CN111175263B - Multi-photon fluorescence microscopic imaging system and imaging method - Google Patents
Multi-photon fluorescence microscopic imaging system and imaging method Download PDFInfo
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Abstract
The invention provides a multi-photon fluorescence microscopic imaging system, which comprises a tunable femtosecond laser, a pulse compressor, a beam expander, a direction adjusting unit, a light beam conversion unit, a resonance scanning galvanometer, a focusing unit, a spatial dispersion unit, a light condensing unit and a reflecting unit, wherein the pulse compressor, the beam expander, the direction adjusting unit, the light beam conversion unit, the resonance scanning galvanometer and the focusing unit are sequentially arranged along a laser output light path of the tunable femtosecond laser; the device comprises a reflection unit, a tunable femtosecond laser, a resonance scanning galvanometer and a controller, and is characterized by further comprising a sample stage which is positioned on one side of the reflection unit and can carry a sample to be tested, a microscope objective which is positioned between the sample stage and the reflection unit, an image acquisition unit which is positioned on the other side of the reflection unit relative to the sample stage, and the controller which is respectively connected with the tunable femtosecond laser, the resonance scanning galvanometer and the image acquisition unit. The invention also provides an imaging method based on the imaging system, and the imaging system can realize rapid imaging by combining space-time focusing, and can improve the resolution and effectively inhibit imaging distortion.
Description
Technical Field
The invention relates to the technical field of fluorescence microscopic imaging, in particular to a novel space-time focusing multi-photon fluorescence microscopic imaging system, and also relates to a multi-photon fluorescence microscopic imaging method based on the system.
Background
Fluorescence microscopy imaging has gained widespread use and widespread acceptance as an indispensable observation tool in the biomedical field. At present, the commonly used fluorescence microscopic imaging technology is a single photon laser scanning confocal microscope, which is excited by a light focusing point and uses a pinhole to filter the influence of optical signals outside a focal domain on imaging, so as to effectively improve resolution and signal-to-noise ratio. However, the light excitation wavelength adopted by single photon fluorescence microscopy is mainly in the range of visible light band (405nm-647nm), the biological sample has high absorption and high light scattering to the visible light band, so that the application of the single photon microscopy imaging technology in biological imaging, especially in the deep and high resolution imaging field is limited.
Compared with a single photon microscopic imaging technology, the multi-photon microscopic imaging uses near-infrared wavelength light as an excitation light source, can effectively reduce the influence of phototoxicity, photobleaching and other factors, and can more accurately penetrate and position the deep fluorescent probe. However, although the multiphoton combined laser scanning confocal microscopic imaging technology can be observed more deeply, imaging distortion still remains a significant problem in the technology due to the complex refractive index distribution of biological tissues. In addition, the existing laser scanning confocal microscopy technology generally has the problems of complex structure, inconvenient operation, low imaging speed, low resolution and the like, and is still difficult to meet the requirement of deep imaging of turbid samples such as biological tissues and the like.
As a new microscopic imaging mode, a time-focusing multiphoton fluorescence microscopic imaging technology has been proved to be effective in removing imaging distortion since the present century, but the imaging method also has the problems of the requirement of a femtosecond laser with high single pulse energy, high instrument cost, large light damage to a sample, uneven light illumination, low longitudinal resolution and the like.
Disclosure of Invention
In view of the above, the present invention is directed to a multi-photon fluorescence microscopy imaging system, which can achieve fast imaging, improve resolution, and effectively suppress imaging distortion.
In order to achieve the purpose, the technical scheme of the invention is realized as follows:
a multi-photon fluorescence microscopic imaging system comprises a tunable femtosecond laser, a pulse compressor, a beam expander, a direction adjusting unit, a light beam converting unit, a resonance scanning galvanometer, a focusing unit, a spatial dispersion unit, a light condensing unit and a reflecting unit, wherein the pulse compressor, the beam expander, the direction adjusting unit, the light beam converting unit, the resonance scanning galvanometer, the one-dimensional focusing unit, the spatial dispersion unit, the light condensing unit and the reflecting unit are sequentially arranged along a laser output light path of the tunable femtosecond laser;
the multi-photon fluorescence microscopic imaging system also comprises a sample stage which is positioned on one side of the reflecting unit and can carry a sample to be tested, a microscope objective which is positioned between the sample stage and the reflecting unit, an image acquisition unit which is positioned on the other side of the reflecting unit relative to the sample stage, and a controller which is respectively connected with the tunable femtosecond laser, the resonance scanning galvanometer and the image acquisition unit; the reflecting unit reflects the converged light beam to emit to the sample stage through the microscope objective, and is also arranged to transmit fluorescence emitted from the sample stage for collection by the image collecting unit.
Furthermore, a diaphragm unit capable of switching on and off the laser output is arranged between the tunable femtosecond laser and the pulse compression unit.
Further, the diaphragm unit adopts an electric control aperture diaphragm.
Furthermore, the center wavelength of the pulse laser output by the tunable femtosecond laser is between 700 and 1000nm, the repetition frequency is 80MHz, the pulse width is 150fs, and the laser single pulse energy is 13-40 nJ.
