WO2023029471A1 - Multi-modal nonlinear microscopic imaging system - Google Patents

Abstract

A multi-modal nonlinear microscopic imaging system, comprising a signal collection apparatus and an imaging processing terminal (31), wherein the signal collection apparatus comprises: an excitation light path module (101) for arranging and exciting laser light in different nonlinear modals at different times; a sample-bearing sheet (36) for bearing a sample; a focusing module (102) for focusing the laser light from the excitation light path module onto the sample on the sample-bearing sheet (36); a transmission-light processing module (103) for processing transmission light that passes through the sample from an excitation light path, and detecting an excited Raman signal; and a reflected-light processing module (104) for detecting reflected light that is generated by the sample on the sample-bearing sheet (36) via the excitation light path, so as to obtain a detection result.

Description

A Multimodal Nonlinear Microscopic Imaging System technical field

The invention relates to the field of optical microscopic imaging, in particular to a multi-mode non-linear microscopic imaging system.

Background technique

Nonlinear optical imaging techniques, mainly including second harmonic, third harmonic, two-photon excited fluorescence, three-photon excited fluorescence, and coherent Raman scattering, have become powerful tools for studying complex biochemical processes. As an imaging method to detect the microenvironment of biological tissues, fluorescence lifetime microscopy provides another dimension of information for biomedical research.

technical problem

Since various nonlinear optical imaging techniques require different excitation conditions, coherent Raman microscopy imaging needs to use narrow-linewidth picosecond lasers and optical parametric oscillator laser sources to obtain good spectral resolution, and picosecond light sources lead to two-photon The low excitation efficiency of excitation fluorescence and harmonic imaging leads to poor imaging signal-to-noise ratio and contrast, further affecting imaging resolution.

For two-photon fluorescence lifetime microscopy imaging, the low excitation efficiency makes photon accumulation slow, resulting in severe photobleaching and photodamage, which seriously affects the accuracy of fluorescence lifetime measurement. In addition, both coherent Raman microscopy and fluorescence lifetime microscopy require multiple scanning imaging to obtain Raman spectra and fluorescence lifetime decay curves. Due to the synchronization of excitation conditions and image acquisition, the simultaneous acquisition and microscopic imaging of nonlinear imaging such as coherent Raman and two-photon imaging and fluorescence lifetime imaging have not yet been realized.

technical solution

The main purpose of the present invention is to provide a multi-modal non-linear microscopic imaging system, aiming to solve the problem of synchronous acquisition and microscopic imaging of non-linear imaging such as coherent Raman and two-photon imaging and fluorescence lifetime imaging in the prior art technical problem.

Beneficial effect

To achieve the above object, the present invention provides a multi-modal non-linear microscopic imaging system, which includes a signal acquisition device and an imaging processing terminal. The signal acquisition device includes: an excitation optical path module, which is used to arrange and excite different non-linear Laser in linear mode; sample carrier sheet, used to carry samples; focusing module, used to focus the laser light excited by the excitation optical path module to the sample on the sample carrier sheet; transmitted light processing module, used to process the laser light The optical path passes through the transmitted light of the sample carrier to detect the stimulated Raman signal; the reflected light processing module is used to detect the reflected light generated by the sample on the sample carrier through the excitation optical path to obtain the detection result.

Wherein, the excitation optical path module includes: a femtosecond laser, used to generate at least two beams of femtosecond-level laser light; a first half-wave plate, used to change the linear polarization angle of a beam of femtosecond laser generated laser light; the first polarization division A beam splitter is used to divide the laser light passing through the first half-wave plate into two beams, and adjust the laser power in combination with the first half-wave plate; the second half-wave plate is used to change the laser beam generated by another femtosecond laser The linear polarization angle; the second polarization beam splitter is used to adjust the laser power passing through the second half-wave plate in combination with the second half-wave plate; the first time delay unit is used to adjust the splitting of the first polarization beam splitter The optical path of a beam of laser light emitted by the first polarizing beam splitter; the first glass rod is used to chirp and broaden the other beam of laser light split by the first polarizing beam splitter into a picosecond beam; the second glass rod is used to A beam of laser light that passes through the second polarization beam splitter is chirped and broadened into a picosecond beam; the third polarization beam splitter is used to combine the laser light that passes through the first glass rod and the light that passes through the first time delay unit The laser is polarized coupled; the second time delay unit is used to adjust the optical path of the laser light path coupled by the third polarization beam splitter, so as to realize Raman frequency shift spectral scanning; The intensity of the laser light from the second glass rod is modulated; the first dichroic mirror is used to couple the laser light passing through the second time delay unit and the laser light modulated by the electro-optic modulator into one beam, and transmit it to the focusing module .

