Frequency-labeled double-photon and three-photon synchronous excitation single-pixel detection microscopic imaging system

Abstract

The invention discloses a frequency-labeled double-photon and three-photon synchronous excitation single-pixel detection microscopic imaging system. The system comprises a GHz magnitude high repetition frequency femtosecond pulse laser light source, a frequency marking module, a pulse compression 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. According to the invention, on the basis of a single-wavelength high-repetition-frequency femtosecond laser, multi-photon imaging is respectively carried out on different markers through two kinds of wavelength lasers which are simultaneously generated by radio frequency programming and soliton self-translation, so that time and space synchronous multi-dimensional microscopic observation is realized; the problem that time-space accurate synchronous explanation of multi-dimensional information cannot be achieved in an existing multi-modal imaging technology is solved, and the method has the advantages of being high in speed, convenient to use, low in cost and the like.

Description

Technical Field

[0001] The present invention relates to the fields of multiphoton biomedical imaging and femtosecond laser technology, and particularly to a frequency-labeled dual- and triple-photon synchronous excitation single-pixel detection microscopy imaging system. Background Art

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

[0003] When multiphoton imaging labels biological tissues with fluorescent dyes, the information that can be obtained in a single scan is relatively limited. Existing technologies for simultaneously performing two-photon and three-photon imaging mostly collect and process the signal lights of the two separately, and cannot achieve the effect of synchronous imaging. Moreover, the light sources used for imaging are not wavelength-tunable according to requirements (Li S.-Q. et al. New advances in biomedical applications of multiphoton imaging technology. Acta Phys. Sin. 69, 228702 (2020)). If different fluorescent dyes can be precisely synchronously excited in time and space, different biological tissue structures labeled with fluorescence based on multiphoton imaging can be collected simultaneously, and more accurate multi-dimensional biological information can be obtained, which is the key to comprehensively explaining biological problems. Such an imaging system can more comprehensively master the internal connections of biological tissues, more accurately analyze complex concurrent medical problems, and provide stronger technical support for biomedical research. Summary of the Invention

[0004] The problems to be solved by the present invention are: the time to obtain multi-dimensional information is relatively long, and the information obtained in a single scan is limited.

[0005] The frequency-labeled dual- and triple-photon synchronous excitation single-pixel detection microscopy imaging system proposed by the present invention will provide a solution to the above problems. Two kinds of laser beams with different wavelengths are simultaneously generated by radio frequency programmable and soliton self-translation based on a single-wavelength high-repetition-rate femtosecond laser to perform multiphoton imaging on different markers respectively.

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

[0007] Frequency-labeled dual- and triple-photon synchronous excitation single-pixel detection microscopy system, including a GHz-level high-repetition-rate femtosecond pulsed laser source, a frequency-labeling module, a pulse compression and amplification module, a pulse modulation module, an intensity-dependent 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 pulsed laser source are frequency-labeled by the reference label signal generated by the frequency-labeling module to obtain a first laser pulse train and a second laser pulse train with the reference label signal, and the first laser pulse train and the second laser pulse train are sequentially subjected to energy amplification and pulse width compression through the pulse compression and amplification module;

[0009] The pulse intensity modulation module performs intensity modulation on the first laser pulse train and the second laser pulse train after energy amplification and pulse width compression, injects them into the intensity-dependent wavelength conversion module, generates a set wavelength redshift through the nonlinear effect and then enters the imaging module; the imaging module performs synchronous scanning dual-photon and triple-photon excitation fluorescence imaging on the sample, and the light signal generated during the imaging process enters the single-pixel optical signal acquisition component to form an electrical signal, and the electrical signal is sent to the data processing component to be synchronously demodulated and processed in combination with the reference label radio frequency signal generated by the frequency-labeling module to obtain a biological image with high resolution and multi-contrast mechanism based on single-pixel detection.

[0010] Further, the GHz-level high-repetition-rate femtosecond pulsed laser source is an all-fiber femtosecond pulsed laser, which is used as the initial light source and emits pulsed laser with a repetition rate of gigahertz level suitable for biological imaging.

[0011] Further, the frequency-labeling module includes a programmable terminal, an arbitrary waveform generator, a radio frequency signal amplifier, and a first optical modulator connected in sequence;

[0012] The programmable terminal controls the arbitrary waveform generator to send a reference standard frequency signal M1, which is amplified by the radio frequency signal amplifier and then loaded on the first optical modulator to realize the frequency labeling of laser pulses at different times, and modulates the laser pulses generated by the GHz-level high-repetition-rate femtosecond pulsed laser source into a first laser pulse train and a second laser pulse train with periodic cyclic frequency labels of f1 and f2 respectively; at this time, the first laser pulse train and the second laser pulse train; the first laser pulse train and the second laser pulse train have a central wavelength of λ0.