Further, the output laser spectral bandwidth of the tunable femtosecond laser is adjustable within the range of 7nm-22 nm.
Further, the amplification factor of the beam expander is between 8 and 10.
Further, the direction adjusting unit adopts an achromatic half-wave plate and enables the polarization direction of the laser line to be rotated by 90 degrees.
Further, the beam conversion unit adopts a phase plate, and the phase plate phase distribution has a functional form
Where gamma is the phase modulation depth, d is the spatial period,
is the initial phase.
Further, the resonance scanning galvanometer is a one-dimensional scanning galvanometer and scans along the direction orthogonal to the one-dimensional flat-top light beam.
Furthermore, the focusing unit adopts a cylindrical lens, and the focusing unit performs one-dimensional focusing in a direction parallel to the scanning direction of the resonance scanning galvanometer.
Further, the spatial dispersion unit adopts a blazed grating.
Further, the light condensing unit adopts a condensing lens.
Further, the reflecting unit adopts a dichroic mirror.
Further, the image acquisition unit adopts a camera, and the controller adopts a computer pre-installed with a set program.
Furthermore, the microscope objective adopts a flat field achromatic microscope objective.
Furthermore, a band-pass filter and a tube lens are arranged between the reflection unit and the image acquisition unit in sequence along the fluorescence emergent direction.
Further, the pulse compressor comprises a first reflector for reflecting the incident laser, a first dispersion prism and a second dispersion prism which are oppositely arranged and through which the reflected laser of the first reflector passes in sequence, a 0-degree reflector for returning the laser emitted from the second dispersion prism through the second dispersion prism and the first dispersion prism, and a second reflector for reflecting the returned laser of the first dispersion prism to form the output laser; the interval between the first dispersion prism and the second dispersion prism is adjustable, and the 0 ° reflecting mirror is set to open the interval between the laser beams going back and forth through the first dispersion prism and the second dispersion prism.
Furthermore, the microscope objective is arranged on the lifting platform to carry out three-dimensional scanning on a sample to be detected carried on the sample stage.
Furthermore, a plurality of reflecting mirrors for changing the direction of the light path are arranged on the laser output light path between the tunable femtosecond laser and the reflecting unit.
Furthermore, the reflecting mirror comprises a reflecting mirror a and a reflecting mirror b which are respectively arranged at two sides of the light beam conversion unit, a reflecting mirror c which is positioned between the resonance scanning galvanometer and the focusing unit, and a reflecting mirror d which is positioned between the spatial dispersion unit and the light condensing unit.
Meanwhile, the invention also provides an imaging method based on the multi-photon fluorescence microscopic imaging system, and the imaging method comprises the following steps:
s1, changing the space dispersion unit into a reflector, and placing an autocorrelator in front of the microscope objective;
s2, starting the tunable femtosecond laser, and adjusting the laser to enable the pulse laser width measured by the autocorrelator to reach a set threshold value;
s3, turning off the tunable femtosecond laser, switching the spatial dispersion unit back, and taking away the autocorrelator;
s4, starting the tunable femtosecond laser, and adjusting the direction adjusting unit to enable the polarization direction of the laser line to rotate by 90 degrees;
s5, performing one-dimensional scanning on the laser along the orthogonal direction by using the resonance scanning galvanometer, and performing one-dimensional focusing on the laser by the focusing unit in the direction parallel to the scanning direction to form a linear beam;
s6, adjusting the light beam conversion unit, and observing the linear light beam intensity distribution at the front end of the spatial dispersion unit or the sample stage until a flat-top light beam with uniform light intensity is formed;
s7, adjusting the spatial dispersion unit to make the first-order diffraction efficiency of the laser after spatial dispersion by the spatial dispersion unit meet a set threshold, and making the laser collected by the light-collecting unit and reflected by the reflection unit into the microscope objective;
s8, adjusting the laser spectral bandwidth of the tunable femtosecond laser to a set threshold value, and enabling the spatial dispersion laser to completely fill the pupil of the microscope objective along the orthogonal x axis and y axis;
s9, placing the sample to be tested on the sample stage, exciting the sample to generate fluorescence by using the laser from the microscope objective, opening the image acquisition unit to acquire a fluorescence image, and generating an acquired image through the controller.
Further, the imaging method further includes:
s10, moving the microscope objective lens by the elevating platform to three-dimensionally scan the sample with the image acquisition unit, and generating a three-dimensional image by the controller.
Compared with the prior art, the invention has the following advantages:
the invention can adjust the laser pulse width by a tunable femtosecond laser, can control the pulse width by a pulse compressor, can convert a fundamental mode Gaussian beam output by the laser into a one-dimensional flat-top beam by using a beam conversion unit, scans the laser by a resonance scanning galvanometer to form ultrafast uniform illumination, focuses the laser beam into a one-dimensional linear beam along the scanning direction by using a focusing unit, spatially disperses the laser spectrum components by using a spatial dispersion unit, collects all the laser frequency components by using a light condensing unit and re-forms femtosecond pulse laser on the focal plane of a microscope objective, can excite a sample at a sample stage by using the femtosecond pulse laser to generate fluorescence, and can realize multi-photon fluorescence microscopic imaging of space-time focusing by using the femtosecond pulse laser to enter an image acquisition unit through a reflection unit.
