Laser scanning microscope comprising electric high-order modulation extraction module

Abstract

A non-linear laser scanning microscope comprising an electric high-order modulation extraction module (400, 500). The present invention relates to a method for enhancing image contrast / sign-to-noise ratio on the basis of a non-linear signal of a first-order modulation (1M), and in particular to after a photon-to-electron conversion process, using a photoelectric detector (200) to extract a frequency of the first-order modulation (1M) to obtain a better sign-to-noise ratio, thereby improving the image contrast / sign-to-noise ratio in imaging of the laser scanning microscope. When the image contrast / signal-to-noise ratio is improved to a certain extent, the image collection time can be shortened, and the imaging depth is further expanded, such that an image obtained by means of the first-order modulation (1M) has better quality than an image obtained without signal modulation.

Description

Laser scanning microscope with electrical high-order modulation extraction module Technical Field

[0001] The present invention relates to a modulation technology for a coherent / incoherent nonlinear optical microscope (or coherent / incoherent nonlinear laser scanning microscope), and more particularly to a laser scanning microscope with an electrical high-order modulation extraction module, and a technology and system for enhancing image contrast / signal-to-noise ratio by using the electrical high-order modulation extraction module. Background Art

[0002] Nonlinear optical imaging, a rapidly developing optical imaging technique in recent years, is a new tool for detecting biological structures and providing durable tracking and labeling. Based on various nonlinear optical principles, photons generate nonlinear signals at their focal point within biological samples. This method offers advantages such as high three-dimensional resolution and minimal damage to biological samples, avoiding many of the inherent drawbacks of microscopy. It is an ideal in vivo imaging method and holds broad application prospects in biomedical research.

[0003] With the advent of Colin Sheppard's use of scanning optical microscopy for nonlinear microscopy studies of crystals, coherent nonlinear laser scanning microscopy (C-NLSM), including second harmonic generation laser scanning microscopy (SHG-LSM), third harmonic generation laser scanning microscopy (THG-LSM), coherent anti-Stokes Raman scattering laser scanning microscopy (CARS-LSM), stimulated Raman scattering laser scanning microscopy (SRSLSM), and incoherent nonlinear laser scanning microscopy (IC-NLSM), including two-photon fluorescence laser scanning microscopy (2PF-LSM) and three-photon fluorescence laser scanning microscopy (3PF-LSM), have become mature commercial devices for materials characterization and a wide range of biomedical applications, thanks to the inherent optical sectioning capability without the need for a confocal aperture, deeper imaging depth than traditional optical microscopy techniques, and high structural specificity provided by the interaction of polarized laser light with tissue.

[0004] To improve image contrast and signal-to-noise ratio in C-NLSM and IC-NLSM, methods include increasing laser power, increasing image acquisition time, or using shorter laser pulses. However, increasing laser power carries the risk of sample damage. Increasing image acquisition time sacrifices image speed. Furthermore, the pulse width of short-pulse lasers has certain limitations.

[0005] Summary of the Invention

[0006] To address the aforementioned issues, the present invention proposes a novel method for enhancing the contrast and signal-to-noise ratio (SNR) of C-NLSM and IC-NLSM images by utilizing first-order modulation (1M) of a nonlinear signal in the frequency domain. This method employs an electrical high-order modulation extraction module (EHMO) on the microscope. Specifically, it employs a down-conversion circuit or a dual-phase demodulator to convert the 1M information components to a DC frequency via a down-conversion circuit. The primary purpose of this technology is to address the fact that conventional LSM uses DC imaging for DAQ. To maintain consistency with conventional LSM DAQ imaging formats, the present invention utilizes the 1M information components, processed by a down-conversion circuit or a bidirectional demodulator, to generate DC signals. Therefore, during DAQ imaging, the 1M information is used to achieve a higher contrast and signal-to-noise ratio, shortening image acquisition time and further extending imaging depth.

[0007] In accordance with the above objectives, the present invention first provides an electrical high-order modulation extraction module, comprising:

[0008] A DC blocking filter is used to filter out the DC signal on the RF sinusoidal signal output by the photodetector;

[0009] A function generator for providing a local oscillation frequency, wherein the local oscillation frequency is an integer multiple of the laser scanning frequency;

[0010] A mixer, one end of which receives the RF sinusoidal signal output by the DC blocking filter, and the other end of which receives the local oscillation frequency output by the function generator and outputs an intermediate frequency signal; wherein,

[0011] The intermediate frequency signal is a first-order or high-order modulated (1M or HOMs) signal down-modulated to a DC coordinate position, and the intermediate frequency signal is transmitted to a DAQ program for pixel reconstruction.

[0012] In accordance with the above objectives, the present invention further provides an electrical high-order modulation extraction module comprising:

[0013] A DC blocking filter is used to filter out the DC signal on the RF sinusoidal signal output by the photodetector;

[0014] A first splitter, for separating the radio frequency sinusoidal signal passing through the DC blocking filter into a first optical path and a second optical path;

[0015] A function generator for providing a local oscillation frequency, wherein the local oscillation frequency is an integer multiple of the laser scanning frequency;

[0016] A second splitter is used to separate the local oscillation frequency into an orthogonal optical path and an in-phase optical path;

[0017] A first mixer, one input end of which receives the sine wave signal of the first optical path and another input end of which receives the orthogonal optical path, and outputs a first intermediate frequency signal, wherein the first intermediate frequency signal is a first-order modulated (1M) signal down-modulated to a DC coordinate position;

[0018] The second mixer receives the sine wave signal of the first optical path at one input terminal and receives the in-phase optical path at the other input terminal, and outputs a second intermediate frequency signal; wherein the second intermediate frequency signal is a first-order modulated (1M) signal down-modulated to a DC coordinate position; wherein,

[0019] After the first intermediate frequency signal and the second intermediate frequency signal are transmitted to an operator for operation, the calculated root mean square signal is transmitted to a DAQ program for pixel reconstruction.

