CN112113940A - Fluorescence imaging device and fluorescence imaging method - Google Patents
Fluorescence imaging device and fluorescence imaging method Download PDFInfo
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Abstract
The application is applicable to the technical field of fluorescence imaging, and provides a fluorescence imaging device and a fluorescence imaging method, wherein the fluorescence imaging device comprises a laser generator for generating laser, and a frequency modulation device, a scanning galvanometer device and a fluorescence microscopic device which are sequentially arranged along the optical path of the laser; the frequency modulation equipment comprises a coupling lens, a polarization-maintaining large mode field optical fiber, a decoupling lens and a first optical filter which are sequentially arranged along the light path of the laser. Compared with the traditional scheme of using the photonic crystal fiber, the laser is more easily coupled to the polarization-maintaining large-mode-field optical fiber, and the polarization-maintaining large-mode-field optical fiber has the advantages of simple and reliable structure, lower price, large market holding amount and capability of greatly reducing the cost of the fluorescence imaging device.
Description
Technical Field
The present disclosure relates to the field of fluorescence imaging technologies, and in particular, to a fluorescence imaging apparatus and a fluorescence imaging method.
Background
Multi-Photon Microscopy (MPM) is a nonlinear optical imaging technique in which a fluorescent molecule can simultaneously absorb two or more photons under irradiation of long-wavelength light with high Photon density, then transition from a low energy state to a high energy state, and transition to the low energy state again during decay to emit fluorescence. The multi-photon microscopic imaging can be divided into two-photon microscopic imaging, three-photon microscopic imaging, four-photon microscopic imaging and the like according to the number of photons which need to be absorbed simultaneously when the same fluorescence signal is excited. Compared with a single photon fluorescence microscope, the multiphoton microscope has the greatest advantage of stronger penetrating capability to living tissues, and is particularly suitable for imaging deep living tissue structures, such as the brain of a living animal.
There are different modes of multiphoton microscopy imaging at different excitation light wavelengths, where three-photon microscopy imaging with excitation light in the 1700nm wavelength window (approximately 1600nm to 1840nm) enables maximum imaging depth. The physical explanation for this phenomenon is that the attenuation of laser light in the 1700nm wavelength window in subcutaneous tissue (including attenuation by absorption and attenuation by scattering) is minimal; moreover, background fluorescence intensity generated at the surface of the living tissue in three-photon microscopic imaging is lower than that in two-photon microscopic imaging.
In the three-photon microscopic imaging technology, a laser with a 1700nm wavelength window is usually obtained by increasing the laser intensity of a femtosecond laser light source, for example, a mode-locked laser technology, an optical parameter oscillation technology, an optical parameter amplification technology and the like are adopted, and a Soliton Self-Frequency Shift effect (SSFS) of the laser is required in the schemes to obtain the high-energy femtosecond pulse laser. The soliton self-frequency shift effect is a nonlinear optical phenomenon in the anomalous dispersion optical fiber, the wavelength of the femtosecond soliton pulse can be subjected to red shift by utilizing the soliton self-frequency shift effect, and then laser emitted by a commercial 1550nm femtosecond laser light source is changed into laser with a 1700nm wavelength window. Due to the energy of solitons and the effective mode field area A of the optical fibereffProportional, photonic crystal fibers have very large effective mode field areas (up to several thousand square microns) and are capable of lasing in the 1700nm wavelength window with soliton energies greater than 100nJAnd the energy requirement of deep tissue multi-photon microscopic imaging is met. However, this also leads to the prior art that the light source for multiphoton microscopy relies heavily on photonic crystal fibers, which have the problem of difficult optical coupling and are very expensive.
Disclosure of Invention
The application aims to provide a fluorescence imaging device and aims to solve the technical problem that laser coupling of traditional multi-photon micro-fluorescence equipment is difficult.
The fluorescence imaging device comprises a laser generator for generating laser, and a frequency modulation device, a scanning galvanometer device and a fluorescence microscopic device which are sequentially arranged along the optical path of the laser; the frequency modulation equipment comprises a coupling lens, a polarization-maintaining large mode field optical fiber, a decoupling lens and a first optical filter which are sequentially arranged along the light path of the laser.