The tunable femtosecond laser is used, the laser wavelength and the spectral bandwidth are adjustable, the space-time focusing effect in space-time focusing multiphoton fluorescence microscopic imaging can be effectively controlled, meanwhile, the femtosecond laser is subjected to one-dimensional wave front shaping through the light beam conversion unit, namely the phase plate, the simple and effective conversion of the basic mode Gaussian beam into the one-dimensional flat-top light beam can be realized, the light beam is scanned through the one-dimensional focusing of the cylindrical lens and the single-axis galvanometer perpendicular to the cylindrical lens, the uniform light excitation of a wide field can be realized, the two are combined, the space-time focusing can be combined to achieve the axial resolution of a near diffraction limit, the axial optical resolution can be effectively improved on the basis of keeping the imaging depth, the imaging resolution is improved, the imaging speed is improved, and the imaging distortion can be effectively inhibited.
In addition, the invention can realize the integration of the laser, the resonance scanning galvanometer and the image acquisition unit, is uniformly controlled by computer programming, and can also improve the operability and stability of the system.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate an embodiment of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 is a schematic view of an imaging system according to an embodiment of the present invention;
FIG. 2 is a schematic diagram showing the configuration of a spectral bandwidth adjusting portion of a pulse in a tunable femtosecond laser;
FIG. 3 is a schematic diagram of a pulse compressor according to an embodiment of the present invention;
FIG. 4 is a phase plate phase modulation spatial distribution diagram according to an embodiment of the present invention;
FIG. 5 is a comparison graph of fluorescence intensity distributions excited by a Gaussian beam before the phase plate is added and a flat-top beam after the phase plate is added, wherein the two beams generate a linear beam on the focal plane of the microscope objective;
FIG. 6 is a graph of three-dimensional fluorescence imaging with Gaussian beam (top) and flat-top beam (bottom) excitation;
description of reference numerals:
the device comprises a tunable femtosecond laser, a 2-diaphragm unit, a 3-pulse compressor, a 4-beam expander, a 5-direction adjusting unit, a 6-beam converting unit, a 7-resonance scanning galvanometer, a 8-focusing unit, a 9-spatial dispersion unit, a 10-light condensing unit, an 11-reflecting unit, a 12-microobjective, a 13-sample stage, a 14-band-pass filter, a 15-tube lens, a 16-image acquisition unit, a 17-controller, a 18-first prism, a 19-second prism, a 20-slit structure, a 21-0-degree reflector, a 22-first reflector, a 23-first dispersion prism, a 24-second dispersion prism, a 25-second reflector and a 26-third reflector.
Detailed Description
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.
The embodiment relates to a multi-photon fluorescence microscopic imaging system which is used for multi-photon fluorescence microscopic imaging of space-time focusing, can realize quick imaging, and can improve imaging resolution and effectively inhibit imaging distortion.
Referring to fig. 1, in overall construction, the multiphoton fluorescence microscopic imaging system of the present embodiment includes a tunable femtosecond laser 1, and a pulse compressor 3 and a beam expander 4 which are sequentially arranged on a laser output optical path of the tunable femtosecond laser 1, a direction adjusting unit 5 for changing the polarization direction of the laser line, a beam converting unit 6 for converting the laser light into a one-dimensional flat-top beam, a resonance scanning galvanometer 7 for one-dimensional scanning of the converted one-dimensional flat-top beam, a focusing unit 8 for one-dimensional focusing of the scanned beam to form a line-shaped beam, and a spatial dispersion unit 9 for spatial separation of spectral components of the line-shaped beam, a light-gathering unit 10 for gathering the spectral components of the spatially separated light beams, and a reflecting unit 11 positioned behind the light-gathering unit 10 and capable of reflecting the gathered light beams.
In addition, the imaging system of the embodiment further includes a sample stage 13 located on one side of the reflection unit 11 and capable of carrying a sample to be measured, a microscope objective 12 located between the sample stage 13 and the reflection unit 11, an image acquisition unit 16 located on the other side of the reflection unit relative to the sample stage 13, and a controller 17 connected to the tunable femtosecond laser 1, the resonant scanning galvanometer 7, and the image acquisition unit 16, respectively.
Specifically, the center wavelength of the pulsed laser output by the tunable femtosecond laser 1 of the present embodiment is adjustable between 700 and 1000nm, the repetition frequency is 80MHz, the pulse width is 150fs, and the laser single pulse energy is 13-40 nJ. When the tunable femtosecond laser 1 works, the output laser beam mode of the tunable femtosecond laser 1 is a fundamental mode Gaussian and is generally vertical (y-axis) linearly polarized light, and the spectral bandwidth of the output laser of the tunable femtosecond laser 1 can be controlled and adjusted within the range of 7nm to 22nm by adjusting the laser.