[0020] In accordance with the above objectives, the present invention provides a method for adjusting a laser scanning microscope imaging image using an electrical high-order modulation extraction module, comprising:

[0021] Provide a module manufactured by an electrical high-order modulation extraction module, and connect the module to a laser scanning microscope and a servo terminal;

[0022] Obtaining and judging whether the laser repetition frequency is the same as the LO signal frequency is performed by obtaining the laser repetition frequency of the laser scanning microscope and the LO signal frequency generated by the function generator through the information displayed on the servo terminal, and judging whether the LO signal frequency is a multiple of the laser repetition frequency;

[0023] Correcting the LO signal frequency is to adjust the LO signal frequency output by the function generator through the servo terminal so that the LO signal frequency and the laser repetition frequency are in a multiple relationship when the LO signal frequency and the laser repetition frequency are not in a multiple relationship;

[0024] The module calculates the high-order modulation signal to obtain a down-modulated high-order modulation signal, and transmits the down-modulated high-order modulation signal to the DAQ program configured in the servo terminal. The DAQ program then combines the image signal with the scanning signal into an image bit by bit.

[0025] The imaging quality is adjusted by the observer selecting a down-modulated signal (1M, 2M, 3M, ...) one by one and transmitting the down-modulated signal to the DAQ, which is then imaged by the DAQ program.

[0026] To achieve the aforementioned objectives, the present invention extracts the frequency of first-order modulation (1M) to achieve a better signal-to-noise ratio, significantly improving the image contrast and signal-to-noise ratio in laser scanning microscopy imaging. This improvement in image contrast and signal-to-noise ratio can shorten image acquisition time and further extend imaging depth, resulting in images obtained with first-order modulation (1M) having better quality than those obtained without signal modulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of a commercial nonlinear scanning microscope (NLSM)

[0028] FIG2 is a schematic diagram of the structure of the electrical high-order modulation extraction module of the present invention

[0029] FIG3a is a position signal of point A in FIG2 according to the present invention.

[0030] FIG3b is a position signal of point B in FIG2 according to the present invention.

[0031] FIG3c is a position signal of point C in FIG2 according to the present invention.

[0032] FIG3d is a position signal of point D in FIG2 according to the present invention.

[0033] FIG3e is a position signal of point E in FIG2 according to the present invention.

[0034] FIG4a is a schematic diagram of the photodetector of the present invention converting photons into electrical signals with frequency response.

[0035] FIG4b is a schematic diagram of the output electrical signal of the photodetector after multiplying the photoelectric conversion signal-to-noise ratio curve of FIG4a with incident light of different orders according to the present invention.

[0036] FIG4c is a schematic diagram of the product of frequency domain electrical signals and signal-to-noise ratios of different orders of the present invention.

[0037] FIG5 is a schematic diagram of contrast and imaging speed of the present invention

[0038] FIG6 is a schematic structural diagram of another embodiment of the electrical high-order modulation extraction module of the present invention

[0039] FIG. 7 is a schematic diagram of a 3D two-photon fluorescence image of a nonlinear microscope of the present invention.

[0040] FIG8a is a three-dimensional schematic diagram of the electrical high-order modulation extraction module product of the present invention

[0041] FIG8b is a side view of the electrical high-order modulation extraction module product of the present invention

[0042] FIG9 is a flow chart of the present invention using the electrical high-order modulation extraction module to obtain imaging

[0043] [Description of symbols] 100 laser device, 105 laser light source, 110 scanning mirror, 130 tube lens, 140 dichroic beam splitter group, 150 focusing lens group, 160 objective lens, 170 sample carrier, 200 photodetector (PD), 210 electrical signal terminal, 300 servo terminal, 400 / 500 electrical high-order modulation extraction module, 410 DC blocking filter (DCBF), 415 / 455 splitter, 420 / 425 RF mixer, 430 / 457 phase shifter, 440 / 441 / 443 amplifier, 450 function generator, 460 / 462 / 464 tunable low-pass filter (Tunable-LPF), 470 operator, 800 electrical high-order modulation extraction module, 810 / 820 connection terminal, 830 band switch, 831 button DETAILED DESCRIPTION

[0044] The present invention will be described in detail below with reference to the embodiments. Those skilled in the art will readily appreciate other advantages and features of the present invention after reading this disclosure. However, the present invention may also be implemented or applied as other embodiments, and various details in this specification may be modified and changed from different viewpoints and based on different applications without departing from the spirit of the present invention. In the following description, abbreviations are used to replace specific terms. For example, 1M stands for first-order modulation, NLSM (Non-liner laser scanning microscopy) stands for nonlinear laser scanning microscopy, C-NLSM (coherent nonlinear laser scanning microscopy) stands for coherent nonlinear laser scanning microscopy, IC-NLSM (incoherent nonlinear laser scanning microscopy) stands for incoherent nonlinear laser scanning microscopy, DAQ (data acquisition card) stands for data acquisition card, HOM (higher-order modulation) stands for high-order modulation, DCBR (DC-block filter) stands for DC-block filter, TLPF (tunable low-pass filter) stands for tunable low-pass filter, PD (Photo-Detector) stands for photodetector, PMT (photomultiplier tube) stands for high-speed photomultiplier tube, PDT (pixel dwell time) stands for pixel dwell time, SHG (Second-Harmonic Generation) stands for second harmonic generation, and SNR (signal-to-noise ratio) stands for signal-to-noise ratio.