In one embodiment of the present application, the fluorescence microscopy apparatus includes a dichroic mirror disposed on an optical path of the laser light reflected by the scanning galvanometer apparatus, an objective lens assembly disposed on an optical path of the laser light reflected by the dichroic mirror, and an imaging assembly disposed on the dichroic mirror in a direction away from the objective lens assembly.
In an embodiment of the present application, the imaging assembly includes a second optical filter disposed in a direction of the dichroic mirror away from the objective lens assembly, a third optical filter and a first optical sensor sequentially disposed on an optical path of the laser light after passing through the second optical filter, and a fourth optical filter and a second optical sensor sequentially disposed on an optical path of the laser light after being reflected by the second optical filter.
In an embodiment of the present application, the first optical filter and the third optical filter both use long-pass filters, and the second optical filter and the fourth optical filter both use band-pass filters.
In an embodiment of the present application, the frequency modulation apparatus further includes a fiber support for carrying the polarization maintaining large mode field fiber, and an adjustment platform connected to the fiber support.
In an embodiment of the present application, the fluorescence imaging apparatus further includes a beam expanding collimator set disposed on an optical path between the first optical filter and the fluorescence microscopy device.
It is another object of the present application to provide a fluorescence imaging method comprising the steps of:
obtaining optical fiber parameters: simulating and calculating parameters of the polarization maintaining large-mode-field optical fiber;
an imaging system is set up: processing the polarization maintaining large mode field optical fiber and building a fluorescence imaging device;
acquiring fluorescence imaging: and carrying out imaging examination on the sample by adopting the fluorescence imaging device to obtain the image parameters of the sample.
In an embodiment of the present application, the step of obtaining the optical fiber parameter specifically includes: acquiring the pulse width and wavelength of the laser generator, the characteristic wavelength of the sample, and the effective mode field area and the nonlinear refractive index of the polarization-maintaining large-mode-field optical fiber; and substituting the pulse width and the wavelength of the laser generator, the characteristic wavelength of the sample, the effective mode field area and the nonlinear refractive index of the polarization-maintaining large-mode-field optical fiber into a frequency domain transmission equation, and simulating and calculating the length of the polarization-maintaining large-mode-field optical fiber.
In one embodiment of the present application, the frequency domain transmission equation is:
wherein the content of the first and second substances,
complex amplitudes representing the frequency domain;
representing a non-linearity parameter; beta (omega) represents the transmission constant of the polarization maintaining large-mode-field optical fiber; beta is a1(ω0) Group for representing transmission of laser in polarization-maintaining large mode field optical fiberThe inverse of the velocity; FT represents Fourier transform; r (t) represents the nonlinear response function of the polarization maintaining large mode field fiber, and R (t) satisfies:
wherein f isRFractional contribution, τ, representing delayed Raman response1And τ1The time parameter representing the delayed raman response, Θ (t) represents the hervesseld step function, and (t) represents the dirac function.
In an embodiment of the present application, before the step of obtaining the fluorescence parameter, a step of fluorescence labeling is further included, where the step of fluorescence labeling specifically includes: injecting a retrovirus into the sample, the retrovirus having a gene including a gene fragment capable of being expressed as a fluorescent protein; alternatively, a protein fluorescent dye is injected into the sample.