In this case, the spectral bandwidth adjustment of the tunable femtosecond laser 1 can be generally shown in fig. 2, where fig. 2 shows the main components of the resonant cavity for spectral bandwidth adjustment in the laser, and also shows the optical path diagram for laser spectral bandwidth adjustment. In detail, when the spectral bandwidth needs to be adjusted, the center wavelength and the spectral bandwidth of the femtosecond laser can be controlled by controlling the angle of the prism pair formed by the first prism 18 and the second prism 19 in the resonant cavity and the slit width of the slit structure 20.
The specific operation of the angle and the slit width of the prism pair is generally realized by using relevant electromechanical moving parts in the laser and utilizing relevant programs preset in the controller 17. At this time, based on the required target value, the controller 17 controls the rotation angle of the rotary table cooperating with the first prism 18 and the second prism 19, so as to control the laser output center wavelength, and the controller 17 controls the linear displacement of the partial structure in the slit structure 20, so as to change the slit width, so as to control the spectral width of the output pulse laser.
In fig. 2, a 0 ° mirror 21 is used for the primary return of the laser, which is arranged generally parallel to the slit structure 20 as a whole. In this embodiment, to ensure safe operation of the laser system, as a further preferred embodiment, an aperture unit 2 capable of switching on and off laser output may be optionally disposed between the tunable femtosecond laser 1 and the pulse compression unit 3. The diaphragm unit 2 generally adopts an electric control aperture diaphragm, and the transmission of the laser output by the tunable femtosecond laser 1 to downstream components can be controlled through the opening and closing of the diaphragm unit.
The pulse compressor 3 of the present embodiment is mainly used for pre-compressing the pulse width of the femtosecond laser, and pre-compensating the group velocity dispersion introduced by the system optical element to the femtosecond laser, and through the setting of the pulse compressor 3, the present embodiment also enables the femtosecond laser to reach the sample while maintaining the low-chirp pulsed laser, and the low-chirp pulsed laser generally adjusts the chirp to make the focal planes of the time focusing and the empty focusing coincide.
Meanwhile, one configuration of the above-described pulse compressor 3 is shown in fig. 3, and includes a first reflecting mirror 22 for reflecting incident laser light, a first dispersion prism 23 and a second dispersion prism 24 which are arranged to face each other and through which the reflected laser light of the first reflecting mirror 22 passes in order, a second reflecting mirror 25 which returns the laser light emitted from the second dispersion prism 24 through the second dispersion prism 24 and the first dispersion prism 23, and a third reflecting mirror 26 which reflects the returned laser light of the first dispersion prism 23 to form output laser light of the pulse compressor 3.
The distance between the first dispersion prism 23 and the second dispersion prism 24, which are arranged oppositely, is set to be adjustable, and the adjustment of the distance can be achieved by causing one or both of the two dispersion prisms to be translated. The second reflecting mirror 25 is a 0 ° mirror, and is also provided so as to be spaced apart from the laser light passing through the first dispersion prism 23 and the second dispersion prism 24.
In this case, the "back and forth", that is, as shown in fig. 3, the incident laser beam is reflected by the first reflecting mirror 22, reflected by the first and second dispersion prisms 23 and 24 to the second reflecting mirror 25, and reflected by the second reflecting mirror 25, reflected by the second and first dispersion prisms 24 and 23 to the third reflecting mirror 26, to form the outgoing laser beam, which is called "back". The "pitch" can be achieved by tilting the second mirror 25 at a pitch angle of 0.2 ° and the distance between the two laser beams is about 5 mm.
When the pulse compressor 3 of the present embodiment is used, the pulse laser output from the tunable femtosecond laser 1 sequentially enters the first dispersion prism 23 and the second dispersion prism 24 through the first reflector 22, the gap between the two dispersion prisms determines the pulse group velocity dispersion, and the pulse width before the femtosecond laser enters the microscope objective 12 is the same as the pulse width of the laser output from the tunable femtosecond laser 1 by adjusting the gap, so that the group velocity dispersion approaches zero when the femtosecond laser with the group velocity dispersion reaches the back focal plane of the microscope objective 12.
The magnification of the beam expander 4 of the present embodiment is set to be between 8 and 10, that is, the diameter of the laser beam can be expanded from 1.0mm to 8mm to 10mm by using the beam expander 4, and the magnification of the beam expander 4 can be selected to be 8 times, for example, in the specific implementation.
The direction adjusting unit 5 of this embodiment specifically uses an achromatic half-wave plate, and the achromatic half-wave plate, that is, a half-wave plate, can rotate the linear polarization direction of the laser by 90 °, so that the linear polarization direction of the laser is changed from vertical (y-axis) polarization to horizontal (x-axis) polarization.
In this embodiment, the beam conversion unit 6 specifically employs a phase plate, and the phase plate is used to shape the laser beam, so that the beam passes through the phase plate and then forms a flat-topped beam in the x-axis direction before the spatial dispersion unit 9. In detail, the phase plate phase distribution of the present embodiment has a functional form
Wherein gamma is the phase modulation depth, d is the spatial period,
an exemplary structure of a phase plate for initial phase based on the phase distribution is shown on the left side of fig. 4, and in particular design, the phase plate can be formed by using an ultraviolet fused quartz substrate and coated with a near-infrared band antireflection film. The phase plate structure can be manufactured by magnetron sputtering, wet etching or photoetching and the like, and the phase plate adopting the structure has the characteristics of small thermal expansion coefficient and high transmissivity.