[0013] Further, the pulse compression and amplification module includes an all-fiber amplifier and a pair of gratings connected in sequence. First, the laser pulse train is amplified in energy through the all-fiber amplifier, and then the pulse width is compressed through the pair of gratings.

[0014] Further, in the pulse intensity modulation module, it includes a programmable terminal, an arbitrary waveform generator, a radio frequency signal amplifier, and a second optical modulator connected in sequence;

[0015] The programmable terminal controls the arbitrary waveform generator to send the required radio frequency signal. After being amplified by the radio frequency signal amplifier, it is loaded on the second optical modulator to perform intensity modulation on the first laser pulse train and the second laser pulse train after energy amplification and pulse width compression. The optical intensity of the first laser pulse train with the frequency marked as f1 is modulated to I1, and the optical intensity of the second laser pulse train with the frequency marked as f2 is modulated to I2. The central wavelengths of the first laser pulse train and the second laser pulse train after optical intensity modulation are λ0.

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

[0017] The intensity-dependent wavelength conversion module uses the intensity-dependent nonlinear effect to synchronously generate a set wavelength redshift for the first laser pulse train and the second laser pulse train after optical intensity modulation output by 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.). The central wavelength of the first laser pulse train with the intensity of I1 moves to λ1, corresponding to the frequency marked as f1; the central wavelength of the second laser pulse train with the intensity of I2 moves to λ2, corresponding to the frequency marked as f2.

[0018] Further, the imaging module includes a relay optical path, an objective lens, a dichroic mirror, and a sample; the synchronous two-color laser pulses are collimated by the relay optical path and then transmitted through the dichroic mirror and focused on the sample by the objective lens to perform two-photon excitation and three-photon excitation on the sample in time and space synchronization. The optical signal excited from the sample returns to the dichroic mirror and is reflected and output to a single-pixel optical signal acquisition component.

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

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

[0021] Further, the frequency marking module, the pulse intensity modulation module, and the data processing component 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 the existing technologies, the beneficial effects of the present invention are as follows:

[0025] Based on a single-wavelength high-repetition-rate femtosecond laser, the present invention simultaneously generates two-wavelength lasers through radio frequency programmability and soliton self-translation to perform multi-photon imaging on different markers respectively, realizing time and space synchronous multi-dimensional microscopic observation, solving the problem that the multi-dimensional information of the existing multi-modal imaging technologies cannot be accurately synchronously interpreted in time and space, and having the advantages of fast speed, convenience, low cost, etc., providing strong support for complex biomedical problems. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

[0030] Figure 5 It is a schematic diagram of single-pixel point detection in an embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0031] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention.

[0032] Currently, the mainstream multi-photon imaging includes two-photon imaging and three-photon imaging. According to the specific requirements of medical imaging and the selected dyes, the intensity of two pulses can be controlled through the pulse intensity modulation module to control the wavelength conversion range, and the 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, in a two-photon - three-photon imaging mode, a frequency-tagged two- and three-photon synchronous excitation single-pixel detection microscopy system, such as Figure 1 shown, includes a GHz-order high-repetition-rate femtosecond pulsed laser light source 101, a frequency tagging module 102, a pulse compression and amplification module 103, a pulse modulation module 104, an intensity-dependent 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-order high-repetition-rate femtosecond pulsed laser light source 101 are frequency-tagged by the reference tagging signals generated by the frequency tagging module 102 to obtain a first laser pulse train and a second laser pulse train with reference tagging signals, and the first laser pulse train and the second laser pulse train are sequentially subjected to energy amplification and pulse width compression by the pulse compression and amplification module 103;

[0035] The pulse intensity modulation module 104 performs intensity modulation on the first laser pulse train and the second laser pulse train after energy amplification and pulse width compression, injects them into the intensity-dependent wavelength conversion module 105, generates a set wavelength redshift through non-linear effects and then enters the imaging module 106; the imaging module 106 performs synchronous scanning two-photon and three-photon excitation fluorescence imaging on the sample, the optical signals generated during the imaging process enter the single-pixel optical signal acquisition component 107 to form electrical signals, and the electrical signals are sent to the data processing component 108 for synchronous demodulation processing in combination with the reference tagging signals generated by the frequency tagging module 102 to obtain a biological image based on single-pixel detection with high resolution and multi-contrast mechanisms.