The fluorescence imaging device or the fluorescence imaging method provided by any embodiment of the application has at least the following beneficial effects:
according to the fluorescence imaging device or the fluorescence imaging method provided by any embodiment of the application, the self-phase modulation effect of laser in the optical fiber is utilized, so that after the laser generates frequency shift in the polarization maintaining large-mode-field optical fiber, the frequency of the laser is selected by using the first optical filter, and then the laser with corresponding frequency and wavelength is obtained, so that the laser scanning confocal fluorescence microscopy is used. For example, the polarization maintaining large mode field optical fiber is combined with self-phase modulation, and can convert 1550nm laser into high-energy femtosecond laser pulse with a 1700nm wavelength window, so that fluorescence microscopic imaging with the depth of 1500 μm on the surface of a sample is realized. According to the fluorescence imaging device or the fluorescence imaging method provided by any embodiment of the application, the laser of the frequency shift can be obtained by utilizing the self-phase modulation of the polarization-maintaining large-mode-field optical fiber, compared with the traditional scheme of using the photonic crystal optical fiber, the laser can be coupled to the polarization-maintaining large-mode-field optical fiber more easily, the polarization-maintaining large-mode-field optical fiber is simple and reliable in structure, lower in price and large in market holding amount, and the cost of the fluorescence imaging device or the fluorescence imaging method can be greatly reduced.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a schematic diagram of a fluorescence imaging apparatus provided in an embodiment of the present application;
FIG. 2 is a schematic flow chart of a fluorescence imaging method provided by an embodiment of the present application;
FIG. 3 is a schematic flow chart of a fluorescence imaging method provided by another embodiment of the present application;
fig. 4 is a diagram illustrating the effect of imaging the surface of the sample by using the fluorescence imaging apparatus and the fluorescence imaging method provided in the present application.
Reference numerals referred to in the above figures are detailed below:
1-a laser generator; 2-a frequency modulation device; 21-a coupling lens; 22-polarization maintaining large mode field optical fiber; 23-a decoupling lens; 24-a first filter; 3-scanning galvanometer equipment; 4-fluorescence microscopy equipment; 41-dichroic mirror; 42-objective lens assembly; 43-a second filter; 44-a third filter; 45-a first light sensor; 46-a fourth filter; 47-a second light sensor; 5-a half-wave plate; 6-a beam expanding collimating lens group; 7-a mirror; 8-sample.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.
It will be understood that when an element is referred to as being "secured to" or "disposed on" another element, it can be directly or indirectly secured to the other element. When an element is referred to as being "connected to" another element, it can be directly or indirectly connected to the other element. The terms "upper", "lower", "left", "right", "front", "rear", "vertical", "horizontal", "top", "bottom", "inner", "outer", and the like indicate orientations or positions based on the orientations or positions shown in the drawings, and are for convenience of description only and not to be construed as limiting the technical solution. The terms "first", "second" and "first" are used merely for descriptive purposes and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features. The meaning of "plurality" is two or more unless specifically limited otherwise.
In order to explain the technical solutions of the present application, the following detailed descriptions are made with reference to specific drawings and examples.
Referring to fig. 1, an embodiment of the present application provides a fluorescence imaging apparatus, including a laser generator 1 for generating laser, and a frequency modulation device 2, a scanning galvanometer device 3, and a fluorescence microscopy device 4 sequentially disposed along an optical path of the laser; the frequency modulation device 2 includes a coupling lens 21, a polarization maintaining large mode field optical fiber 22, a decoupling lens 23, and a first filter 24, which are sequentially disposed along the optical path of the laser light.
Specifically, the fluorescence imaging apparatus provided in the present embodiment operates as follows:
laser generated by the laser generator 1 sequentially passes through the frequency modulation device 2 and the scanning galvanometer device 3, irradiates the fluorescence microscopy device 4, and is used as a light source of laser scanning confocal fluorescence microscopy by the fluorescence microscopy device 4. When laser passes through the frequency Modulation device 2, the laser is first coupled into the polarization maintaining large mode field fiber 22 by the coupling lens 21, and Self-phase Modulation (SPM) occurs in the polarization maintaining large mode field fiber 22; then, the laser is decoupled and combined to irradiate the first optical filter 24 when passing through the decoupling lens 23, and the first optical filter 24 can screen the wave band of the self-phase modulated laser, remove stray light in the laser which interferes with fluorescence imaging, that is, only retain light with the wavelength for enabling the sample 8 to generate fluorescence. The laser passing through the first optical filter 24 irradiates the fluorescence microscopy device 4 under the control of the scanning galvanometer device 3, and then laser scanning confocal fluorescence microscopy is carried out on the sample 8.