In practical applications, the values of the parameters may be selected according to different situations, and when γ is 1.05 and d is 7.1 in this embodiment, the corresponding phase distribution is shown on the right side of fig. 4. After passing through the phase plate, the femtosecond laser generates three-level beams including 0 level and +/-1 level by diffraction, and the three-level beams are transmitted at a transmission distance lDAfter that, the adjacent light beam interval is lambda2lD/2d2Wherein λ is the center wavelength of the femtosecond laser, and the spectral bandwidth of the femtosecond laser is Ω, and the time coherence length is λ2And/2 pi omega. Therefore, when the distance between the light beams is larger than the time coherence length, the intensity of the three-level light beams is superposed to form a flat-top light beam in the x-axis direction.
The present embodiment generates a flat-topped beam with a phase plate, which can also triple the field of view while providing uniform illumination. Meanwhile, it should be noted that the phase plate of this embodiment converts the fundamental mode gaussian beam output by the tunable femtosecond laser 1 into a one-dimensional flat-top beam, and still has the fundamental mode gaussian mode along the orthogonal direction of the one-dimensional flat-top beam.
In this embodiment, the resonant scanning galvanometer 7 is a one-dimensional scanning galvanometer, and the scanning angle is 20 °, the scanning frequency is 8kHZ, and the scanning is performed along a direction orthogonal to the one-dimensional flat-top beam, that is, the y-axis direction. Since the linear light beam formed by one-dimensional focusing of the condensing unit 8 is also linear and is excited on the focal plane of the microscope objective lens 12, the resonance scanning galvanometer 7 is arranged for scanning, so that the light beam can be excited by wide-field illumination.
The focusing unit 8 of the present embodiment specifically uses a cylindrical lens, which may have a focal length of 500mm, for example, and performs one-dimensional focusing in a direction parallel to the scanning direction of the resonant scanning mirror 7, thereby forming a linear beam in the x-axis direction. At this time, it should be noted that the linear beam is a beam formed by using a cylindrical lens to condense the gaussian beam along a single axis direction (the spherical lens is two-dimensionally condensed in x and y, and the cylindrical lens is condensed along x or y axes), and the one-dimensional flat-topped beam is a beam along a direction in which the cylindrical lens is not condensed.
The spatial dispersion unit 9 of the present embodiment specifically employs a blazed grating, and as an implementation manner, the grating pitch may be 1200/mm, and the spatial dispersion may be along the first-order diffraction light direction, and may have a first-order diffraction efficiency of 80% to spatially separate the femtosecond laser spectrum components.
The light-collecting unit 10 of the present embodiment may specifically employ a light-collecting lens to collect the spectral components of the spatially separated femtosecond laser light. The reflecting unit 11 can reflect the collected light beam to emit to the sample stage 13 through the microscope objective 12, and the reflecting unit 11 can also transmit the fluorescence emitted from the sample stage 13 for being collected by the image collecting unit 16. Specifically, the reflecting unit 11 may be a dichroic mirror, and the dichroic mirror may reflect the collected laser light at an angle of 45 degrees into the microscope objective 12 to excite the sample to be detected loaded on the sample stage 13, so that the sample emits fluorescence, and meanwhile, the dichroic mirror may also transmit the fluorescence emitted by the sample to be detected, so that the fluorescence is collected by the image collecting unit 16.
Specifically, the microscope objective 12 of the present embodiment may be a flat field achromatic microscope objective, for example, a water immersion microscope objective with a magnification of 10x and a numerical aperture of 0.45 may be selected, and in practical implementation, the microscope objective 12 may be disposed on a lifting platform to perform three-dimensional scanning on a sample to be measured mounted on the sample stage 13. Among them, the elevating platform for carrying the microscope objective 12 generally adopts an electric elevating platform, and for example, 2000 μm focus lens scanning system ND72Z2LAQ of PI company can be adopted. When the microscope is in work, the electric lifting platform is mainly used for carrying the microscope objective 12 in an imaging system, a sample to be detected is fixed on the sample table 13, and the microscope objective 12 is moved up and down through the electric lifting platform, so that the depth of the focal plane of the objective in the sample can be controlled. Therefore, when the camera moves to a depth position, the camera exposes and collects a frame, and images at different depths are sequentially superposed to finally reconstruct a three-dimensional image.
The image capturing unit 16 of the present embodiment may be a camera, and the controller 17 may be a computer pre-installed with a setting program. Further, as a preferred embodiment, a band-pass filter 14 and a tube lens 15 arranged in this order in the fluorescence emission direction can also be provided between the reflection unit 11 and the image pickup unit 16. The bandpass filter 14 allows the fluorescence to pass through and cut off the excitation light, and the tube lens 15 images the fluorescence on the surface of the image acquisition unit 16, i.e., the detector of the camera.