Wherein, the first time delay unit includes: a first mirror and a second mirror, and are symmetrically placed on the displacement stage; the first mirror is used to reflect a beam of laser light split by the first polarization beam splitter to the The second reflector; the second reflector is used to reflect the laser light reflected by the first reflector to the third polarization beam splitter; the second time delay unit includes: a third reflector, a fourth reflector , the fifth reflector and the sixth reflector are both used to reflect laser light, the fourth reflector and the fifth reflector are symmetrically placed on the displacement stage, and the laser light coupled by the third polarization beam splitter passes through the third polarized beam splitter in sequence reflective mirror, fourth reflective mirror, fifth reflective mirror and sixth reflective mirror, transmitted to the first dichroic mirror.

Wherein, the focusing module includes: a scanning galvanometer, a scanning lens, a sleeve lens, and an objective lens; the laser light excited by the excitation optical path module is focused on the sample through the scanning galvanometer, scanning lens, sleeve lens, and objective lens in sequence. Samples on a carrier sheet.

Wherein, the transmitted light processing module includes: a condenser, a first lens, a third filter, a photodiode and a lock-in amplifier; the transmitted light passes through the condenser, the first lens and the filter in sequence, and is captured by the The photodiode detects and generates a first photoelectric signal, and the lock-in amplifier is used to demodulate the first photoelectric signal to generate a stimulated Raman signal.

Wherein, the objective lens is also used to collect reflected light; the reflected light processing module includes: a second lens, a beam splitter, a first filter, a second filter, a multi-wavelength splitting module, and a multi-channel photomultiplier tube , a time-correlated single photon counting module, a second dichroic mirror, a third dichroic mirror, and a photomultiplier tube; the second dichroic mirror is arranged between the sleeve lens and the objective lens for Reflecting the nonlinear signal generated by the excitation light path through the sample to the second lens, and transmitting the excitation light to prevent it from reaching the second lens; the second lens is used to focus the reflected light reflected by the second dichroic mirror The beam splitter is used to split the reflected light focused by the second lens, one beam is transmitted to the first filter, and the other beam is transmitted to the third dichroic mirror; the multi-wavelength light splitting module It is used to perform fluorescence optical dispersion on the reflected light passing through the first filter to realize the spatial distribution of different wavelengths; the multi-channel photomultiplier tube is used to detect the difference of the reflected light passing through the multi-wavelength splitting module. Fluorescent signals in different bands; the time-correlated single-photon counting module is used to count photons of the fluorescent signals to realize multi-wavelength fluorescence lifetime detection; the photomultiplier tube is used to transmit to the third dichroic mirror, and pass through The reflected light of the second optical filter is used to detect the harmonic signal.

Wherein, the system further includes: an acquisition card; the acquisition card is used to control the scanning oscillating mirror of the focusing module and the acquisition of the lock-in amplifier output signal and the PMT output signal of the transmitted light processing module, and realize the scanning oscillating The scanning of the mirror is synchronized with the acquisition of the acquisition card, and the pixel pulse, row pulse and frame pulse of the scanning signal are synchronously output, and the three-way pulse of the pixel pulse, row pulse and frame pulse is transmitted to the reflected light processing module. A time-correlated single-photon counting module to realize the synchronization of fluorescence lifetime signal acquisition and scanning; and control the second time delay unit of the excitation optical path module to realize spectral scanning during the stimulated Raman imaging process.

Wherein, the number of channels of the multi-wavelength spectroscopic module and the multi-channel photomultiplier tube is 1-16, so as to realize the fluorescence lifetime detection of 1-16 bands.