[0045] First, please refer to Figure 1, which is a schematic diagram of a laser scanning microscope (LSM). As shown in Figure 1, the LSM includes a laser device 100, which provides a laser light source 105. A scanning mirror 110 converts the laser light source 105 into a light scanning signal. A tube lens 130 is used in conjunction with an objective lens 160. Together, the two provide a highly accurate correction for the aberrations of the objective lens. A dichroic beam splitter assembly 140 allows the scanning light to pass through the objective lens 160 and generate a nonlinear signal from the sample, which is then reflected or scattered to a high-speed photodetector (PD) 200. A focusing lens assembly 150, a smooth-surfaced focusing lens structure, specifically a monolithic composite Fresnel lens, is used to focus the reflected light from the sample onto the PD 200. The objective lens 160 focuses the light passing through the tube lens 130 onto the sample on the sample carrier 170. A high-speed photodetector (PD) 200 converts the optical signal passing through the focusing lens assembly 150 into an electrical signal. The photodetector (PD) 200 can be a high-speed photomultiplier tube (PMT) or an avalanche photodiode (APD), but the present invention is not limited thereto. An electrical high-order modulation extraction module 400 / 500 processes the electrical signal generated by the PD 200 through a down-conversion circuit or a dual-phase demodulator to provide a sample image signal that converts the first-order modulated (1M) image signal into a DC signal. A servo device 300 receives the first-order modulated (1M) signal transmitted by the photodetector (PD) 200 and, through the imaging process of the data acquisition card (DAQ) in the servo device 300, gradually combines the image signal with the scan signal into an image. In the present invention, which uses a laser scanning microscope (LSM) to construct an image, the laser device 100 is a femtosecond sapphire laser with a pulse width of 140 fs and a repetition rate of ~80.2 MHz. Furthermore, the photodetector (PD) 200 converts nonlinear photons into electrical signals and collects the weak SHG signal, where the electrical signal is a radio frequency (RF) sinusoidal wave signal.

[0046] Next, please refer to Figures 2 and 3, where Figure 2 is a schematic diagram of the electrical high-order modulation extraction module of the present invention, and Figure 3 is a schematic diagram of the electrical signals at each node corresponding to Figure 2. As shown in Figure 2, the electrical high-order modulation extraction module 400 receives the electrical signal transmitted by the photodetector (PD) 200, and each sampling frequency (0, fs, 2fs, ...) in the electrical signal contains the spatial information of the same sample. In other words, the photodetector (PD) 200 can simultaneously collect DC, 1M, 2M and 3M as well as high-order modulation (HOM). The signal output by the photodetector (PD) 200 is at point A in Figure 2, and the signal corresponding to point A is shown in Figure 3a. In addition, the 1M, 2M, and 3M (all referred to as high-order modulation) mentioned in the present invention refer to several times the laser repetition rate. For example, when the laser pulses 50 million times per second, meaning the laser repetition rate is 50 MHz, the center frequency of 0 MHz is 0 Hz, the center frequency of 1 MHz is 50 MHz, the center frequency of 2 MHz is 100 MHz, and the center frequency of 3 MHz is 150 MHz. At this point, if the bandwidth of the photodetector (PD) 200 is limited to 80 MHz, the 0 MHz and 1 MHz signals can be fully detected, while the 2 MHz and 3 MHz signals will be significantly attenuated. Therefore, the 1 MHz signal is optimal, as shown in Figure 4c.

[0047] Next, the electrical high-order modulation extraction module 400 includes: a direct current blocking filter (DCBF) 410 for filtering out the DC signal from the radio frequency (RF) sinusoidal signal output by the photodetector (PD) 200. The DCBF 410 uses a passband of 50 MHz to 26.5 GHz to filter out the DC signal. Therefore, the corresponding signal at point B in FIG2 is shown in FIG3b. An RF mixer 420, one end of which is connected to receive the RF sinusoidal signal output by the DCBF 410 (i.e., FIG3b). A function generator 450 is used to provide a local oscillator (LO) signal, for example, a single adjustable output frequency between 250 kHz and 4 GHz or a laser pulse. This LO signal is located at point C in FIG2, and the corresponding signal is shown in FIG3c. When the LO signal is provided to the other input of RF mixer 420, RF mixer 420 outputs an intermediate frequency (IF) signal. The IF signal is located at point D in Figure 2, and the corresponding signal at point D is shown in Figure 3d. This IF signal is the sum and difference frequencies of the RF sinusoidal signal and the LO signal, and is proportional to the product of the RF sinusoidal signal and the LO signal. For example, when the LO signal frequency is equal to the laser pulse repetition frequency (as shown in Figure 3c), the first-order modulated (1M) signal can be down-modulated to the DC coordinate after passing through mixer 420, as shown in Figure 3d, indicating that the first-order modulated (1M) signal can be selected for imaging in the LSM. It is important to note that since the output frequency of the LO signal at point C in Figure 2 can be an integer multiple of the laser pulse repetition frequency, a specific HOM (High-Order Modulation) (HOM) can be selected for imaging, such as 1M, 2M, 3M, or other high-order modulation (HOM), as shown at point D in Figure 2. In principle, pixels from different modulation orders can be obtained by sequentially changing the frequency of the LO signal until the bandwidth of the LO signal or the mixer is saturated. Obviously, in an embodiment of the present invention, the LO signal frequency of the function generator 450 can be selected as an integer multiple of the laser pulse repetition frequency. Therefore, the function generator 450 can provide various high-order modulation frequencies. In addition, after the function generator 450 generates the LO signal, it can be selectively passed through a phase shifter 430 to shift the relative angle difference between the LO signal and the RF signal to a set angle difference. For example, by setting the phase shifter 430 to a 90-degree shifter, a sine wave can be converted into a cosine wave.