In order to facilitate the arrangement of each component in the system, in a preferred implementation of this embodiment, a plurality of mirrors capable of changing the direction of the optical path may be disposed on the laser output optical path between the tunable femtosecond laser 1 and the reflection unit 11, so as to implement the component arrangement mode shown in fig. 1, for example. At this time, as shown in fig. 1, the mirrors provided specifically include a mirror a and a mirror b disposed at both sides of the beam conversion unit 6, a mirror c disposed between the resonance scanning galvanometer 7 and the focusing unit 8, and a mirror d disposed between the spatial dispersion unit 9 and the condensing unit 10.
Of course, instead of using four mirrors a, b, c, and d as described above, different numbers of mirrors and arrangement distributions of mirrors may be selected based on actual implementation, and the arrangement of each mirror does not affect the operation of each optical component.
As an application example, in an actual implementation of the imaging system of this embodiment, manufacturers and models of some of the optical components that can be used in the imaging system are as follows:
tunable femtosecond laser: spectra-physics, Mai Tai HP;
achromatism half-wave plate Thorlabs, AHWP 05M-980;
resonance scanning galvanometer: cambridge technology, CRS 8 KHz;
cylindrical lenses Thorlabs, LJ1144 RM-B;
blazed gratings Thorlabs, GR 25-1208;
condensing lenses Thorlabs, AC 508-500-B;
a dichroic mirror Chroma, ZT775sp-2 p;
zeiss objective 421747-;
a sample stage, Marzhauser, SCAN IM 130x 100;
a band-pass filter is Chroma, ET600/50 m;
a camera: andor, iXon 888.
In addition, for other components not listed above, the pulse compressor 3 can be self-assembled, and the two dispersion prisms therein can use the Brewster's angle dispersion prism Newport:10SF10 for ultrashort pulses. The beam expander 4 can also be self-assembled and for example can take the form of a double lens combination, or the beam expander 4 can also be commercially available as an existing product that meets the requirements of use. The phase plate can be customized based on the illustration, and the tube lens 15 is the inherent configuration of the microscope and can be used directly.
When the multi-photon fluorescence microscopy imaging system of the embodiment is used for imaging, the method specifically comprises the following steps:
firstly, replacing a blazed grating with a reflector, placing an autocorrelator in front of a microscope objective, then starting the tunable femtosecond laser 1, opening an electrically controlled aperture diaphragm, and adjusting the tunable femtosecond laser 1 to enable the pulse laser width measured by the autocorrelator to reach a set threshold (for example, 80 fs). Then, the tunable femtosecond laser 1 is closed, the blazed grating is replaced, the autocorrelator is taken away, the tunable femtosecond laser 1 is opened, and the achromatic half-wave plate is adjusted to enable the polarization direction of the laser line to rotate 90 degrees.
Then, the laser light rotated in the direction is one-dimensionally scanned by the resonance scanning galvanometer 7, and is one-dimensionally focused by the cylindrical lens in a direction parallel to the scanning to form a linear beam. Then, the phase plate is adjusted again, and the intensity distribution of the linear light beam is observed at the front end of the blazed grating or the sample stage 13 until a flat-top light beam with uniform light intensity is formed. Wherein, for observing the intensity distribution of the linear light beam in front of the blazed grating, the blazed grating can be replaced by a camera to observe the light intensity, and a neutral density optical filter can be used for attenuating the light intensity to prevent the supersaturation of the camera imaging.
Observing the intensity distribution of the linear light beam at the sample stage 13, uniformly coating rhodamine B dye on a cover glass, air-drying, and then placing the cover glass on the sample stage 13, so that laser excites the dye through a microscope objective and images. The images obtained by the two modes are analyzed by image processing software to obtain light intensity distribution, and when the light intensity difference of the linear light beam along the long axis direction of the light beam except for 10% areas at two ends is within 5%, and the deviation of the two-photon fluorescence intensity is within 10%, the linear light beam can be basically regarded as a flat-top light beam with uniform light intensity. The linear flat-top beams at the two positions of the front end of the blazed grating and the sample stage 13 are similar in shape but different in size, and the size ratio between the linear flat-top beams is the focal length ratio between the condenser lens 10 and the microscope objective lens 12.
Then, the blazed grating angle is adjusted, so that the first-order diffraction efficiency of the femtosecond laser after spatial dispersion of the blazed grating meets a set threshold (for example, 80%) and has higher first-order diffraction efficiency, and at this time, for example, a laser power meter can be used to measure the ratio of the first-order diffraction of the laser emitted from the blazed grating to the laser intensity before incidence to the blazed grating, so as to determine whether the ratio is 80%. Then, the laser light is collected by the condenser lens and reflected by the dichroic mirror into the microscope objective 12, and the laser spectral bandwidth of the tunable femtosecond laser 1 is adjusted to a set threshold (for example, 21nm) so that the spatially dispersed laser light completely fills the pupil of the microscope objective 12 along both the orthogonal x-axis and y-axis.
Finally, a sample to be detected is packaged by upper and lower cover glass and then is placed on a sample table 13, laser from a microscope objective 12 is used for exciting the sample to generate fluorescence, the fluorescence is emitted to a camera through the microscope objective 12, a dichroic mirror, a band-pass filter 14 and a lens barrel lens 15, the camera is opened for fluorescence image acquisition, and a fluorescence acquisition image can be generated through a computer.