Wherein, the laser in the excitation optical path module outputs at least two laser beams at the same time, both of which have a pulse width of 100 fs, and the wavelength of one laser beam is 1040 nm, which is transmitted to the second half-wave plate, and the wavelength of the other laser beam is 680 - 1300 nm, transmitted to the first half-wave plate.

Wherein, the scanning galvanometer includes: a resonant mirror in the X direction and a galvanometer in the Y direction, or a first galvanometer in the X direction and a second galvanometer in the Y direction.

The invention provides a multi-modal nonlinear microscopic imaging system, which has the beneficial effect of being able to use the excitation optical path module to arrange and excite lasers of different nonlinear modes at different times, and when the acquisition process of various imaging modes is synchronized , which can realize multi-modal microscopic imaging at the same time, so as to accurately obtain multi-dimensional and multi-parameter information of the sample, and greatly improve the imaging speed.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention. For those skilled in the art, other drawings can also be obtained according to these drawings without creative work.

Fig. 1 is a schematic block diagram of the structure of a multi-modal non-linear microscopic imaging system according to an embodiment of the present application;

FIG. 2 is a schematic structural diagram of a multi-modal nonlinear microscopic imaging system according to an embodiment of the present application.

BEST MODE FOR CARRYING OUT THE INVENTION

In order to make the purpose, features and advantages of the present invention more obvious and understandable, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described The embodiments are only some of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative efforts belong to the protection scope of the present invention.

Please refer to FIG. 1 , which is a multimodal nonlinear microscopic imaging system, including: a signal acquisition device and an imaging processing terminal 31 , the signal acquisition device is used to collect imaging signals, and the processing terminal 31 is used to perform imaging according to the imaging signals.

The signal acquisition device includes: an excitation optical path module 101, a sample carrier 36, a focusing module 102, a transmitted light processing module 103, and a reflected light processing module 104; the excitation optical path module 101 is used to arrange and excite lasers of different nonlinear modes at different times The sample carrier 36 is used to carry the sample; the focusing module 102 is used to focus the laser light excited by the excitation optical path module 101 to the sample on the sample carrier 36; The transmitted light is used to detect the stimulated Raman signal; the reflected light processing module 104 is used to detect the reflected light generated by the sample on the sample carrier 36 through the excitation light path to obtain the detection result.

In this embodiment, the transmitted light processing module 103 and the reflected light processing module 104 are electrically connected to the processing terminal 31, and the processing terminal 31 is based on the Stimulated Raman signal detected by the transmitted light processing module 103 and the detected signal obtained by the reflected light processing module 104. As a result, image reconstruction is performed.

The multi-modal nonlinear microscopic imaging system provided in this embodiment can use the excitation optical path module 101 to arrange and excite lasers of different nonlinear modes at different times, and when the acquisition process of various imaging modes is synchronized, it can At the same time, multi-modal microscopic imaging is realized, so as to accurately obtain multi-dimensional and multi-parameter information of the sample, and greatly improve the imaging speed.

In this embodiment, the terminals described in this application may include mobile phones, tablet computers, notebook computers, palmtop computers, personal digital assistants (Personal Digital Assistant, PDA for short), portable media players (Portable Media Player, PMP for short), ), navigation devices, wearable devices, smart bracelets and other mobile terminals, as well as fixed terminals such as digital TVs and desktop computers.

In one embodiment, the excitation optical path module 101 includes: a femtosecond laser 1, a first half-wave plate 2, a first polarization beam splitter 4, a second half-wave plate 3, a second polarization beam splitter 5, a first time Delay unit 8 , first glass rod 6 , second glass rod 7 , third polarizing beam splitter 9 , second time delay unit 10 , electro-optic modulator 11 and first dichroic mirror 12 .