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

[0037] In one embodiment, the frequency tagging 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 an electro-optic modulator.

[0038] As Figure 2As shown, the programmable terminal controls the arbitrary waveform generator to send the reference marker radio frequency signal M1. After being amplified by the radio frequency signal amplifier, it is loaded onto the first optical modulator to achieve frequency marking of laser pulses at different times, modulating the laser pulses generated by the GHz-order high-repetition-rate femtosecond pulse laser source 101 into the first laser pulse train and the second laser pulse train with frequency markings of f1 and f2 respectively in a periodic cycle; at this time, the first laser pulse train and the second laser pulse train; the central wavelengths of the first laser pulse train and the second laser pulse train are λ0.

[0039] As Figure 2 shown, there is a corresponding relationship between the wavelength redshift generated by subsequent wavelength conversion and the marking frequency. The laser pulse trains with frequency markings of f1 and f2 respectively correspond to the laser pulses with subsequent central wavelengths redshifted to λ1 and λ2, and the reference marker signal M1 used for marking is used for final demodulation to generate the imaging result.

[0040] In one embodiment, the pulse compression and amplification module 103 includes a fully fiber optic amplifier and a pair of gratings connected in sequence. First, the fully fiber optic amplifier is used to amplify the energy of the laser pulse train, and then the pulse width is compressed by the pair of gratings.

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

[0042] The programmable terminal controls the arbitrary waveform generator to send the required radio frequency signal. After being amplified by the radio frequency signal amplifier, it is loaded onto the second optical modulator to perform intensity modulation on the first laser pulse train and the second laser pulse train after energy amplification and pulse width compression, modulating the optical intensity of the first laser pulse train with frequency marking f1 to I1, and modulating the optical intensity of the second laser pulse train with frequency marking f2 to I2. The central wavelengths of the first laser pulse train and the second laser pulse train after optical intensity modulation are λ0.

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

[0044] The intensity-dependent wavelength conversion module 105 synchronously generates a set wavelength redshift for the first and second laser pulse trains output by the pulse intensity modulation module 104 by using intensity-dependent nonlinear effects (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.). Since the soliton self-frequency shift causes the wavelength of the laser pulse to shift towards the long-wavelength direction, different intensities of laser pulses passing through the photonic crystal fiber will produce different nonlinear effects. Therefore, the amplitudes of the two intensities of laser pulses shifting towards the long-wavelength direction are different. In one embodiment, the intensity-dependent wavelength conversion module 105 shifts the central wavelength of the first laser pulse train with intensity I1 to λ1, where λ1 = 1200 nm, and the corresponding frequency is marked as f1; the central wavelength of the second laser pulse train with intensity I2 is shifted to λ2, where λ2 = 1300 nm, and the corresponding frequency is marked as f2, as Figure 4 shown, to generate synchronous two-color laser pulses.

[0045] In one embodiment, the imaging module 106 includes a relay optical path, an objective lens, a dichroic mirror, and a sample; the synchronous two-color laser pulses with central wavelengths λ1 and λ2 are collimated by the relay optical path and then transmitted through the dichroic mirror and focused on the sample by the objective lens to perform time- and space-synchronized two-photon excitation and three-photon excitation on the sample, as Figure 5 shown, and the optical signal excited from the sample returns to the dichroic mirror and is reflected and output to the single-pixel optical signal acquisition component 107.

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

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

[0048] Furthermore, the frequency marking module 102, the pulse intensity modulation module 104, and the data processing component 108 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 manners of the present invention, but the implementation manners of the present invention are not limited by the said embodiments and test examples. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement manners and are all included in the protection scope of the present invention.