[0048] The electrical high-order modulation extraction module 400 may further include an amplifier 440 and a tunable low-pass filter (T-LPF) 460. Once the LO signal frequency is determined, these filters are used to remove some IF frequencies at point D. For example, when the LO signal frequency is equal to the laser pulse repetition frequency, the T-LPF 460 removes IF frequencies exceeding the laser pulse repetition frequency, leaving only the first-order modulation (1M) signal at the coordinate with a frequency of 0 (i.e., DC), as shown at point E in Figure 2. If the LO signal frequency is twice the laser pulse repetition frequency, the T-LPF 460 removes IF frequencies beyond 2M, leaving only the second-order modulation (2M) signal at the DC coordinate. Finally, the selected first-order modulation (1M) signal or other modulated signal is sent to the DAQ in the servo terminal 300 for pixel reconstruction.

[0049] Next, please refer to Figure 4, which is a schematic diagram of the operating principle of the pixel contrast / signal-to-noise ratio enhancement mechanism used in the present invention. This principle is applicable to both coherent and noncoherent nonlinear microscopes. As shown in Figure 4a, a schematic diagram of the photodetector (PD) 200 converting photons into electrical signals with a frequency response is shown. The curve labeled 1 represents the gain of the photodetector (PD) 200 when converting photons into electrons, the curve labeled 2 represents the noise generated by the photodetector (PD) 200 when converting photons into electrons, and the curve labeled 3 represents the frequency response of the photodetector (PD) 200 when converting photons into electrons relative to the signal-to-noise ratio.

[0050] Next, in Figure 4b, due to the inherent noise characteristics of the mode-locked laser, optical signals of different orders (DC, 1M, 2M, 3M...) (represented by the lighter colors at the top of the signal waveform) carry different optical noise (represented by the darker colors at the bottom of the signal waveform). The noise carried by the DC optical signal is much greater than that of the 1M, 2M, and 3M signals. Next, in Figure 4b, the photoelectric conversion signal-to-noise ratio curve (labeled 3 in Figure 4a) is multiplied by the incident light of different orders (DC, 1M, 2M, 3M...) to obtain the electrical signal output by the photodetector (PD) 200.

[0051] Next, as shown in Figure 4c, the converted electrical signals of different orders (DC, 1M, 2M, 3M, etc.) that can be used for imaging are clearly the product of the frequency-domain electrical signals of different orders and the signal-to-noise ratio. The imaging electrical signal comprises a noise background (lower layer) and a signal (upper layer). It is particularly important to note that higher-order converted electrical signals have smaller values. Therefore, to maximize the performance of the photon-electron conversion process and achieve a higher signal-to-noise ratio, it is crucial to select the most appropriate electrical signal modulation sequence for the microscope's DAQ circuit. For example, in embodiments of the present invention, a first-order modulated (1M) electrical signal can be selected as the imaging signal, thereby achieving the optimal signal-to-noise ratio. It is worth noting that conventional LSM DAQ uses DC imaging. To maintain consistency with the conventional LSM DAQ imaging format, the present invention processes the 1M modulated information at DC after processing by the down-conversion circuit 400 or the dual-phase demodulator circuit 500, as shown in Figures 3d or 3e. Therefore, when DAQ imaging is performed, it uses information from first-order modulation (1M) with a higher image contrast / signal-to-noise ratio. Because DAQ uses an imaging process to combine scanned image signals bit by bit into a single image, improving image contrast or reducing the signal-to-noise ratio is crucial for imaging quality. In a preferred embodiment of the present invention, a technique for improving image contrast is employed, downmodulating the first-order modulation (1M) information to a DC level. This achieves relatively good image contrast and improves the contrast / signal-to-noise ratio.

[0052] Obviously, the converted signal passing through point B in Figure 2 (i.e., with relatively low noise) may not be strong enough to be used for subsequent data reception. Therefore, an additional amplifier is required to amplify the converted signal. The amplified converted signal can then be passed through a low-frequency filter to filter out the additional noise. Therefore, when the converted signal passing through point E is provided to the DAQ, the image contrast / signal-to-noise ratio can be improved.

[0053] Next, an actual sample is imaged and analyzed using a nonlinear laser scanning microscope (NSLM) equipped with an electrical high-order modulation extraction module to illustrate the effect of the present invention in using high-order modulation (HOM) technology to improve the image contrast / signal-to-noise ratio.

[0054] The tissue sample used for imaging in the present invention is entangled muscle collagen tissue extracted from mice. Continuing with Figure 5, it shows SHG tissue imaging matrices with different (electrical) modulation orders, PDT, and TLPF bandwidths. These matrices are constructed using different PDT (0.4μs, 1μs, and 5μs), modulation orders (1M-3M), and DC. In the preferred embodiment of the present invention, all images share the same color bar and dynamic range. Scale bar = 100μm.

[0055] Please refer to Figure 5, which shows an image matrix of SHG modulation signals from different PDTs at different modulation orders. To clearly demonstrate that a 1M (sampling frequency 80 MHz) signal provides the best image contrast, the image columns corresponding to the different modulation orders shown in Figure 2(f) are arranged below Figure 5. The dynamic range of Figure 5 is normalized within the range of 0-17,000. Clearly, in each column of Figure 5, image contrast increases with increasing PDT. Furthermore, each row of Figure 5 shows that, regardless of PDT, the image obtained from 1M exhibits better contrast than other images obtained from DC and HOM (N = 2, 3). The results shown in the image matrix of Figure 5 demonstrate the consistency of the working principle of the present invention, which optimizes the high-frequency modulation order to improve image quality. Furthermore, Figure 5 also shows that slower scanning results in higher image contrast and clearer images.