Wherein, the femtosecond laser 1 is used to produce at least two femtosecond-level lasers; the first half-wave plate 2 is used to change the linear polarization angle of a beam of femtosecond laser 1; the first polarization beam splitter 4 is used to The laser light passing through the first half-wave plate 2 is divided into two beams, and the laser power is adjusted in combination with the first half-wave plate 2; the second half-wave plate 3 is used to change the linear polarization angle of the laser light generated by the other femtosecond laser 1; The second polarization beam splitter 5 is used to adjust the laser power passing through the second half-wave plate 3 in combination with the second half-wave plate 3; the first time delay unit 8 is used to adjust a beam split by the first polarization beam splitter 4 The optical path of the laser light path; the first glass rod 6 is used to broaden another beam of laser chirp split by the first polarizing beam splitter 4 into a picosecond beam; the second glass rod 7 is used to A beam of laser light chirped by the beam device 5 is broadened into a picosecond beam; the third polarizing beam splitter 9 is used to polarize the laser light passing through the first glass rod 6 and the laser light passing through the first time delay unit 8; The second time delay unit 10 is used to adjust the optical path of the laser light path coupled by the third polarization beam splitter 9 to realize Raman frequency shift spectral scanning; the electro-optic modulator 11 is used to pass through the second glass rod 7 according to the modulation signal The intensity of the laser beam is modulated; the first dichroic mirror 12 is used to couple the laser beam passing through the second time delay unit 10 and the laser beam modulated by the electro-optic modulator 11 into one beam, and transmit it to the focusing module 102 .

In this embodiment, after the femtosecond laser 1 synchronously outputs two femtosecond pulsed lasers, the first half-wave plate 2 and the second half-wave plate 3 change the linear polarization angles of the lasers respectively, and then the two lasers pass through the first and second half-wave plates respectively. A polarizing beam splitter 4 and a second polarizing beam splitter 5 adjust the power, wherein the laser beam passing through the first polarizing beam splitter 4 is divided into two beams, and the laser beams divided into two beams are all femtosecond-level lasers, One beam passes through the first time delay unit 8 and then the second time delay unit 10 to adjust the propagation time of the laser beam, and the other beam passes through the first glass rod 6 and is chirped and broadened into picosecond-level laser light. The laser beams merge at the third polarizing beam splitter 9 and are coupled into a beam of laser light by the third polarizing beam splitter 9; the laser light passing through the second polarizing beam splitter 5 passes through the second glass rod 7 and is chirped The laser beam broadened to a picosecond level is then passed through the electro-optic modulator 11 to modulate the intensity of the excitation light; the three-way laser beam of the pixel pulse, row pulse and frame pulse after passing through the third polarizing beam splitter 9 and the electro-optic modulator 11 is The first dichroic mirror 12 converges and is coupled by the first dichroic mirror 12 into a beam of laser light, which is transmitted to the focusing module 102 .

Wherein, when the second glass rod 7 broadens femtosecond-level laser chirp to picosecond-level laser, the picosecond pulse width depends on the length of the glass rod and the dispersion coefficient of the glass rod, and grating pairs, prism pairs can be used , optical fiber, etc. to achieve linear chirping of femtosecond pulses. In different cases, the glass rod can be replaced to perform different linear chirping of the laser.

In this embodiment, the multimodal nonlinear microscopic imaging system further includes a signal generator 24, the signal generator 24 is electrically connected to the electro-optical modulator 11, and is used to generate a modulation signal, and transmit the modulation signal to the electro-optic modulator 11 The electro-optic modulator 11 is used for the intensity modulation of the Stokes light, and its modulation signal is generated by a signal generator 24, and the modulation frequency is 20 MHz, which is amplified by a voltage amplifier to obtain a larger modulation depth; in addition, before the electro-optic modulator 11 Equipped with a half-wave plate and a quarter-wave plate, the half-wave plate is used to change the angle between the linearly polarized light and the optical axis to be 45°, and the circularly polarized light after passing through the quarter-wave plate to reduce the required modulation signal voltage amplitude.

In one embodiment, the first time delay unit 8 includes: a first reflector and a second reflector; the first reflector is used to reflect a beam of laser light split by the first polarization beam splitter 4 to the second reflector mirror; the second mirror is used to reflect the laser light reflected by the first mirror to the third polarizing beam splitter 9 .