Self-phase modulation is a nonlinear optical effect generated by high-energy ultrashort-pulse laser in media such as optical fibers, and is essentially a four-wave mixing process. In the self-phase modulation process, two photons are annihilated at the original frequency of the laser to generate two new photons, the frequency is respectively blue-shifted (the wavelength is shortened, the energy is increased) and red-shifted (the wavelength is lengthened, the energy is reduced), the spectrum of the laser is broadened, and new spectral components, namely side lobes, appear on the short wavelength side and the long wavelength side. For example, for three-photon laser scanning confocal fluorescence microscopy, the laser with the wavelength window of 1700nm can be obtained and separated from a 1550nm laser light source by using the self-phase modulation effect, and the three-photon laser scanning confocal fluorescence microscopy is used for the sample 8.
The fluorescence imaging device provided by the embodiment has at least the following beneficial technical effects:
the fluorescence imaging apparatus provided in this embodiment utilizes the self-phase modulation effect of laser in the optical fiber, so that after the laser generates a frequency shift in the polarization maintaining large mode field optical fiber 22, the frequency of the laser is selected by using the first optical filter 24, and then the laser with corresponding frequency and wavelength is obtained, so as to be used for laser scanning confocal fluorescence microscopy. For example, the polarization maintaining large mode field fiber 22, in combination with self-phase modulation, can convert 1550nm laser light into 1700nm wavelength window high-energy femtosecond laser pulses, thereby achieving 1500 μm depth fluorescence microscopy on the surface of the sample 8. The fluorescence imaging device provided by the embodiment can obtain the laser with frequency shift by utilizing the self-phase modulation of the polarization-maintaining large-mode-field optical fiber 22, compared with the traditional scheme using a photonic crystal optical fiber, the laser is easier to couple to the polarization-maintaining large-mode-field optical fiber 22, and the polarization-maintaining large-mode-field optical fiber 22 has simple and reliable structure, lower price and large market holding amount, and can greatly reduce the cost of the fluorescence imaging device.
As a specific scheme of this embodiment, the laser generator 1 may adopt a 1550nm laser generator 1, and the laser with a wavelength window of 1700nm can be obtained through further filtering by the self-phase modulation effect of the polarization-maintaining large mode field optical fiber 22; the pulse frequency of the laser output by the laser generator 1 is 1MHz, the pulse width is 556fs, and the laser is linearly polarized light, and a half-wave plate 5 can be arranged between the laser generator 1 and the frequency modulation device 2, so that the polarization of the laser is aligned with the main shaft of the polarization-maintaining large-mode-field optical fiber 22; the first filter 24 is a 1575nm long-pass filter to filter short-wave components generated by blue-shift of the laser light passing through the polarization-maintaining large-mode-field fiber 22.
Referring to fig. 1, in an embodiment of the present application, the fluorescence microscopy apparatus 4 includes a dichroic mirror 41 disposed on an optical path of the laser light reflected by the scanning galvanometer apparatus 3, an objective lens assembly 42 disposed on an optical path of the laser light reflected by the dichroic mirror 41, and an imaging assembly disposed on a direction of the dichroic mirror 41 away from the objective lens assembly 42. The scanning galvanometer device 3 can control the laser to scan on the surface of the sample 8, and the objective lens assembly 42 controls the imaging depth of the surface of the sample 8 through the adjustment of the focal length of the objective lens assembly.
As a specific scheme of this embodiment, the dichroic mirror 41 diagonally reflects the laser light reflected by the scanning mirror device 3 at an angle of 45 °, and the laser light is reflected by the dichroic mirror 41 to the objective lens assembly 42, and then irradiates the sample 8 and excites the sample 8; the sample 8 generates fluorescence after being excited, the fluorescence passes through the objective lens assembly 42 and then irradiates the dichroic mirror 41 again, the dichroic mirror 41 has selectivity for the transmission of light, and the fluorescence can pass through the dichroic mirror 41 and be received by the imaging assembly.
Referring to fig. 1, in an embodiment of the present application, the imaging assembly includes a second filter 43 disposed on the dichroic mirror 41 in a direction away from the objective lens assembly 42, a third filter 44 and a first optical sensor 45 sequentially disposed on a light path of the laser light after passing through the second filter 43, and a fourth filter 46 and a second optical sensor 47 sequentially disposed on a light path of the laser light after being reflected by the second filter 43.