[0056] The present invention has successfully demonstrated that the first-order modulation (1M) frequency extracted by the electrical high-order modulation extraction module 400 can significantly improve image quality. Furthermore, in order to apply the electrical high-order modulation extraction module to processing optical signals or imaging other fine surfaces, such as for use in industrial imaging inspection, there is a pressing need for the electrical high-order modulation extraction module to simultaneously process non-coherent optical signals. Next, the present invention further discloses an electrical high-order modulation extraction module capable of processing both coherent and non-coherent optical signals.

[0057] Next, please refer to Figure 6, which is a schematic diagram of another embodiment of the electrical high-order modulation extraction module of the present invention. As shown in Figure 6, the electrical high-order modulation extraction module 500 can be a dual-phase demodulator. When the photodetector (PD) 200 receives an incoherent or coherent optical signal, such as fluorescence or harmonic generation, it converts the coherent and incoherent optical signals into electrical signals carrying various modulated sinusoidal waves. The sinusoidal wave frequency in the electrical signal is the same as the laser repetition rate or a multiple thereof, and the modulated electrical signal carries spatial information of the same sample. In other words, the photodetector (PD) 200 can simultaneously collect DC, 1M, 2M, 3M, and other high-order modulation (HOM) signals. Next, the electrical high-order modulation extraction module 500 receives and extracts the high-order modulated electrical signal (1M, 2M, or 3M) transmitted by one group of photodetectors (PD) 200, wherein the signal output by the photodetector (PD) 200 is at point A in FIG6 , and the corresponding signal at point A is shown in FIG3a .

[0058] Next, the electrical high-order modulation extraction module 500 includes a direct current blocking filter (DCBF) 410, which removes the DC signal and its transmitted modulation signal from the radio frequency (RF) sinusoidal signal output by the photodetector (PD) 200, allowing 1M, 2M, 3M, and other high-order modulation (HOM) signals to pass through simultaneously. The DCBF 410 can also be replaced with an appropriate bandpass filter (BPF). This bandpass filter not only removes the DC signal and its transmitted modulation signal, but also selectively passes 1M, 2M, 3M, or other high-order modulation (HOM) signals. The modulated signal passing through the DCBF 410 then passes through a splitter 415, which separates the 1M, 2M, 3M, and other high-order modulation (HOM) signals into two optical paths (B1 and B2). The corresponding signals of B1 and B2 are shown in Figure 3b. Then, the B1 optical path transmits the B1 signal to the receiving end of the first RF mixer 420, and the B2 optical path transmits the B2 signal to the receiving end of the second RF mixer 425. In the embodiment of the present invention, both the B1 and B2 signals are Asin(2πf s t+θ), where A is the amplitude, f s It is the sampling frequency, which is the carrier frequency on 1M, 2M, 3M or other high-order modulation (HOM), and is also an integer multiple of the laser repetition rate.

[0059] A sine wave signal generator 450 provides a local oscillator (LO) signal with a frequency equal to or a multiple of the laser repetition rate. This signal is used to select 1M, 2M, 3M, or other high-order modulation (HOM) for nonlinear microscopy imaging. The LO signal can be generated using a simple electrical signal generator or by using the femtosecond laser 100 shown in Figure 1. This signal is generated by splitting a portion of the generated femtosecond pulse laser light into an electrical pulse signal converted by a photodetector and then applying an appropriate bandpass filter (BPF). The LO signal passes through a splitter 455, which separates the LO signal into two optical paths (C1 and C2). Path C1 passes through a phase shifter 457. When phase shifter 457 is set to a 90-degree shifter, it converts the sine wave into a quadrature cosine wave signal. The corresponding LO signal for path C1 is shown in Figure 3c. The C2 optical path is a sinusoidal signal in phase. In the embodiment of the present invention, the C1 signal is Bcos(2πf r t), where B is the amplitude, f r is the pulse center frequency. The C2 signal is Bsin(2πf r t), where B is the amplitude, f r is the pulse center frequency.

[0060] One input of the first RF mixer 420 receives a sine wave signal from the B1 optical path, such as a sine wave signal, while the other input of the first RF mixer 420 receives a sine wave signal from the C1 optical path, such as a cosine wave signal. After processing by the first RF mixer 420, a first intermediate frequency (IF) signal is output. The IF signal is located at point D1 in Figure 6 , and the signal corresponding to point D1 is shown in Figure 3d . The first IF signal is the sum and difference frequencies of the RF sine wave signal and the LO signal. Furthermore, the first IF signal is proportional to the product of the RF sine wave signal and the LO signal. For example, when the LO signal frequency is equal to twice the laser pulse repetition frequency (as shown in Figure 3c ), the first-order modulated (1M) signal can be down-modulated to a DC coordinate position after passing through the first mixer 420, as shown in Figure 3d , indicating that this first-order modulated (1M) signal can be selected for imaging in the LSM. It should be noted that since the output frequency of the LO signal in FIG6 can be an integer multiple of the laser pulse repetition frequency, a specific HOM can be selected for imaging, such as 1M, 2M, 3M or other high-order modulation (HOM).

