Frequency-tagged synchronous two-photon and three-photon excitation single-pixel detection microscopic imaging system

Abstract

Disclosed in the present invention is a frequency-tagged synchronous two-photon and three-photon excitation single-pixel detection microscopic imaging system. The system comprises a GHz-level high-repetition-rate femtosecond pulse laser source, a frequency tagging module, a pulse compression and amplification module, a pulse modulation module, a nonlinear wavelength conversion module, an imaging module, a single-pixel optical signal acquisition component and a data processing component. In the present invention, multi-photon imaging of different labels is performed on the basis of each of the laser beams at two wavelengths simultaneously generated by a single-wavelength high-repetition-rate femtosecond laser by means of radio frequency programmability and soliton self-frequency shift, so as to realize temporally and spatially synchronized multi-dimensional microscopic observation, thereby solving the problem in existing multimodal imaging techniques of it not being possible to realize the temporally and spatially precisely synchronized interpretation of multi-dimensional information, and achieving the advantages of high speed, operational convenience, low cost, etc.

Description

Frequency-labeled dual- and three-photon synchronous excitation single-pixel detection microscopic imaging system Technical Field

[0001] This invention relates to the fields of multiphoton biomedical imaging and femtosecond laser technology, and particularly to a frequency-marked dual- or triphoton synchronous excitation single-pixel detection microscopic imaging system. Background Technology

[0002] With the development of neuromedicine and the widespread application of fluorescent proteins, multiphoton imaging technology has become a powerful tool in biomedical research due to its advantages such as low invasiveness, strong penetration, high spatial resolution, and high selectivity. It plays a significant role in the study of deep brain tissue structure and physiological function, neurological diseases, and cancer pathology. Currently, two-photon excitation fluorescence microscopy is the most widely used multiphoton imaging technique. In recent years, three-photon excitation fluorescence microscopy has also been developing rapidly and has enormous application potential in the biomedical field.

[0003] When using multiphoton imaging to label biological tissues with fluorescent dyes, the information obtained in a single scan is relatively limited. Existing simultaneous two-photon and three-photon imaging techniques mostly collect and process the signals from both separately, failing to achieve synchronous imaging. Furthermore, the light source used for imaging is not wavelength-tunable (Li S.-Q. et al. New advances in biomedical applications of multiphoton imaging technology. Acta Phys. Sin. 69, 228702 (2020)). If several different fluorescent dyes could be excited simultaneously and precisely in time and space, different fluorescently labeled biological tissue structures could be acquired simultaneously using multiphoton imaging, obtaining more precise, multi-dimensional biological information—a key to comprehensively explaining biological problems. Such imaging systems can provide a more comprehensive understanding of the intrinsic connections within biological tissues, more accurately resolve complex concurrent medical problems, and provide stronger technical support for biomedical research. Summary of the Invention

[0004] The problem this invention aims to solve is that acquiring multi-dimensional information takes a long time and the information acquired in a single scan is limited.

[0005] The frequency-marked dual and three-photon synchronous excitation single-pixel detection microscopic imaging system proposed in this invention will provide a solution to the above problems. Based on a single-wavelength high-repetition-rate femtosecond laser, two wavelength lasers generated simultaneously by radio frequency programmability and soliton self-translation are used to perform multiphoton imaging on different markers.

[0006] The objective of this invention is achieved by at least one of the following technical solutions.

[0007] The frequency-marked dual- or three-photon synchronous excitation single-pixel detection microscopic imaging system includes a GHz-level high-repetition-rate femtosecond pulse laser source, a frequency marking module, a pulse compression and amplification module, a pulse modulation module, an intensity-correlated wavelength conversion module, an imaging module, a single-pixel optical signal acquisition component, and a data processing component connected in sequence.

[0008] The laser pulses generated by the GHz-level high repetition rate femtosecond pulse laser source are marked by a frequency marking module that generates a reference marking signal to mark the frequency of the laser pulses, resulting in a first laser pulse train and a second laser pulse train with the reference marking signal. The first laser pulse train and the second laser pulse train are then amplified in energy and compressed in pulse width sequentially by a pulse compression and amplification module.

[0009] The pulse intensity modulation module modulates the intensity of the first and second laser pulse trains after energy amplification and pulse width compression, injects them into the intensity-related wavelength conversion module, and generates a set wavelength redshift through nonlinear effects before entering the imaging module. The imaging module performs synchronous scanning of the sample using two-photon and three-photon excitation fluorescence imaging. The light signal generated during the imaging process enters the single-pixel light signal acquisition component to form an electrical signal. The electrical signal is sent to the data processing component and, combined with the reference marker radio frequency signal generated by the frequency marking module, synchronously demodulates and processes it to obtain a high-resolution, multi-contrast biological image based on single-pixel detection.