Specifically, the objective lens assembly 42 has an optical axis, and the sample 8, the dichroic mirror 41, the second filter 43, the third filter 44, and the first optical sensor 45 are all disposed along the optical axis of the objective lens assembly 42, wherein the sample 8 is disposed on one side of the objective lens assembly 42, and the dichroic mirror 41, the second filter 43, the third filter 44, and the first optical sensor 45 are sequentially disposed on the other side of the objective lens assembly 42. The second filter 43 is a reflective filter, which can allow light in a partial waveband to pass through and reflect the rest of the light; the second filter 43 is disposed at a predetermined angle to the optical axis of the objective lens assembly 42, and the fourth filter 46 and the second optical sensor 47 are sequentially disposed on the optical path of the fluorescence of the sample 8 after being reflected by the second filter 43.
It should be understood that the optical axis in the embodiment of the present application refers to an optical axis of an ideal optical system in which the objective lens assembly 42 is located, that is, a straight line in which a direction in which light rays are not deflected in the ideal optical system in which the objective lens assembly 42 is located. The explanation applies to the description relating to the optical axis in the embodiments of the present application, and the concept of the optical axis is only for the purpose of briefly and clearly illustrating the mechanical structure of the fluorescence microscopy apparatus in the embodiments of the present application, and should not be interpreted as a limitation to the present technical solution.
Referring to fig. 1, in an embodiment of the present application, the first filter 24 and the third filter 44 both use long-pass filters, and the second filter 43 and the fourth filter 46 both use band-pass filters. Thus, the first filter 24 and the third filter 44 can pass light having a wavelength greater than a preset wavelength and block light having a wavelength less than the preset wavelength; the second filter 43 and the fourth filter 46 can pass light of a specific wavelength band and block light of other wavelength bands.
As a specific scheme of this embodiment, when the laser generator 1 employs a 1550nm laser generator 1 and laser with a wavelength window of 1700nm (such as 1603nm laser) needs to be obtained for three-photon fluorescence microscopic imaging, the first optical filter 24 employs a 1575nm long-pass optical filter, the second optical filter 43 employs a 630nm band-pass reflective optical filter, the third optical filter 44 employs a 593nm long-pass optical filter, and the fourth optical filter 46 employs a 535nm band-pass optical filter; the first photosensor 45 employs a gallium arsenide phosphide (GaAsP) photomultiplier Tube (PMT) for collecting a three-photon fluorescence signal of the sample 8, and the second photosensor 47 employs a gallium arsenide (GaAs) photomultiplier Tube for collecting a third harmonic signal of the sample 8.
In one embodiment of the present application, the frequency modulation device 2 further comprises a fiber support (not shown) for carrying the polarization maintaining large mode field fiber 22, and an adjustment platform (not shown) connected to the fiber support. The optical fiber support plays a role in protecting the polarization maintaining large mode field optical fiber 22, meanwhile, the bending of the polarization maintaining large mode field light is avoided, the preset self-phase modulation effect of the polarization maintaining large mode field optical fiber 22 can be ensured, and the broadening of a laser spectrum is realized. Specifically, the optical fiber holder may employ a three-dimensional motion platform for adjusting the positional relationship between the optical fiber and the coupling lens 21.
Referring to fig. 1, in an embodiment of the present application, the fluorescence imaging apparatus further includes a beam expanding collimator set 6, the beam expanding collimator set 6 is disposed on an optical path between the first optical filter 24 and the fluorescence microscopy device 4, and the beam expanding collimator set 6 is configured to perform beam expanding collimation on the laser.
Optionally, the fluorescence imaging device may further include several mirrors 7 to realize the folding of the light path; it should be noted that the half-wave loss and the polarization problem at the mirror 7 need to be taken into account after the mirror 7 is arranged.
