast resonant mirror. The line synchronization signal (H-sync) and the sinusoidal motion trajectory tend to have a user-adjustable phase delay. Signal 12 of fig. 9 shows the result of the line sync signal (H-sync) after digitization, which corresponds to signal H of fig. 8. There is also a user settable phase delay between signal 11 and signal 12 of figure 9 equal to a positive integer multiple of the pixel clock signal p.

The trough or peak of the fast resonant mirror sinusoidal motion trajectory of signal 10 of fig. 9 and signal 12 have a user adjustable phase delay.

When the modulation methods described in

embodiments

1, 2 and 3 are generated by electronic hardware, such as FPGA, DSP or other electronic devices, the electronic hardware can directly detect the rising edge or the falling edge of the pulse of the signal 12 in fig. 9.

When the modulation method of embodiment 1 described above is generated by electronic hardware, such as FPGA, DSP or other electronic device, a digital counter is established in the electronic hardware. The value of each increment of the digital counter corresponds to one pixel clock p.

Modulation signal 13 in fig. 9 shows that the falling edge of the signal 12 pulse starts clearing and counting. Once the counter has accumulated 131 the preset value, the modulation signal starts to toggle for turning on the light source, see fig. 9, part 132. Portion 132 of fig. 9 corresponds to the image window of the resonant mirror forward scan. The size of the image window is set by the user. After the forward scanned image window ends, the counter flips the modulation signal to turn off the light source until the resonant mirror reverse scanned image window arrives, see section 133 of FIG. 9. The size of portion 133 of fig. 9 is set by the user. After 133 of fig. 9 is finished, the counter continues to flip the modulation signal to turn on the light source until the end of data sampling of the image window 134 of the reverse scan.

According to the requirement of the user, 132 and 134 of fig. 9 can be opened fully, only 132 can be opened, and only 134 can be opened. This situation corresponds to the situation of fig. 3A, 3B and 3C, respectively.

The method described above phase locks the light source modulation signal fig. 9 signal 13 to the pixel clock signal p and the line synchronization signal h of the ADC using electronic hardware counter technology to avoid image jitter.

Fig. 9 shows a hardware constant 131 for adjusting the forward scan image shift amount, which is set once. Fig. 9, 133, is also a hardware constant for adjusting the reverse scan image shift amount, which is set at once.

To generate the light source modulation signal of the second modulation method in embodiment 2, the generation of the modulation signal is similar to that of fig. 9. But here an analog-to-digital converter (DAC) with a sufficiently high resolution needs to be applied to generate the analog signal modulation AOM. The analog signal curves are consistent with those of fig. 4A, 4B, and 4C within the

sampling windows

132, 134. The 131 offset of fig. 9 needs to be readjusted according to the delay of the AOM.

Combining the implementation steps of the first modulation method in embodiment 1 and the second modulation method in embodiment 2, the third modulation method in embodiment 3, that is, the light source modulation signal shown in fig. 6C, can be generated.

The digitized line sync signal h of fig. 8 and the digitized field sync signal v of fig. 8 described above are used to phase lock the light source modulation signal of the inventive method four described above.

Fig. 10 shows the process of generating the light source modulation signal of the fourth modulation method in embodiment 4 described above.

As shown in fig. 10, the fast oscillating mirror scanning window 11 generates a line synchronizing pulse signal every scanning period (forward scanning section 111, reverse scanning section 112), and the drive signal for the slow scanning mirror is increased by one step by the electronic counter and the analog-to-digital converter DAC. The drive signal for the slow scan mirror is divided into a forward scan segment 121 and a reverse scan segment 122. Forward scan segment 121 is associated with data acquisition, and reverse scan segment 122 causes the slow scan mirror to quickly return to the initial position for forward scanning. The forward segment 121 and the reverse segment 122 may also be equally spaced so that the image acquisition module samples data from both the forward scan and the reverse scan of the slow scan mirror.

An electronic counter may also be used to generate a light source modulation signal in the slow scan direction, which is cleared at the moment the falling (or rising) edge of the field sync pulse (V-sync) arrives. The unit of this counter is the row sync pulse (H-sync). Counting the number of accumulated line syncs, i.e. the number of lines, starts immediately after the offset 141 set by the user, while the modulation signal is inverted. In the image capture area 142, i.e., the number of rows that each frame of image needs to be captured, the modulation signal turns on the light source. Once image capture area 142 is finished, the modulated signal is flipped to turn off the light source to area 143.

The slow scan direction light source modulation signal 14 generated in fig. 10 ensures that the light source is turned on only in the image acquisition region specified by the user and that the image acquisition module is turned off when no data is being sampled.

After applying the above-described light source modulation method two and method three, the integration algorithm of fig. 11 is implemented for the de-sinusoidal warping of the image. In fig. 11, in the slow operation stage of the resonant mirror, which is indicated by

time periods

11, 12, and 13, the light source is directly turned off, and the image acquisition module does not acquire an image. In the middle part of the sinusoid, the linear spatial domain Δ x_iBy a non-linear time domain at_iThe covered pixel values are accumulated. Also, the linear spatial domain Δ x_jBy a non-linear time domain at_jThe covered pixel values are accumulated. The linear and non-linear are described herein as being relative. In the sample space, each pixel falls in the sample space being scanned non-linearly if the pixel clock (time domain) is considered linear. In the scanned sample space, the image after sinusoidal distortion correction is linear, such as Δ x of FIG. 11_iAnd Δ x_jBut corresponding pixel interval Δ t of the sample (time) space_iAnd Δ t_jIs non-linear.

The integration process in fig. 11 is usually performed by a CPU of the host, an FPGA, or a Graphics Processing Unit (GPU). In this embodiment, the integration process is performed in a CPU of an Intel PC, for example.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention.

Claims

1. A light source modulation method of a confocal scanning microscope is characterized by comprising the following steps:

simultaneously carrying out power modulation on the forward scanning sampling window and the reverse scanning sampling window of the fast resonance mirror at the output end of the light source; or the power of the output end of the light source is modulated in the forward scanning sampling window; or the power of the output end of the light source is modulated in the reverse scanning sampling window.

2. The confocal scanning microscope light source modulation method according to claim 1, characterized in that the light source power modulator is an acousto-optic modulator AOM.

3. The method for modulating the light source of the confocal scanning microscope according to claim 2, wherein the power of the emitting end of the light source is nonlinearly modulated by an acousto-optic modulator (AOM) to obtain the radiant quantity distribution of the light source in the sample space:

I′(x)＝ΔI(x)/Δx＝k·P₀/ω

wherein: Δ I (x) is the amount of radiation received per unit spatial dimension of the scan sample; Δ x is position; k is the attenuation coefficient of the AOM and is a constant; p₀Is the transmitting end power of the light source; ω is the angular velocity of the fast resonant mirror.

4. A light source modulation method of a confocal scanning microscope is characterized by comprising the following steps:

5. The confocal scanning microscope light source modulation method according to claim 4, characterized in that the light source power modulator is an acousto-optic modulator AOM.

6. The method for modulating the light source of the confocal scanning microscope according to claim 5, wherein the power of the emitting end of the light source is nonlinearly modulated by the acousto-optic modulator AOM to obtain the radiation distribution of the light source in the sample space:

I′(x)＝ΔI(x)/Δx＝k·P₀/ω

wherein: Δ i (x) is the amount of radiation received per unit spatial dimension of the scan sample, x being the position; Δ x is a position offset; k is the attenuation coefficient of the AOM and is a constant; p₀Is the transmitting end power of the light source; ω is the angular velocity of the fast resonant mirror.