The first reflecting mirror and the second reflecting mirror are symmetrically placed on the displacement stage, and the optical path of the laser light can be adjusted through the first reflecting mirror and the second reflecting mirror, thereby adjusting the transmission time of the laser light.

The second time delay unit 10 includes: a third reflector, a fourth reflector, a fifth reflector and a sixth reflector, all used to reflect laser light, the fourth reflector and the fifth reflector are symmetrically placed on the displacement stage, The laser light reflected by the first time delay unit 8 is transmitted to the first dichroic mirror 12 through the third mirror, the fourth mirror, the fifth mirror and the sixth mirror in sequence.

In this embodiment, the principle of the second time delay unit 10 is the same as that of the first time delay unit 8, and also uses two reflectors to adjust the optical path of the laser, thereby adjusting the transmission time of the laser; wherein, the first time The positions of the two mirrors of the delay unit 8 and the second time delay unit 10 can be adjusted to increase or decrease the laser light path length of the first time delay unit 8 or the second time delay unit 10 and adjust the transmission time of the laser.

In one embodiment, the focusing module 102 includes: a scanning galvanometer 13, a scanning lens 14, a sleeve lens 15, and an objective lens 17; the laser light excited by the excitation optical path module 101 passes through the scanning galvanometer 13, the scanning lens 14, and the sleeve lens 15 in sequence. And the objective lens 17 focuses on the sample on the sample holder 36.

After the two-way lasers synchronously output by the femtosecond laser 1 pass through the third polarization beam splitter 9 and the electro-optic modulator 11 respectively, the two-way lasers converge at the first dichroic mirror 12 and are absorbed by the first dichroic mirror 12 After being coupled into a beam of laser light, it is transmitted to the scanning galvanometer 13, and then through the scanning lens 14, the sleeve lens 15 and the objective lens 17, the laser light is focused on the sample carried by the carrier sheet. During the laser focusing process, the scanning lens 14 and the sleeve lens The tube lens 15 expands the laser beam so that its size is equal to the size of the entrance pupil of the objective lens 17, and then a part of the laser light, that is, the transmitted light passes through the sample and the carrier, and the laser and the sample are backscattered by the nonlinear signal of the excitation beam Reflected, that is, the reflected light is reflected by the sample to the objective lens 17.

In one embodiment, the transmitted light processing module 103 includes: a condenser 18, a first lens 20, a third filter 21, a photodiode 22 and a lock-in amplifier 23; the transmitted light passes through the condenser 18, the first lens 20 and the filter in sequence The light sheet is detected by the photodiode 22 to generate a first photoelectric signal, and the lock-in amplifier 23 is used to demodulate the first photoelectric signal to generate a stimulated Raman signal.

In this embodiment, after the laser beam is irradiated on the sample carried by the carrier sheet, the transmitted light is detected by the photodiode 22 after passing through the condenser lens 18, the first lens 20 and the third filter 21, and then resolved by the lock-in amplifier 23. During this process, the condenser lens 18 collects the forward scattering signal passing through the sample, the first lens 20 focuses the detection beam to the photosensitive surface of the detector, and the third filter 21 is used to filter out To detect light beams other than light, the photodiode 22 realizes the detection of the light beams, and the lock-in amplifier 23 realizes the demodulation of Raman signals.

When the condenser lens 18 collects the forward scattering signal passing through the sample, the numerical aperture of the condenser lens 18 is greater than or equal to the numerical aperture of the excitation objective lens 17; The first lens 20 is a large-size lens, and the number of the first lenses 20 can also be increased to improve the spot focusing effect; a high OD filter is used between the first lens 20 and the detector to filter out the modulated light beam, which can Increase the number of filters to reduce the transmittance of the modulated beam.

In one embodiment, the objective lens 17 is also used to collect reflected light; the reflected light processing module 104 includes: a second lens 25, a beam splitter 32, a first filter 26, a second filter 34, a multi-wavelength light splitting module 27. A multi-channel photomultiplier tube 28, a time-correlated single photon counting module 29, a second dichroic mirror 16, a third dichroic mirror 33, and a photomultiplier tube 35.