[0061] One input of the second RF mixer 425 receives a sine wave signal, e.g., a sinusoidal signal, from the B2 optical path, while the other input of the second RF mixer 425 receives a sine wave signal, e.g., a sinusoidal signal, from the C2 optical path. The second RF mixer 425 then outputs a second intermediate frequency (IF) signal. This second IF signal is located at point D2 in Figure 6 , and the corresponding signal at point D2 is shown in Figure 3d . The second IF signal is the sum and difference frequencies of the RF sine wave signal and the LO signal, and is proportional to the product of the RF sine wave signal and the LO signal. For example, when the LO signal frequency is equal to the laser pulse repetition frequency (as shown in Figure 3c ), the first-order modulated (1M) signal can be down-modulated to a DC coordinate after passing through the second mixer 425, as shown in Figure 3d , indicating that this first-order modulated (1M) signal can be selected for imaging in the LSM. It should be noted that since the output frequency of the LO signal in FIG6 can be an integer multiple of the laser pulse repetition frequency, a specific HOM can be selected for imaging, such as 1M, 2M, 3M or other high-order modulation (HOM).

[0062] Next, the signals at points D1 and D2 are passed through LPF 462 and LPF 464 to filter out the IF frequencies that are down-modulated outside the DC coordinate. The two filtered signals X and Y are then sent to operator 470 for operation to obtain a signal R, where R = (X 2 +Y 2 ) 0.5 The operator 470 can be implemented by hardware circuits or software programs. In a preferred embodiment, the operator 470 can be configured in the servo terminal 300 to calculate the R signal through the DAQ program. For example, when the frequency of the LO signal is equal to the laser pulse repetition frequency, that is, when f s =f r When , LPF 462 and LPF 464 will filter out the IF frequency that exceeds the laser pulse repetition frequency, leaving only the first-order modulation (1M) signal at the coordinate with a frequency of 0 (i.e., DC-direct current). At this time, the signals at the X and Y positions in FIG6 are shown in FIG3d. Among them, the signal at the X position is The signal at the Y position is And θ is the phase difference between the laser and the nonlinear signal. Finally, the operator 470 calculates a R=(X 2 +Y 2 ) 0.5 The signal is then reconstructed pixel by pixel using the DAQ program.

[0063] For coherent nonlinear signals, the down-conversion circuit of FIG2 or the dual-phase demodulator circuit of FIG6 can be used to extract signals with a relatively good signal-to-noise ratio at 1M, 2M, 3M or other high-order modulation (HOM). In non-coherent nonlinear signals, since θ is the phase difference between the laser and the nonlinear signal, it will change with time and space. Unlike coherent nonlinear signals, θ is a fixed value and can be compensated by using the fixed phase shifter of the phase shifter 430 in FIG2. Therefore, it is necessary to use the circuit of FIG6 and R=(X 2 +Y 2 ) 0.5 Signal calculation to extract the amplitude of the true nonlinear signal.

[0064] Finally, please refer to Figure 7, which is a schematic diagram of a 3D two-photon fluorescence image obtained by the nonlinear microscope of the present invention. As shown in Figure 7, using the R = (X² + Y²)0.5 signal of the embodiment, the LSM achieves the following imaging effects. For example, the original 3D two-photon fluorescence image (using a DC signal) from the nonlinear microscope is shown in Figure 7(a), while the 3D two-photon fluorescence image (using a DC signal) obtained using the 1M signal extracted by the dual-phase demodulator circuit of Figure 6 is shown in Figure 7(b). At a depth of 100μm, the original (DC) 2D slice image is shown in Figures 7(c) and 7(e), while the 2D slice image at a depth of 100μm using the 1M signal extracted by the circuit of Figure 6 is shown in Figures 7(d) and 7(f). Figures 7(c) and 7(d) are plotted using a linear scale. It can be seen that after passing through the circuit of Figure 6, the signal is enhanced by approximately 3-4 times. To demonstrate the image noise reduction achieved by this method, Figures 7(e) and 7(f) can be log-scaled and the image noise represented using black and white grayscale. Clearly, the use of a 1M high-order modulation term significantly reduces overall noise by approximately 3-4 times. The black and red lines in Figures 7(f) and 7(h) represent the signal improvement and noise reduction at different image depths using the original microscope (DC, black line) and using this method (1M, red line), respectively. The signal improvement ratios shown in Figure 7(f) and the noise reduction ratios shown in Figure 7(h) are then translated to Figure 7(i). Figure 7(i) shows that the signal-to-noise ratio of two-photon fluorescence microscopy images at all depths has improved by approximately an order of magnitude (10 times).

[0065] The electrical high-order modulation extraction module 500 disclosed herein, which processes both coherent and non-coherent optical signals, can simultaneously process non-coherent optical signals to extract first-order modulation (1M) signals. Therefore, when the electrical high-order modulation extraction module 500 uses the extracted first-order modulation (1M) signals as LSM imaging information, image quality can be significantly improved. This allows the electrical high-order modulation extraction module 500 to be applied to fluorescence signal processing and other fine surface imaging applications.