One specific embodiment is provided below:
referring to fig. 1, in the present embodiment, the fluorescence imaging apparatus includes a laser generator 1 for generating laser, and a half-wave plate 5, a frequency modulation device 2, a reflecting mirror 7, a scanning galvanometer device 3, a beam expanding collimator set 6, and a fluorescence microscopic device 4, which are sequentially disposed along an optical path of the laser; the frequency modulation device 2 comprises a coupling lens 21, a polarization maintaining large mode field optical fiber 22, a decoupling lens 23 and a first optical filter 24 which are sequentially arranged along the optical path of the laser, the frequency modulation device 2 further comprises an optical fiber support for bearing the polarization maintaining large mode field optical fiber 22, and an adjusting platform which is connected with the optical fiber support to adjust the position relation between the polarization maintaining large mode field optical fiber 22 and the coupling lens 21; the fluorescence microscopy apparatus 4 includes an objective lens assembly 42 having an optical axis, and a dichroic mirror 41, a second optical filter 43, a third optical filter 44, and a first optical sensor 45, and a fourth optical filter 46 and a second optical sensor 47, which are disposed along the optical axis of the objective lens assembly 42, the sample 8 being disposed on one side of the objective lens assembly 42, the dichroic mirror 41, the second optical filter 43, the third optical filter 44, and the first optical sensor 45 being disposed in this order on the other side of the objective lens assembly 42; the second filter 43 is a reflective filter, which can allow light in a partial waveband to pass through and reflect the rest of the light; the second filter 43 is disposed at a predetermined angle to the optical axis of the objective lens assembly 42, and the fourth filter 46 and the second optical sensor 47 are sequentially disposed on the optical path of the fluorescence of the sample 8 after being reflected by the second filter 43.
Specifically, the laser generator 1 adopts a 1550nm laser generator 1, the pulse frequency of the laser output by the laser generator 1 is 1MHz, the pulse width is 556fs, the laser is linearly polarized light, the effective mode field area of the polarization-maintaining large-mode-field optical fiber 22 is 616 square microns, the length is 13.5cm, and the nonlinear refractive index n is2＝2.6x10-20m2Therefore, after passing through the polarization maintaining large mode field optical fiber 22, laser generates a side lobe with a wavelength of 1603nm due to the self-phase modulation effect; further, the first optical filter 24 is a 1575nm long-pass optical filter, the second optical filter 43 is a 630nm band-pass reflective optical filter, the third optical filter 44 is a 593nm long-pass optical filter, and the fourth optical filter 46 is a 535nm band-pass optical filter; the first light sensor 45 is a gallium arsenide phosphide photomultiplier for collecting the three-photon fluorescence signal of the sample 8, and the second light sensor 47 is a gallium arsenide photomultiplier for collecting the third harmonic signal of the sample 8.
Referring to fig. 4, it can be seen that the fluorescence imaging apparatus provided in the present embodiment can perform fluorescence imaging on a depth within 1500 μm below a surface layer of a sample.
Referring to FIG. 2, another objective of the present application is to provide a fluorescence imaging method, comprising the following steps:
s1: obtaining optical fiber parameters: simulating and calculating parameters of the polarization maintaining large-mode-field optical fiber 22;
s2: an imaging system is set up: processing the polarization maintaining large mode field optical fiber 22 and building a fluorescence imaging device;
s3: acquiring fluorescence imaging: the sample 8 is subjected to an imaging examination using a fluorescence imaging device, and image parameters of the sample 8 are obtained.
The fluorescence imaging method provided by the embodiment has at least the following beneficial technical effects:
in the fluorescence imaging method provided in this embodiment, the self-phase modulation effect of the laser in the optical fiber is utilized, so that the frequency of the laser is selected by using the optical filter after the laser generates a frequency shift in the polarization maintaining large mode field optical fiber 22, and then the laser with corresponding frequency and wavelength is obtained for laser scanning confocal fluorescence microscopy. For example, the polarization maintaining large mode field fiber 22, in combination with self-phase modulation, can convert 1550nm laser light into 1700nm wavelength window (e.g., 1603nm wavelength laser light) high energy femtosecond laser pulses to achieve 1500 μm depth fluorescence microscopy on the surface of the sample 8. In the fluorescence imaging method provided by this embodiment, the self-phase modulation of the polarization-maintaining large mode field fiber 22 is used to obtain the frequency-shifted laser, and compared with the conventional scheme using a photonic crystal fiber, the laser is more easily coupled to the polarization-maintaining large mode field fiber 22, and the polarization-maintaining large mode field fiber 22 has a simple and reliable structure, a lower price and a large market holding amount, and the fluorescence imaging method provided by this embodiment can greatly reduce the cost of fluorescence imaging.