Wherein, the second dichroic mirror 16 is arranged between the sleeve lens 15 and the objective lens 17, and is used for reflecting the nonlinear signal generated by the excitation light through the sample to the second lens 25, and transmitting the excitation light to prevent it from reaching the second lens 25 The second lens 25 is used to focus the reflected light reflected by the second dichroic mirror 16; the beam splitter 32 is used to split the reflected light focused by the second lens 25, and one beam is transmitted to the first filter 26, The other beam is transmitted to the third dichroic mirror 33; the multi-wavelength splitting module 27 is used to perform fluorescence optical dispersion on the reflected light passing through the first optical filter 26, so as to realize the spatial distribution of different wavelengths; the multi-channel photomultiplier tube 28 is used to detect the fluorescent signals of different bands of the reflected light passing through the multi-wavelength spectroscopic module 27; the time-correlated single-photon counting module 29 is used to count the photons of the fluorescent signals to realize multi-wavelength fluorescence lifetime detection; the photomultiplier tube 35 is used The harmonic signal is detected on the reflected light transmitted to the third dichroic mirror 33 and passed through the second filter 34 .

In this embodiment, the reflected light is collected by the objective lens 17. After passing through the second dichroic mirror 16, the fluorescent signal in the reflected light is focused by the lens, and then split by the beam splitter 32. In this embodiment, the reflected light The light is divided into two beams, one beam of reflected light reaches the multi-wavelength light splitting module 27 after passing through the first optical filter 26, and is detected by the multi-channel photomultiplier tube 28, and the time-correlated single photon counting module 29 counts the detected reflected light, Realize the detection of fluorescence lifetime; another beam of reflected light passes through the third dichroic mirror 33 to split the fluorescence signal in different bands, and then passes through the second filter 34 and then is detected by the photomultiplier tube 35 to detect the harmonic signal probing.

In this embodiment, the time-correlated single photon counting module 29 (Time-Correlated Single Photon Counting, TCSPC) can realize fluorescence lifetime measurement, and different first filter 26 and second filter 34 can be replaced to realize two-photon, For second harmonic, anti-Stokes and other imaging, when the second dichroic mirror 16 performs beam splitting, it transmits long-wavelength excitation light and reflects short-wavelength fluorescence signals.

In one embodiment, the number of channels of the multi-wavelength spectroscopic module 27 and the multi-channel photomultiplier tube 28 is 1-16, so as to realize fluorescence lifetime detection in 1-16 bands.

In the present embodiment, multi-wavelength spectroscopic module 27 gathers multi-channel photomultiplier tube 28 and can carry out the fluorescence lifetime detection of 16 bands; Fluorescence lifetime detection of 16 bands, each channel can realize fluorescence lifetime detection of one band.

In other embodiments, before the second dichroic mirror 16, a combination of at least one group of dichroic mirrors, optical filters and photomultiplier tubes can also be provided for beam splitting of different wavelength bands of fluorescent signals, and can also realize Second harmonic, third harmonic, coherent anti-Stokes, etc. imaging.

Therefore, the imaging of the multimodal nonlinear microscopic imaging system provided in this embodiment includes but not limited to stimulated Raman microscopic imaging, two-photon excited fluorescence, second harmonic and other nonlinear imaging.

In one embodiment, the system also includes: an acquisition card 30; the acquisition card 30 is used to control the scanning galvanometer 13 of the focusing module 102 and the acquisition of the lock-in amplifier 23 output signal and the PMT output signal of the transmitted light processing module 103, and realize The scanning of the scanning galvanometer 13 is synchronized with the acquisition of the acquisition card 30, and the pixel pulse, row pulse and frame pulse of the scanning signal are synchronously output, and the three-way pulse of the pixel pulse, row pulse and frame pulse is transmitted to the reflected light processing module 104 The time-correlated single-photon counting module 29 is used to realize the synchronization of fluorescence lifetime signal acquisition and scanning; and in the stimulated Raman imaging process, the second time delay unit 10 of the excitation optical path module 101 is controlled to realize spectral scanning.

In this embodiment, when the acquisition card 30 and the scanning galvanometer 13 are triggered synchronously, the synchronization of fluorescence lifetime signal acquisition and scanning can be realized, thereby realizing the detection of multispectral fluorescence lifetime.