[0066] Next, please refer to Figure 8, which is a schematic diagram of a product having an electrical high-order modulation extraction module according to the present invention. Figure 8a is a perspective schematic diagram of the electrical high-order modulation extraction module, while Figure 8b is a top schematic diagram of the electrical high-order modulation extraction module. As shown in Figure 8a, the down-conversion circuit of Figure 2 and the dual-phase demodulator of Figure 6 are fabricated on a circuit board (not shown). The circuit board is then placed in a geometrically shaped module 800 to form a portable module 800 product. A connector 820 on the housing of module 800 can be connected to the electrical signal from the photodetector (PD) 200 to transmit the electrical signal to the DC blocking filter (DCBF) 410. Another connector 810 on the housing of module 800 can be connected to the servo terminal 300 to transmit the first-order modulated (1M) signal to the DAQ program in the servo terminal 300 for pixel reconstruction. Module 800 is further configured with a band switch 830 connected to the function generator 450 shown in Figures 2 and 6. Band switch 830 adjusts the LO signal output by function generator 450. The LO signal provides a sine wave signal that is equal to or a multiple of the laser repetition frequency. The adjusted LO signal is combined with the electrical signal from photodetector (PD) 200 through RF mixer 420 to extract 1M, 2M, 3M, or other high-order modulation (HOM) as an imaging signal. For example, when the laser repetition frequency is 50MHz, adjusting the LO signal to approximately 50MHz allows the complete extraction of the 1M signal when the bandwidth of photodetector (PD) 200 is 80MHz, thus providing the DAQ imaging signal. Obviously, when different LSMs use different photodetectors (PD) 200, although the photodetectors (PD) 200 may have different bandwidths, the corresponding laser repetition frequencies can be adjusted through the band switch 830 of the present invention, and the present invention does not impose any restrictions.

[0067] When module 800 manufactured by the electrical high-order modulation extraction module of the present invention is connected to an LSM, either connection terminal 810 or connection terminal 820 on module 800 can be connected to the electrical signal of the servo terminal 300 or the photodetector (PD) 200, while the other end can be connected via a single wire to complete a closed loop connection. For example, after connection terminal 810 on module 800 is connected to the servo terminal 300, a single wire can be used to connect connection terminal 820 to the electrical signal terminal 210 of the photodetector (PD) 200. Of course, in another embodiment, two wires can be used to connect module 800 to the LSM and the servo terminal 300, with one wire connecting connection terminal 810 to the servo terminal 300 and the other wire connecting connection terminal 820 to the photodetector (PD) 200. The present invention does not limit the method used to connect module 800 to the LSM, and the method is based on user convenience.

[0068] Obviously, when the module 800 manufactured by the electrical high-order modulation extraction module of the present invention is connected to the LSM and the servo terminal 300, the following steps must be followed. FIG9 shows the imaging process of the module 800 using the electrical high-order modulation extraction module of the present invention. To achieve optimal imaging, the LSM imaging operation steps include:

[0069] Step 910 : The module 800 manufactured by the electrical high-order modulation extraction module is connected to the LSM and the servo terminal 300 .

[0070] Step 930, obtaining the laser repetition frequency and the LO signal frequency and determining whether the LO signal frequency is a multiple of the laser repetition frequency, uses information displayed on the servo terminal 300 to obtain the laser repetition frequency of the LSM and the LO signal frequency of the function generator 450. Next, a determination is made as to whether the LO signal frequency is a multiple of the laser repetition frequency. The determination method can be user-defined and is not limited by the present invention.

[0071] Step 950 calibrates the LO signal frequency of function generator 450. If the LO signal frequency of function generator 450 is different from the laser repetition frequency, servo terminal 300 adjusts the LO signal frequency of function generator 450 so that the LO signal frequency is the same as or a multiple of the laser repetition frequency. The method for adjusting the LO signal frequency can be user-defined, and the present invention does not limit the calibration method.

[0072] Step 970 provides a high-order modulation signal to the DAQ. When the LO signal frequency of the function generator 450 is a multiple of the laser pulse repetition frequency, the module 800 manufactured by the electrical high-order modulation extraction module completes the operation and obtains the down-modulated high-order modulation signal. After that, the down-modulated high-order modulation signal is transmitted to the DAQ program configured in the servo terminal. The DAQ program combines the image signal with the scanning signal bit by bit into an image.

[0073] Step 990, adjusting the imaging quality, is achieved by the observer adjusting the button 831 on the band switch 830 to select the LO signal frequency as a down-converted signal (1M, 2M, 3M, ...), and transmitting the down-converted signal to the DAQ, which is imaged by the DAQ program.

[0074] Therefore, through the above method, the present invention can view the imaging results of each down-converted signal (1M, 2M, 3M, ...) on the Sever 300 and obtain the best imaging image on the display of the Sever 300.

[0075] According to the above, the laser scanning microscope with an electrical high-order modulation extraction module provided by the present invention can achieve the following advantages, including:

[0076] 1) The electrical high-order modulation extraction module used is simple in structure and is conducive to adapting to other forms of coherent or incoherent nonlinear optical imaging techniques, such as third harmonic generation (THG), coherent anti-Stokes Raman scattering (CARS), stimulated Raman scattering (SRS) microscopy, two-photon fluorescence microscopy (2PF), and three-photon fluorescence microscopy (3PF).

[0077] 2) The configuration using the electrical high-order modulation extraction module processes the electrical signal only at the detection end, so it offers many possibilities for combining existing contrast enhancement methods, such as: using near-infrared wavelengths to reduce light scattering in tissue, immersing the sample in a clearing agent, and adaptive optics for wavefront shaping to further improve pixel contrast and signal-to-noise ratio.

[0078] 3) The currently used photodetector (PD) 200 has an 80 MHz bandwidth, which severely attenuates the 2M and 3M signals. Therefore, in a preferred embodiment of the present invention, the use of a GaAsP photodetector (PD) 200 and a high-bandwidth transimpedance amplifier (hundreds of MHz or >1 GHz) will help maintain higher amplitude 2M and 3M signals, thereby providing a better signal-to-noise ratio.

[0079] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of the rights of the present invention. At the same time, the above description should be clear and implementable to those with ordinary knowledge in the relevant technical field. Therefore, other equivalent changes or modifications that do not depart from the concepts disclosed in the present invention should be included in the scope of the patent application.