Referring to fig. 3, in an embodiment of the present application, the step of obtaining the fiber parameter specifically includes: s10: acquiring the pulse width and wavelength of the laser generator 1, the characteristic wavelength of the sample 8, and the effective mode field area and the nonlinear refractive index of the polarization-maintaining large-mode-field optical fiber 22; and substituting the pulse width and the wavelength of the laser generator 1, the characteristic wavelength of the sample 8, the effective mode field area and the nonlinear refractive index of the polarization-maintaining large-mode-field optical fiber 22 into a frequency domain transmission equation, and simulating and calculating the length of the polarization-maintaining large-mode-field optical fiber 22.
According to the relevant parameters of the laser generator 1, the needed relevant parameters of the laser and the propagation behavior of the laser in the polarization maintaining large mode field optical fiber 22, the parameters such as the length of the polarization maintaining large mode field optical fiber 22 and the like can be obtained through simulation calculation; and then, the polarization-maintaining large mode field fiber 22 can be processed according to the parameters, so that after the laser passes through the polarization-maintaining large mode field fiber 22, the laser with a required wavelength window can be obtained through self-phase modulation and further filtering.
In one embodiment of the present application, the frequency domain transmission equation comprises:
wherein the content of the first and second substances,
complex amplitudes representing the frequency domain;
representing a non-linearity parameter; β (ω) represents the transmission constant of the polarization maintaining large mode field fiber 22; beta is a1(ω0) Represents the inverse of the group velocity of the laser transmitted through the polarization maintaining large mode field fiber 22; FT represents Fourier transform; r (t) represents the nonlinear response function of the polarization-maintaining large mode field fiber 22, and R (t) satisfies:
wherein f isRFractional contribution, τ, representing delayed Raman response1And τ1The time parameter representing the delayed raman response, Θ (t) represents the hervesseld step function, and (t) represents the dirac function.
In this embodiment, according to the above formula, the relevant parameters of the laser emitted by the laser generator 1 and the specific parameters of the polarization maintaining large mode field fiber 22 can be fitted, and then the polarization maintaining large mode field fiber 22 meeting the requirements can be processed according to the fitting result. For example, the laser generator 1 adopts a 1550nm laser generator 1, and needs to obtain laser with a wavelength window of 1700nm through the self-phase modulation effect of the polarization-maintaining large mode field optical fiber 22 and the filtering of the first optical filter 24, the pulse frequency of the laser output by the laser generator 1 is 1MHz, the pulse width is 556fs, the effective mode field area of the polarization-maintaining large mode field optical fiber 22 is 616 square microns, and the nonlinear refractive index n2＝2.6x10-20m2At the time of/W, it can be seen from the above equation that when the length of the polarization maintaining large mode field fiber 22 is 13.5cm, a red-shifted side lobe at a wavelength of 1603nm can be obtained.
In an embodiment of the present application, before the step of obtaining the fluorescence parameter, a fluorescence labeling step is further included, where the fluorescence labeling step specifically includes: injecting a retrovirus, which has a gene including a gene fragment capable of being expressed as a fluorescent protein, into the sample 8; alternatively, a protein fluorescent dye is injected into the sample 8.
The expression gene of the fluorescent protein can be reversely transcribed to the living body sample 8 by injecting retrovirus into the sample 8, so that the sample 8 can express the fluorescent protein for a long time, and the fluorescent protein can generate fluorescence under the irradiation of laser with a wavelength window of 1700 nm; alternatively, more directly, a protein fluorescent dye may be injected into the sample 8 to dye specific protein molecules in the sample 8, so that the protein molecules can generate fluorescence under the irradiation of the laser with the wavelength window of 1700nm, and the protein molecules capable of generating fluorescence can be obtained in the sample 8 more quickly.