[0010] Furthermore, the GHz-level high repetition rate femtosecond pulse laser source is an all-fiber femtosecond pulse laser, which serves as the initial light source and emits pulse lasers with a repetition rate in the gigahertz range suitable for biological imaging.

[0011] Furthermore, the frequency marking module includes a programmable terminal, an arbitrary waveform generator, an RF signal amplifier, and a first optical modulator connected in sequence;

[0012] Programmable terminal controls arbitrary waveform generator to send reference frequency signal After being amplified by an RF signal amplifier, the signal is applied to the first optical modulator to mark the frequency of laser pulses at different times. This modulates the laser pulses generated by a GHz-level high-repetition-rate femtosecond pulse laser source into periodic, cyclical frequency markers. and The first laser pulse train and the second laser pulse train; at this time, the center wavelengths of the first laser pulse train and the second laser pulse train are... .

[0013] Furthermore, the pulse compression amplification module includes an all-fiber amplifier and a grating pair connected in sequence. First, the laser pulse train is amplified by the all-fiber amplifier, and then the pulse width is compressed by the grating pair.

[0014] Furthermore, the pulse intensity modulation module includes a programmable terminal, an arbitrary waveform generator, an RF signal amplifier, and a second optical modulator connected in sequence.

[0015] The programmable terminal controls an arbitrary waveform generator to send the required radio frequency (RF) signal. After being amplified by an RF signal amplifier, the signal is applied to a second optical modulator to modulate the intensity of the first and second laser pulse trains, which have undergone energy amplification and pulse width compression. The frequencies are then marked as follows: The light intensity of the first laser pulse train is modulated to The frequency is marked as The light intensity of the second laser pulse train is modulated to The center wavelengths of the first and second laser pulse trains after intensity modulation are: .

[0016] Furthermore, the intensity-dependent wavelength conversion module includes a lens and a photonic crystal fiber;

[0017] The intensity-dependent wavelength conversion module utilizes an intensity-dependent nonlinear effect to synchronously generate a set wavelength redshift for the first and second laser pulse trains modulated by the light intensity output from the pulse intensity modulation module (W. Wang et al., 'High-speed wavelength-swept femtosecond source from 1055 to 1300 nm using a GHz femtosecond fiber laser', Opt. Lett., OL, vol. 47, no. 7, pp. 1677–1680, Apr. 2022, doi: 10.1364 / OL.449955.), making the intensity... The center wavelength of the first laser pulse train shifts to The corresponding frequency is marked as The strength is The center wavelength of the second laser pulse train shifts to The corresponding frequency is marked as .

[0018] Furthermore, the imaging module includes a relay optical path, an objective lens, a dichroic mirror, and a sample; the synchronous two-color laser pulse is collimated through the relay optical path and transmitted through the dichroic mirror into the objective lens and focused on the sample, performing time- and space-synchronized two-photon and three-photon excitation on the sample, and the light signal excited by the sample returns to the dichroic mirror and is reflected and output to the single-pixel light signal acquisition component.

[0019] Furthermore, the single-pixel optical signal acquisition component has two channels suitable for different wavelengths, used to simultaneously receive optical signals generated by three-photon excitation and two-photon excitation, and convert them into electrical signals.

[0020] Furthermore, the data processing component includes a programmable terminal that synchronously demodulates and processes the collected electrical signals through a reference marker signal generated by a frequency marker module to obtain a high-resolution, multi-contrast biological image based on single-pixel detection.

[0021] Furthermore, the frequency marking module, pulse intensity modulation module, and data processing unit all use the same programmable terminal;

[0022] The frequency marking module and the pulse intensity modulation module use the same arbitrary waveform generator;

[0023] The frequency marking module and the pulse intensity modulation module use the same radio frequency signal amplifier.

[0024] Compared with existing technologies, the beneficial effects of the present invention are as follows:

[0025] This invention utilizes a single-wavelength high-repetition-rate femtosecond laser to simultaneously generate two wavelengths of laser light through radio frequency programmability and soliton self-translation, enabling multiphoton imaging of different markers. This achieves synchronous multidimensional microscopic observation in time and space, solving the problem that existing multimodal imaging technologies cannot achieve precise spatiotemporal synchronous interpretation of multidimensional information. Furthermore, it has advantages such as high speed, convenience, and low cost, providing strong support for complex biomedical problems. Attached Figure Description

[0026] Figure 1 is a schematic diagram of the structure of the frequency-marked dual- and three-photon synchronous excitation single-pixel detection microscopic imaging system in an embodiment of the present invention.