In this case, the image is acquired by fluorescence as obtained by the above method, and the size of the image may be designed to be 10 × 10mm, for example2And the number of image pixels can be designed to be 1024 x 1024 to meet the imaging requirement.
In addition, based on the foregoing, the microscope objective 12 is loaded by the electrically controlled elevating platform, so that the microscope objective 12 can be moved by the elevating platform during the fluorescence microscopic imaging, so as to scan the sample three-dimensionally by the image acquisition unit 16, and finally generate a three-dimensional image by the controller 17.
In this case, the size of the three-dimensional image obtained may be, for example, 800 × 150 μm3Moreover, the inventors have obtained, through specific experiments, the linear laser excitation fluorescence intensity distributions without using a phase plate and with using a phase plate, respectively, as shown in fig. 5. In addition, two three-dimensional fluorescence imaging images are obtained by scanning the laser beam through the resonance scanning galvanometer, and are specifically shown in fig. 6, wherein the upper image is a three-dimensional fluorescence image excited by a gaussian beam when a phase plate is not adopted, and the lower image is a three-dimensional fluorescence image excited by a one-dimensional flat-top beam after the phase plate is adopted. As is apparent from fig. 6, the phase plate is added, that is, the one-dimensional flat-top beam is generated, so that the wide-field uniform illumination can be achieved, that is, the field of view is wide and the laser illumination is uniform, compared with the gaussian beam.
In summary, the imaging system of the embodiment can realize space-time aggregation multiphoton fluorescence microscopic imaging, improve fluorescence imaging speed, reduce image distortion caused by sample light scattering, and achieve one-dimensional uniform light excitation, the system is simple and easy to operate, and only needs to adjust a laser to find out an optimal excitation wavelength and a spectral bandwidth, so that the laser completely fills the clear aperture of the microscope objective 12.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (22)
1. A multi-photon fluorescence microscopy imaging system, characterized by:
the multi-photon fluorescence microscopic imaging system comprises a tunable femtosecond laser (1), a pulse compressor (3), a beam expander (4), a direction adjusting unit (5) for changing the linear polarization direction of the laser, a light beam converting unit (6) for converting the laser into a one-dimensional flat-top light beam, a resonance scanning galvanometer (7) for performing one-dimensional scanning on the converted one-dimensional flat-top light beam, and a focusing unit (8) for performing one-dimensional focusing on the scanned light beam to form a linear light beam, wherein the pulse compressor (3), the beam expander (4), the direction adjusting unit (5) are sequentially arranged along a laser output light path of the tunable femtosecond laser (1), and a spatial dispersion unit (9) for spatially separating spectral components of the linear beam, a condensing unit (10) for condensing the spectral components of the spatially separated beam, and a reflection unit (11) positioned behind the light collection unit (10) and capable of reflecting the collected light beam;
the multi-photon fluorescence microscopic imaging system further comprises a sample stage (13) which is positioned on one side of the reflecting unit (11) and can carry a sample to be tested, a microscope objective (12) which is positioned between the sample stage (13) and the reflecting unit (11), an image acquisition unit (16) which is positioned on the other side of the reflecting unit relative to the sample stage (13), and a controller (17) which is respectively connected with the tunable femtosecond laser (1), the resonant scanning galvanometer (7) and the image acquisition unit (16); the reflecting unit (11) reflects the collected light beams to emit to the sample stage (13) through the microscope objective (12), and the reflecting unit (11) is also arranged to transmit fluorescence emitted from the sample stage (13) for being collected by the image collecting unit (16).
2. The multiphoton fluorescence microscopy imaging system of claim 1, wherein: and a diaphragm unit (2) capable of switching on and off the laser output is arranged between the tunable femtosecond laser (1) and the pulse compression unit (3).
3. The multiphoton fluorescence microscopy imaging system of claim 2, wherein: the diaphragm unit (2) adopts an electric control aperture diaphragm.
4. The multiphoton fluorescence microscopy imaging system of claim 1, wherein: the center wavelength of the pulse laser output by the tunable femtosecond laser (1) is between 700 and 1000nm, the repetition frequency is 80MHz, the pulse width is 150fs, and the laser single pulse energy is 13-40 nJ.
5. The multiphoton fluorescence microscopy imaging system of claim 1, wherein: the output laser spectral bandwidth of the tunable femtosecond laser (1) is adjustable within the range of 7nm-22 nm.
6. The multiphoton fluorescence microscopy imaging system of claim 1, wherein: the amplification factor of the beam expander (4) is between 8 and 10.
7. The multiphoton fluorescence microscopy imaging system of claim 1, wherein: the direction adjusting unit (5) adopts an achromatic half-wave plate and enables the polarization direction of the laser line to be rotated by 90 degrees.
8. The multiphoton fluorescence microscopy imaging system of claim 1, wherein: the beam conversion unit (6) adopts a phase plate, and the phase plate phase distribution has a functional form
Wherein gamma is the phase modulation depth, d is the spatial period,
is the initial phase.