It should be understood that the steps of injecting the retrovirus or the protein fluorescent dye into the sample 8 and the steps of obtaining the optical fiber parameters and setting up the imaging system are parallel and have no precedence relationship.
The above description is only exemplary of the present application and should not be taken as limiting the present application, as any modification, equivalent replacement, or improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (10)
1. A fluorescence imaging device is characterized by comprising a laser generator for generating laser, and a frequency modulation device, a scanning galvanometer device and a fluorescence microscopic device which are sequentially arranged along the optical path of the laser; the frequency modulation equipment comprises a coupling lens, a polarization-maintaining large mode field optical fiber, a decoupling lens and a first optical filter which are sequentially arranged along the light path of the laser.
2. The fluorescence imaging apparatus according to claim 1, wherein said fluorescence microscopy device comprises a dichroic mirror disposed in an optical path of said laser light reflected by said scanning galvanometer device, an objective lens assembly disposed in an optical path of said laser light reflected by said dichroic mirror, and an imaging assembly disposed in a direction of said dichroic mirror away from said objective lens assembly.
3. The fluorescence imaging apparatus according to claim 2, wherein said imaging component comprises a second filter disposed in a direction of said dichroic mirror away from said objective lens component, a third filter and a first photosensor disposed in sequence on an optical path of said laser light after passing through said second filter, and a fourth filter and a second photosensor disposed in sequence on an optical path of said laser light after being reflected by said second filter.
4. The fluorescence imaging apparatus according to claim 3, wherein the first filter and the third filter each employ a long-pass filter, and the second filter and the fourth filter each employ a band-pass filter.
5. The fluorescence imaging apparatus of any of claims 1-4, wherein said frequency modulation device further comprises a fiber support for carrying said polarization maintaining large mode field fiber, and an adjustment platform connected to said fiber support.
6. The fluorescence imaging apparatus of any of claims 1-4, further comprising a set of beam expanding collimators disposed in an optical path between the first filter and the fluorescence microscopy device.
7. A fluorescence imaging method, comprising the steps of:
obtaining optical fiber parameters: simulating and calculating parameters of the polarization maintaining large-mode-field optical fiber;
an imaging system is set up: processing the polarization maintaining large mode field optical fiber and building a fluorescence imaging device;
acquiring fluorescence imaging: and carrying out imaging examination on the sample by adopting the fluorescence imaging device to obtain the image parameters of the sample.
8. The fluorescence imaging method of claim 7, wherein said step of obtaining fiber parameters specifically comprises: acquiring the pulse width and wavelength of the laser generator, the characteristic wavelength of the sample, and the effective mode field area and the nonlinear refractive index of the polarization-maintaining large-mode-field optical fiber; and substituting the pulse width and the wavelength of the laser generator, the characteristic wavelength of the sample, the effective mode field area and the nonlinear refractive index of the polarization-maintaining large-mode-field optical fiber into a frequency domain transmission equation, and simulating and calculating the length of the polarization-maintaining large-mode-field optical fiber.
9. The fluorescence imaging method of claim 8, wherein the frequency domain transmission equation is:
wherein the content of the first and second substances,
complex amplitudes representing the frequency domain;
representing a non-linearity parameter; beta (omega) represents the transmission constant of the polarization maintaining large-mode-field optical fiber; beta is a1(ω0) Representing the inverse of the group velocity of the laser transmitted in the polarization maintaining large mode field optical fiber; FT represents Fourier transform; r (t) represents the nonlinear response function of the polarization maintaining large mode field fiber, and R (t) satisfies:
wherein f isRFractional contribution, τ, representing delayed Raman response1And τ1The time parameter representing the delayed raman response, Θ (t) represents the hervesseld step function, and (t) represents the dirac function.
10. The fluorescence imaging method of claim 7, wherein prior to the step of obtaining fluorescence parameters, further comprising a fluorescence labeling step, the fluorescence labeling step specifically comprising: injecting a retrovirus into the sample, the retrovirus having a gene including a gene fragment capable of being expressed as a fluorescent protein; alternatively, a protein fluorescent dye is injected into the sample.
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