[0027] Figure 2 is a schematic diagram of laser pulse frequency marking in an embodiment of the present invention.

[0028] Figure 3 is a schematic diagram of laser pulse intensity modulation in an embodiment of the present invention.

[0029] Figure 4 is a schematic diagram of laser pulse intensity-related wavelength conversion in an embodiment of the present invention.

[0030] Figure 5 is a schematic diagram of single-pixel detection in an embodiment of the present invention. Embodiments of the present invention

[0031] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0032] Currently, the mainstream multiphoton imaging includes two-photon imaging and three-photon imaging. Depending on the specific requirements of medical imaging and the selected dye, the intensity of the two pulses can be controlled by the pulse intensity modulation module, thereby controlling the range of wavelength conversion, and two-photon-two-photon imaging, two-photon-three-photon imaging, or three-photon-three-photon imaging modes can be selected in sequence.

[0033] In one embodiment, a frequency-marked dual- and three-photon synchronous excitation single-pixel detection microscopic imaging system in a two-photon-three-photon imaging mode, as shown in Figure 1, includes a GHz-level high repetition rate femtosecond pulse laser source 101, a frequency marking module 102, a pulse compression and amplification module 103, a pulse modulation module 104, an intensity-correlated wavelength conversion module 105, an imaging module 106, a single-pixel optical signal acquisition component 107, and a data processing component 108 connected in sequence.

[0034] The laser pulses generated by the GHz-level high repetition rate femtosecond pulse laser source 101 are marked by a reference mark signal generated by a frequency marking module 102 to obtain a first laser pulse train and a second laser pulse train with a reference mark signal. The first laser pulse train and the second laser pulse train are then amplified in energy and compressed in pulse width by a pulse compression and amplification module 103.

[0035] The pulse intensity modulation module 104 modulates the intensity of the first and second laser pulse trains after energy amplification and pulse width compression, injects them into the intensity-related wavelength conversion module 105, and generates a set wavelength redshift through nonlinear effects before entering the imaging module 106. The imaging module 106 performs synchronous scanning of the sample using two-photon and three-photon excitation fluorescence imaging. During the imaging process, the generated light signal enters the single-pixel light signal acquisition component 107 to form an electrical signal. The electrical signal is sent to the data processing component 108 and, combined with the reference marker signal generated by the frequency marker module 102, synchronously demodulates and processes it to obtain a high-resolution, multi-contrast biological image based on single-pixel detection.

[0036] In one embodiment, the GHz-level high-repetition-rate femtosecond pulsed laser source 101 is an 1100nm all-fiber femtosecond pulsed laser with an output laser pulse repetition rate of 1GHz and a pulse width of 100fs. As an initial light source, it can emit laser pulses suitable for biological imaging.

[0037] In one embodiment, the frequency marking module 102 includes a programmable terminal, an arbitrary waveform generator, a radio frequency signal amplifier, and a first optical modulator connected in sequence; the first optical modulator is selected as an electro-optic modulator.

[0038] As shown in Figure 2, the programmable terminal controls the arbitrary waveform generator to send a reference marker radio frequency signal. After being amplified by an RF signal amplifier, the signal is applied to the first optical modulator to mark the frequency of the laser pulses at different times. This modulates the laser pulses generated by the GHz-level high-repetition-rate femtosecond pulse laser source 101 into periodic, cyclical frequency markers. and The first laser pulse train and the second laser pulse train; at this time, the center wavelengths of the first laser pulse train and the second laser pulse train are... .

[0039] As shown in Figure 2, there is a corresponding relationship between the wavelength redshift produced by the subsequent wavelength conversion and the marked frequency, which is marked as follows. and The laser pulse trains correspond to subsequent center wavelengths redshifted to and The laser pulse is used as a reference marker signal for marking. Used for final demodulation to generate imaging results.

[0040] In one embodiment, the pulse compression amplification module 103 includes an all-fiber amplifier and a grating pair connected in sequence. The all-fiber amplifier first amplifies the energy of the laser pulse train, and then the grating pair compresses the pulse width.

[0041] In one embodiment, the pulse intensity modulation module 104 includes a programmable terminal, an arbitrary waveform generator, an RF signal amplifier, and a second optical modulator connected in sequence; the second optical modulator is selected as an acousto-optic modulator.