9. The multiphoton fluorescence microscopy imaging system of claim 1, wherein: the resonance scanning galvanometer (7) is a one-dimensional scanning galvanometer and scans along the direction orthogonal to the one-dimensional flat-top light beam.
10. The multiphoton fluorescence microscopy imaging system of claim 1, wherein: the focusing unit (8) adopts a cylindrical lens, and the focusing unit (8) performs one-dimensional focusing in the direction parallel to the scanning direction of the resonance scanning galvanometer (7).
11. The multiphoton fluorescence microscopy imaging system of claim 1, wherein: the spatial dispersion unit (9) adopts a blazed grating.
12. The multiphoton fluorescence microscopy imaging system of claim 1, wherein: the light-gathering unit (10) adopts a light-gathering lens.
13. The multiphoton fluorescence microscopy imaging system of claim 1, wherein: the reflection unit (11) adopts a dichroic mirror.
14. The multiphoton fluorescence microscopy imaging system of claim 1, wherein: the image acquisition unit (16) adopts a camera, and the controller (17) adopts a computer pre-installed with a set program.
15. The multiphoton fluorescence microscopy imaging system of claim 1, wherein: the microscope objective (12) adopts a flat field achromatic microscope objective.
16. The multiphoton fluorescence microscopy imaging system of claim 1, wherein: a band-pass filter (14) and a tube lens (15) which are sequentially arranged along the fluorescence emergence direction are arranged between the reflection unit (11) and the image acquisition unit (16).
17. The multiphoton fluorescence microscopy imaging system of claim 1, wherein: the pulse compressor (3) comprises a first reflector (22) for reflecting incident laser, a first dispersion prism (23) and a second dispersion prism (24) which are oppositely arranged and through which the reflected laser of the first reflector (22) passes in sequence, a second reflector (25) for returning the laser emitted by the second dispersion prism (24) through the second dispersion prism (24) and the first dispersion prism (23), and a third reflector (26) for reflecting the returned laser of the first dispersion prism (23) to form output laser; the distance between the first dispersion prism (23) and the second dispersion prism (24) is adjustable, and the second reflecting mirror (25) is provided so as to open the distance between the laser beams going back and forth through the first dispersion prism (23) and the second dispersion prism (24).
18. The multiphoton fluorescence microscopy imaging system of claim 1, wherein: the microscope objective (12) is arranged on the lifting platform to carry out three-dimensional scanning on a sample to be detected carried on the sample stage (13).
19. The multiphoton fluorescence microscopy imaging system according to any one of claims 1 to 18, wherein: and a plurality of reflectors for changing the direction of the light path are arranged on the laser output light path between the tunable femtosecond laser (1) and the reflection unit (11).
20. The multiphoton fluorescence microscopy imaging system of claim 19, wherein: the reflecting mirror comprises a reflecting mirror a and a reflecting mirror b which are respectively arranged at two sides of the light beam conversion unit (6), a reflecting mirror c positioned between the resonance scanning galvanometer (7) and the focusing unit (8), and a reflecting mirror d positioned between the spatial dispersion unit (9) and the light condensing unit (10).
21. An imaging method of the multiphoton fluorescence microscopy imaging system according to claim 1, wherein the imaging method comprises the steps of:
s1, changing the space dispersion unit (9) into a reflecting mirror, and placing an autocorrelator in front of the microscope objective;
s2, turning on the tunable femtosecond laser (1) and adjusting the laser to enable the pulse laser width measured by the autocorrelator to reach a set threshold value;
s3, turning off the tunable femtosecond laser (1), replacing the spatial dispersion unit (9) and taking away the autocorrelator;
s4, turning on the tunable femtosecond laser (1), and adjusting the direction adjusting unit (5) to rotate the polarization direction of the laser line by 90 degrees;
s5, one-dimensional scanning is carried out on the laser along the orthogonal direction by using the resonance scanning galvanometer (7), and the laser is one-dimensionally focused by the focusing unit (8) in the direction parallel to the scanning direction to form a linear beam;
s6, adjusting the light beam conversion unit (6), and observing the linear light beam intensity distribution at the front end of the spatial dispersion unit (9) or the sample stage (13) until a flat-top light beam with uniform light intensity is formed;
s7, adjusting the spatial dispersion unit (9) to make the first-order diffraction efficiency of the laser after spatial dispersion by the spatial dispersion unit (9) meet a set threshold, and making the laser collected by the light collection unit (10) and reflected by the reflection unit (11) into the microscope objective;
s8, adjusting the laser spectral bandwidth of the tunable femtosecond laser (1) to a set threshold value, so that the spatial dispersion laser completely fills the pupil of the microscope objective (12) along the orthogonal x axis and y axis;
s9, placing the sample to be tested on the sample stage (13), exciting the sample to generate fluorescence by using the laser from the microscope objective (12), opening the image acquisition unit (16) to acquire a fluorescence image, and generating an acquired image through the controller (17).
22. The method of imaging a multiphoton fluorescence microscopy imaging system as set forth in claim 21, wherein: further comprising:
s10, moving the microscope objective (12) by the elevating platform to three-dimensionally scan the sample with the image acquisition unit (16), and generating a three-dimensional image by the controller (17).
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