In one embodiment, the femtosecond laser 1 of the excitation optical path module 101 outputs at least two laser beams synchronously, with a pulse width of 100 fs. The wavelength is 680 – 1300 nm, transmitted to the first half-wave plate 2.

In this embodiment, one beam of laser light excited by the excitation optical path module 101 is Stokes light (Stokes) with a wavelength of 1040 nm, and the other beam is pump light (Pump) with a wavelength of 680-1300 nm.

In one embodiment, the scanning galvanometer 13 includes: a resonant mirror in the X direction and a galvanometer in the Y direction, or a first galvanometer in the X direction and a second galvanometer in the Y direction.

The scanning galvanometer 13 in this embodiment can realize point scanning in the X direction and the Y direction, and the scanning galvanometer 13 and the sleeve lens 15 construct a telecentric lens system, which can generate almost Constant spot size and thus almost constant image resolution within the sample scanning area; this telecentric lens system can also be realized with a broadband achromat lens set.

In all the embodiments of the present application, in order to facilitate the transmission of the laser light, mirrors can be used to change the direction of the laser light.

In the foregoing embodiments, the descriptions of each embodiment have their own emphases, and for parts not described in detail in a certain embodiment, reference may be made to relevant descriptions of other embodiments.

The above is a description of a multi-modal nonlinear microscopic imaging system provided by the present invention. For those skilled in the art, according to the idea of the embodiment of the present invention, there will be changes in the specific implementation and application range In summary, the contents of this specification should not be construed as limiting the present invention.

Claims (10)