[0042] The programmable terminal controls an arbitrary waveform generator to send the required radio frequency (RF) signal. After being amplified by an RF signal amplifier, the signal is applied to a second optical modulator to modulate the intensity of the first and second laser pulse trains, which have undergone energy amplification and pulse width compression. The frequencies are then marked as follows: The light intensity of the first laser pulse train is modulated to The frequency is marked as The light intensity of the second laser pulse train is modulated to The center wavelengths of the first and second laser pulse trains after intensity modulation are: .

[0043] Furthermore, the intensity-dependent wavelength conversion module 105 includes a lens and a photonic crystal fiber;

[0044] The intensity-dependent wavelength conversion module 105 utilizes intensity-dependent nonlinear effects to synchronously generate a set wavelength redshift for the first and second laser pulse trains after the light intensity modulation output from the pulse intensity modulation module 104 (W. Wang et al., 'High-speed wavelength-swept femtosecond source from 1055 to 1300 nm using a GHz femtosecond fiber laser', Opt. Lett., OL, vol. 47, no. 7, pp. 1677–1680, Apr. 2022, doi: 10.1364 / OL.449955.). Due to soliton self-frequency shift, the wavelength of the laser pulses shifts towards longer wavelengths. Different intensities of laser pulses will produce different nonlinear effects when passing through photonic crystal fibers. Therefore, the magnitude of the shift towards longer wavelengths differs between the two intensities. In one embodiment, the intensity-dependent wavelength conversion module 105 makes the intensity... The center wavelength of the first laser pulse train shifts to , 1200nm, the corresponding frequency is marked as The strength is The center wavelength of the second laser pulse train shifts to , 1300nm, the corresponding frequency is marked as As shown in Figure 4, synchronous dual-color laser pulses are generated.

[0045] In one embodiment, the imaging module 106 includes a relay optical path, an objective lens, a dichroic mirror, and a sample; the center wavelength is and The synchronous two-color laser pulse is collimated through the relay optical path and then transmitted through the dichroic mirror into the objective lens and focused on the sample. The sample is subjected to time- and space-synchronized two-photon and three-photon excitation, as shown in Figure 5. The light signal excited by the sample returns to the dichroic mirror and is reflected and output to the single-pixel light signal acquisition unit 107.

[0046] In one embodiment, the single-pixel optical signal acquisition component 107 is a photomultiplier tube with two channels suitable for different wavelengths, used to simultaneously receive optical signals generated by three-photon excitation and two-photon excitation, and convert them into electrical signals.

[0047] Furthermore, the data processing unit 108 includes a programmable terminal that synchronously demodulates and processes the collected electrical signals through the reference marker signal generated by the frequency marker module 102 to obtain a high-resolution, multi-contrast biological image based on single-pixel detection.

[0048] Furthermore, the frequency marking module 102, the pulse intensity modulation module 104, and the data processing unit 108 all use the same programmable terminal;

[0049] The frequency marking module 102 and the pulse intensity modulation module 104 use the same arbitrary waveform generator;

[0050] The frequency marking module 102 and the pulse intensity modulation module 104 use the same radio frequency signal amplifier.

[0051] The above embodiments are one of the implementation methods of the present invention, but the implementation methods of the present invention are not limited to the embodiments and test examples. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A frequency-marked dual- or three-photon synchronous excitation single-pixel detection microscopic imaging system, characterized in that, It includes a GHz-level high repetition rate femtosecond pulse laser source (101), a frequency marking module (102), a pulse compression and amplification module (103), a pulse modulation module (104), an intensity-related wavelength conversion module (105), an imaging module (106), a single-pixel optical signal acquisition component (107), and a data processing component (108) connected in sequence. The laser pulses generated by the GHz-level high repetition rate femtosecond pulse laser source (101) are marked by a reference mark signal generated by a frequency marking module (102) to obtain a first laser pulse train and a second laser pulse train with a reference mark signal. The first laser pulse train and the second laser pulse train are then amplified in energy and compressed in pulse width by a pulse compression and amplification module (103). The pulse intensity modulation module (104) modulates the intensity of the first and second laser pulse trains after energy amplification and pulse width compression, injects them into the intensity-related wavelength conversion module (105), and generates a set wavelength redshift through nonlinear effects before entering the imaging module (106). The imaging module (106) performs synchronous scanning of the sample with two-photon and three-photon excitation fluorescence imaging. The light signal generated during the imaging process enters the single-pixel light signal acquisition component (107) to form an electrical signal. The electrical signal is sent to the data processing component (108) and, combined with the reference marker signal generated by the frequency marking module (102), synchronously demodulates and processes it to obtain a biological image based on single-pixel detection.