Claims

1. An electrical high-order modulation extraction module for receiving a radio frequency sinusoidal signal output by a photodetector on a laser scanning microscope having a laser scanning frequency, characterized in that The electrical high-order modulation extraction module includes: A DC blocking filter, used for filtering out a DC signal on the radio frequency sinusoidal signal output by the photodetector; a function generator for providing a local oscillation frequency, wherein the local oscillation frequency is an integer multiple of the laser scanning frequency; A mixer, one end of which receives the RF sine wave signal output by the DC blocking filter, and the other end of which receives the local oscillation frequency output by the function generator and outputs an intermediate frequency signal; wherein, The intermediate frequency signal is a first order or other high order modulation (1M or HOMs) signal down modulated to a DC coordinate position, and the intermediate frequency signal is transmitted to a DAQ program for pixel reconstruction.

2. The electrical high-order modulation extraction module as claimed in claim 1, characterized in that: The invention further comprises a phase shifter for shifting the relative angle difference between the LO signal and the RF signal to a set angle difference.

3. The electrical high-order modulation extraction module according to claim 1, characterized in that: The intermediate frequency signal passes through a low pass filter.

4. The electrical high-order modulation extraction module according to claim 3, characterized in that: The intermediate frequency signal passing through the low-pass filter is first amplified by an amplifier and then filtered by the low-pass filter.

5. The electrical high-order modulation extraction module according to claim 1, characterized in that: The down-modulation circuit is formed on a substrate, and the substrate is arranged in a geometric shell to form a portable functional module, and a first connection end and a second connection end are formed at two ends of the functional module, wherein the first connection end is connected to a servo terminal, and the second connection end receives the radio frequency sinusoidal wave signal output by the light detector.

6. An electrical high-order modulation extraction module for receiving a radio frequency sinusoidal signal output by a photodetector on a laser scanning microscope having a laser scanning frequency, characterized in that The electrical high-order modulation extraction module includes: A DC blocking filter, used for filtering out a DC signal on the radio frequency sinusoidal signal output by the photodetector; a first separator, for separating the radio frequency sinusoidal wave signal passing through the DC blocking filter into a first optical path and a second optical path; a function generator for providing a local oscillation frequency, wherein the local oscillation frequency is an integer multiple of the laser scanning frequency; a second separator for separating the local oscillation frequency into an orthogonal optical path and an in-phase optical path; A first mixer, one input end of which receives the sine wave signal of the first optical path, and another input end of which receives the orthogonal optical path, and outputs a first intermediate frequency signal; wherein the first intermediate frequency signal is a first-order modulation (1M) signal down-modulated to a DC coordinate position; A second mixer, one input end of which receives the sine wave signal of the second optical path, and another input end of which receives the in-phase optical path, and outputs a second intermediate frequency signal; wherein the second intermediate frequency signal is a first-order modulation (1M) signal down-modulated to a DC coordinate position; wherein, After the first intermediate frequency signal and the second intermediate frequency signal are transmitted to a computing unit for computing, the calculated root mean square signal is transmitted to a DAQ program for pixel reconstruction.

7. The electrical high-order modulation extraction module according to claim 6, characterized in that: The orthogonal optical path is to pass the local oscillator frequency through a 90 degree phase shifter.

8. The electrical high-order modulation extraction module according to claim 6, characterized in that: The operator is configured in the server terminal.

9. The electrical high-order modulation extraction module according to claim 6, characterized in that: The first intermediate frequency signal passes through a first low-pass filter, and the second intermediate frequency signal passes through a second low-pass filter.

10. The electrical high-order modulation extraction module according to claim 9, characterized in that: The first intermediate frequency signal and the second intermediate frequency signal that pass through the first low-pass filter and the first low-pass filter first pass through a first amplifier and a second amplifier to amplify the first intermediate frequency signal and the second intermediate frequency signal, and then pass through the first low-pass filter and the first low-pass filter for filtering.

11. The electrical high-order modulation extraction module according to claim 6, characterized in that: The dual-phase demodulator circuit is formed on a substrate, and the substrate is arranged in a geometric shell to form a portable functional module, and a first connection end and a second connection end are formed at two ends of the functional module, wherein the first connection end is connected to a servo terminal, and the second connection end receives the radio frequency sine wave signal output by the light detector.

12. The electrical high-order modulation extraction module according to claim 1 or 6, characterized in that: The DC blocking filter is a band pass filter (BPF).

13. A method for adjusting laser scanning microscope imaging using an electrical high-order modulation extraction module, characterized in that include: Providing a module manufactured by an electrical high-order modulation extraction module, and connecting the module with a laser scanning microscope and a servo terminal; Obtaining and judging whether the laser repetition frequency is the same as the LO signal frequency is performed by obtaining the laser repetition frequency of the laser scanning microscope and the LO signal frequency generated by the function generator through information displayed on the servo terminal, and judging whether the LO signal frequency is a multiple of the laser repetition frequency; Correcting the LO signal frequency is to adjust the LO signal frequency output by the function generator through the servo terminal so that the LO signal frequency and the laser repetition frequency are in a multiple relationship when the LO signal frequency and the laser repetition frequency are not in a multiple relationship; Providing a high-order modulation signal is to obtain a down-modulated high-order modulation signal after the operation of the module, and transmit the down-modulated high-order modulation signal to a DAQ program configured in the servo terminal, and the DAQ program performs pixel reconstruction; The imaging quality is adjusted by the observer selecting a down-modulated signal (1M, 2M, 3M, ...) one by one, and transmitting the down-modulated signal to the DAQ, and the DAQ program performs pixel reconstruction one by one.

Images