A multi-modal non-linear microscopic imaging system, comprising a signal acquisition device and an imaging processing terminal, characterized in that the signal acquisition device includes: The excitation optical path module is used to arrange and excite lasers of different nonlinear modes at different times; The sample carrier sheet is used to carry the sample; a focusing module, configured to focus the laser light excited by the excitation optical path module onto the sample on the sample carrier; A transmitted light processing module, configured to process the transmitted light transmitted through the sample carrier by the excitation light path, and detect stimulated Raman signals; The reflected light processing module is used to detect the reflected light generated by the sample on the sample carrier sheet through the excitation light path to obtain the detection result. The multimodal non-linear microscopic imaging system according to claim 1, characterized in that, The excitation light path module includes: A femtosecond laser for generating at least two femtosecond laser beams; The first half-wave plate is used to change the linear polarization angle of the laser light generated by a femtosecond laser; a first polarizing beam splitter, configured to split the laser light passing through the first half-wave plate into two beams, and adjust the laser power in combination with the first half-wave plate; The second half-wave plate is used to change the linear polarization angle of the laser light generated by another femtosecond laser; a second polarization beam splitter, used in combination with a second half-wave plate to adjust the laser power passing through the second half-wave plate; The first time delay unit is used to adjust the optical path of a laser beam split by the first polarization beam splitter; The first glass rod is used to chirp and broaden another laser beam split by the first polarization beam splitter into a picosecond beam; The second glass rod is used to broaden a beam of laser chirp passing through the second polarization beam splitter into a picosecond beam; a third polarization beam splitter, configured to polarize the laser light passing through the first glass rod and the laser light passing through the first time delay unit; The second time delay unit is used to adjust the optical path of the laser light path coupled by the third polarization beam splitter, so as to realize Raman frequency shift spectral scanning; an electro-optic modulator, configured to modulate the intensity of the laser light passing through the second glass rod according to the modulation signal; The first dichroic mirror is used to couple the laser light passing through the second time delay unit and the laser light modulated by the electro-optic modulator into one beam, and transmit it to the focusing module. The multimodal non-linear microscopic imaging system according to claim 2, characterized in that, The first time delay unit includes: a first reflector and a second reflector, and are placed symmetrically on the displacement stage; The first reflector is used to reflect a beam of laser light split by the first polarizing beam splitter to the second reflector; a second mirror, configured to reflect the laser light reflected by the first mirror to the third polarizing beam splitter; The second time delay unit includes: a third reflector, a fourth reflector, a fifth reflector and a sixth reflector, all of which are used to reflect laser light, and the fourth reflector and the fifth reflector are symmetrically placed on the displacement stage, so The laser light coupled by the third polarizing beam splitter passes through the third reflective mirror, the fourth reflective mirror, the fifth reflective mirror and the sixth reflective mirror in sequence, and is transmitted to the first dichroic mirror. The multimodal non-linear microscopic imaging system according to claim 1, characterized in that, The focusing module includes: a scanning galvanometer, a scanning lens, a sleeve lens and an objective lens; the laser light excited by the excitation optical path module is focused on the sample carrier through the scanning galvanometer, scanning lens, sleeve lens and objective lens in sequence sample. The multimodal non-linear microscopic imaging system according to claim 1, characterized in that, The transmitted light processing module includes: a condenser, a first lens, a third filter, a photodiode and a lock-in amplifier; the transmitted light passes through the condenser, the first lens and the filter in sequence, and is captured by the photodiode detecting and generating a first photoelectric signal, and the lock-in amplifier is used to demodulate the first photoelectric signal to generate a stimulated Raman signal. The multimodal non-linear microscopic imaging system according to claim 4, characterized in that, The objective lens is also used to collect reflected light; The reflected light processing module includes: a second lens, a beam splitter, a first filter, a second filter, a multi-wavelength splitting module, a multi-channel photomultiplier tube, a time-correlated single-photon counting module, a second dichroic Chromatic mirror, third dichroic mirror, photomultiplier tube; The second dichroic mirror is arranged between the sleeve lens and the objective lens, and is used to reflect the nonlinear signal generated by the excitation light path through the sample to the second lens, and transmit the excitation light to prevent it from reaching the second lens; The second lens is used to focus the reflected light reflected by the second dichroic mirror; The beam splitter is used to split the reflected light focused by the second lens, one beam is transmitted to the first filter, and the other beam is transmitted to the third dichroic mirror; The multi-wavelength splitting module is used to perform fluorescence optical dispersion on the reflected light passing through the first filter, so as to realize the spatial distribution of different wavelengths; The multi-channel photomultiplier tube is used to detect fluorescence signals of different bands of reflected light passing through the multi-wavelength spectroscopic module; The time-correlated single-photon counting module is used to count the photons of the fluorescent signal to realize multi-wavelength fluorescence lifetime detection; The photomultiplier tube is used to detect harmonic signals from the reflected light transmitted to the third dichroic mirror and passed through the second filter. The multimodal non-linear microscopic imaging system according to claim 1, characterized in that, The system also includes: a collection card; The acquisition card is used to control the acquisition of the scanning galvanometer of the focusing module and the lock-in amplifier output signal and the PMT output signal of the transmitted light processing module, and realize the synchronization of the scanning of the scanning galvanometer and the acquisition of the acquisition card, and Synchronously output the pixel pulse, row pulse and frame pulse of the scanning signal, and transmit the three-way pulse of the pixel pulse, row pulse and frame pulse to the time-correlated single photon counting module of the reflected light processing module to realize the fluorescence lifetime Synchronization of signal acquisition and scanning; and controlling the second time delay unit of the excitation optical path module to realize spectral scanning during the stimulated Raman imaging process. The multimodal non-linear microscopic imaging system according to claim 6, characterized in that, The number of channels of the multi-wavelength spectroscopic module and the multi-channel photomultiplier tube is 1-16, so as to realize the fluorescence lifetime detection of 1-16 bands. The multimodal non-linear microscopic imaging system according to claim 2, characterized in that, The laser of the excitation optical path module outputs at least two beams of excitation light at the same time, both of which have a pulse width of 100 fs. One beam of laser light has a wavelength of 1040 nm, which is transmitted to the second half-wave plate, and the other beam of laser light has a wavelength of 680-1300 nm. , transmitted to the first half-wave plate. The multimodal non-linear microscopic imaging system according to claim 2, characterized in that, The scanning galvanometer includes: a resonant mirror in the X direction and a galvanometer in the Y direction, or a first galvanometer in the X direction and a second galvanometer in the Y direction.