Claims

1. Frequency-tagged two- and three-photon synchronous excitation single-pixel detection microscopy system, characterized in that, It includes a femtosecond pulse laser source (101) with a high repetition rate in the GHz range, a frequency marking module (102), a pulse compression and amplification module (103), a pulse modulation module (104), an intensity-dependent 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 femtosecond pulse laser source (101) with a high repetition rate in the GHz range are frequency marked by the reference marking signals generated by the frequency marking module (102) to obtain a first laser pulse train and a second laser pulse train with reference marking signals. The first laser pulse train and the second laser pulse train are sequentially subjected to energy amplification and pulse width compression by the pulse compression and amplification module (103); The pulse intensity modulation module (104) performs intensity modulation on the first laser pulse train and the second laser pulse train after energy amplification and pulse width compression, injects them into the intensity-dependent wavelength conversion module (105), and generates a set wavelength redshift through non-linear effects and then enters the imaging module (106); The imaging module (106) performs synchronous scanning two-photon and three-photon excitation fluorescence imaging on the sample. The optical signals generated during the imaging process enter the single-pixel optical signal acquisition component (107) to form electrical signals, and the electrical signals are sent to the data processing component (108) for synchronous demodulation processing in combination with the reference marking signals generated by the frequency marking module (102) to obtain a biological image based on single-pixel detection.

2. The frequency-labeled dual- and triple-photon synchronous excitation single-pixel detection microscopy imaging system according to claim 1, wherein: The femtosecond pulse laser source (101) with a high repetition rate in the GHz range 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 (GHz) range suitable for biological imaging.

3. The frequency-labeled dual- and triple-photon synchronous excitation single-pixel detection microscopy imaging system according to claim 1, characterized in that: 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 programmable terminal controls the arbitrary waveform generator to send a reference marking signal M1, which is amplified by the radio frequency signal amplifier and then loaded on the first optical modulator to achieve frequency marking of laser pulses at different times, and modulates the laser pulses generated by the femtosecond pulse laser source (101) with a high repetition rate in the GHz range into a first laser pulse train and a second laser pulse train with periodical cycles and frequency markings of f1 and f2 respectively; The central wavelengths of the first laser pulse train and the second laser pulse train are λ0.

4. The frequency-tagged two- and three-photon synchronous excitation single-pixel detection microscopy imaging system according to claim 1, wherein: The pulse compression and amplification module (103) includes an all-fiber amplifier and a pair of gratings connected in sequence. First, the first and second laser pulse trains are subjected to energy amplification by the all-fiber amplifier, and then the pulse width is compressed by the pair of gratings.

5. The frequency-tagged two- and three-photon synchronous excitation single-pixel detection microscopy imaging system according to claim 3, characterized in that: In the pulse intensity modulation module (104), it includes a programmable terminal, an arbitrary waveform generator, a radio frequency signal amplifier, and a second optical modulator connected in sequence; The programmable terminal controls the arbitrary waveform generator to send the required radio frequency signal. After being amplified by the radio frequency signal amplifier, it is loaded on the second optical modulator to intensity-modulate the first laser pulse train and the second laser pulse train after energy amplification and pulse width compression. The optical intensity of the first laser pulse train with the frequency marked as f1 is modulated to I1, and the optical intensity of the second laser pulse train with the frequency marked as f2 is modulated to I2. The central wavelengths of the first laser pulse train and the second laser pulse train after optical intensity modulation are λ0.

6. The frequency-tagged two- and three-photon synchronous excitation single-pixel detection microscopy 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) uses the intensity-dependent nonlinear effect to synchronously generate a set wavelength redshift for the first laser pulse train and the second laser pulse train intensity-modulated by the pulse intensity modulation module (104), so that the central wavelength of the first laser pulse train with the intensity of I1 moves to λ1, corresponding to the frequency marked as f1; the central wavelength of the second laser pulse train with the intensity of I2 moves to λ2, corresponding to the frequency marked as f2.

7. The frequency-tagged two- and three-photon synchronous excitation single-pixel detection microscopy imaging system according to claim 1, wherein: The imaging module (106) includes a relay optical path, an objective lens, a dichroic mirror, and a sample. The synchronous two-color laser pulses are collimated by the relay optical path and then transmitted through the dichroic mirror and focused on the sample by the objective lens for two-photon excitation and three-photon excitation that are time and space synchronous. The optical signal excited from the sample returns to the dichroic mirror and is reflected and output to the single-pixel optical signal acquisition component (107).

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

9. The frequency-tagged dual- and three-photon synchronous excitation single-pixel detection microscopy imaging system according to claim 1, wherein: The data processing component (108) includes a programmable terminal, which synchronously demodulates and processes the collected electrical signals through the reference marking signal generated by the frequency marking module (102) to obtain a biological image based on single-pixel detection.

10. The frequency-labeled two- and three-photon synchronous excitation single-pixel detection microscopy imaging system according to any one of claims 3, 5, and 9, characterized in that: The frequency marking module (102), the pulse intensity modulation module (104), and the data processing component (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.

Images