2. The frequency-marked dual- or three-photon synchronous excitation single-pixel detection microscopic imaging system according to claim 1, characterized in that: The GHz-level high repetition rate femtosecond pulse laser source (101) is an all-fiber femtosecond pulse laser that serves as the initial light source, emitting pulse lasers with a repetition rate of gigahertz (GHz) suitable for biological imaging.

3. The frequency-marked dual- or three-photon synchronous excitation single-pixel detection microscopic imaging system according to claim 1, characterized in that: The frequency marking module (102) includes a programmable terminal, an arbitrary waveform generator, an RF signal amplifier, and a first optical modulator connected in sequence. Programmable terminal controls arbitrary waveform generator to send reference marker signal After being amplified by an RF signal amplifier, the signal is applied to the first optical modulator to mark the frequency of the laser pulses at different times. This modulates the laser pulses generated by the GHz-level high-repetition-rate femtosecond pulse laser source (101) into periodic cyclical frequency markers. and The first laser pulse train and the second laser pulse train; the center wavelengths of the first laser pulse train and the second laser pulse train are 。 4. The frequency-marked dual- or three-photon synchronous excitation single-pixel detection microscopic imaging system according to claim 1, characterized in that: The pulse compression amplification module (103) includes an all-fiber amplifier and a grating pair connected in sequence. First, the all-fiber amplifier amplifies the energy of the first and second laser pulse trains, and then the grating pair compresses the pulse width.

5. The frequency-marked dual- or three-photon synchronous excitation single-pixel detection microscopic imaging system according to claim 3, characterized in that: The pulse intensity modulation module (104) includes a programmable terminal, an arbitrary waveform generator, an RF signal amplifier, and a second optical modulator connected in sequence. The programmable terminal controls an arbitrary waveform generator to send the required radio frequency (RF) signal. After being amplified by an RF signal amplifier, the signal is applied to a second optical modulator to modulate the intensity of the first and second laser pulse trains, which have undergone energy amplification and pulse width compression. The frequencies are then marked as follows: The light intensity of the first laser pulse train is modulated to The frequency is marked as The light intensity of the second laser pulse train is modulated to The center wavelengths of the first and second laser pulse trains after intensity modulation are: 。 6. The frequency-marked dual- or three-photon synchronous excitation single-pixel detection microscopic imaging system according to claim 5, characterized in that: The intensity-dependent wavelength conversion module (105) includes a lens and a photonic crystal fiber; The intensity-dependent wavelength conversion module (105) utilizes an intensity-dependent nonlinear effect to synchronously generate a set wavelength redshift for the first and second laser pulse trains after the light intensity of the pulse intensity modulation module (104) is modulated, so that the intensity is The center wavelength of the first laser pulse train shifts to The corresponding frequency is marked as The strength is The center wavelength of the second laser pulse train shifts to The corresponding frequency is marked as 。 7. The frequency-marked dual- or three-photon synchronous excitation single-pixel detection microscopic imaging system according to claim 1, characterized in that: The imaging module (106) includes a relay optical path, an objective lens, a dichroic mirror, and a sample. The synchronous two-color laser pulse is collimated through the relay optical path and then transmitted through the dichroic mirror into the objective lens and focused on the sample. The sample is subjected to time- and space-synchronized two-photon excitation and three-photon excitation. The light signal excited by the sample returns to the dichroic mirror and is reflected and output to the single-pixel light signal acquisition component (107).

8. The frequency-marked dual- or three-photon synchronous excitation single-pixel detection microscopic imaging system according to claim 1, characterized in that: The single-pixel optical signal acquisition component (107) has two channels suitable for different wavelengths, which are used to simultaneously receive optical signals generated by three-photon excitation and two-photon excitation, and convert them into electrical signals.

9. The frequency-marked dual- or three-photon synchronous excitation single-pixel detection microscopic imaging system according to claim 1, characterized in that: The data processing unit (108) includes a programmable terminal that synchronously demodulates and processes the collected electrical signals through a reference marker signal generated by a frequency marker module (102) to obtain a biological image based on single-pixel detection.

10. The frequency-marked dual- or three-photon synchronous excitation single-pixel detection microscopic imaging system according to any one of claims 3, 5, and 9, characterized in that: The frequency marking module (102), pulse intensity modulation module (104), and data processing unit (108) use the same programmable terminal; The frequency marking module (102) and the pulse intensity modulation module (104) use the same arbitrary waveform generator; The frequency marking module (102) and the pulse intensity modulation module (104) use the same radio frequency signal amplifier.

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