CN113835207A - Double-objective-lens single-molecule fluorescence microscopic imaging method and device based on three-dimensional illumination modulation - Google Patents
Double-objective-lens single-molecule fluorescence microscopic imaging method and device based on three-dimensional illumination modulation Download PDFInfo
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Abstract
The invention discloses a double-objective-lens single-molecule fluorescence microscopic imaging device based on three-dimensional illumination modulation, which comprises an excitation light path module and an imaging light path module, wherein the excitation light path module comprises the following components in sequential arrangement: a laser emitting a laser beam; the beam splitting and scanning system is used for splitting the light beam into four beams of linearly polarized light which are independently gated or cut off, and scanning the imaging position and changing the optical path difference; the double-objective system is used for dividing the exciting light into two symmetrical groups to interfere on an image surface and collecting fluorescence; the imaging light path module comprises the following components in sequence: the phase modulation system is used for dividing the two paths of fluorescence into four beams of interference light according to s and p polarization and introducing appointed phase delay; a camera for collecting fluorescence intensity signals; and the computer is used for controlling the beam splitting and scanning system and the camera, changing the phase and the direction of the interference fringes, taking a picture, processing the acquired data and obtaining a super-resolution image. The invention also discloses a double-objective lens single-molecule fluorescence microscopic imaging method based on three-dimensional illumination modulation.
Description
Technical Field
The invention belongs to the field of optical super-resolution fluorescence microscopic imaging, and particularly relates to a double-objective-lens monomolecular fluorescence microscopic imaging method and device based on three-dimensional illumination modulation.
Background
In the fields of biomedical imaging and life science research, super-resolution fluorescence imaging is always the focus of research, but is limited by diffraction limit, and the optical resolution is only half wavelength of fluorescence, so that finer structures in cells cannot be resolved. Therefore, in recent decades, scientists have been dedicated to improving the resolution of fluorescence microscopes, and the two main types of super-resolution fluorescence microscopy in recent years are single-molecule positioning technology and structured light illumination technology.
The single molecule positioning technology only enables partial single molecules to emit fluorescence each time by utilizing the sparse luminescence characteristic of special fluorescent dye, and a super-resolution image is reconstructed by shooting a large number of sparse luminescence single molecule pictures for synthesis. For example, in the monomolecular positioning device provided in patent application with publication number CN109407293A, because the luminous monomolecular distribution of a single picture is sparse, the resolution can be improved to 10nm magnitude by using a fitting positioning method, and then super-resolution image reconstruction is realized by using superposition of multiple frames of images. This method requires high density of fluorescent labels, requires special dyes, and is slow to image.
The structured light illumination technology excites a fluorescent sample by modulating illumination light, processes an image frequency spectrum in a Fourier domain, moves high-frequency components which cannot be observed by a common microscope into a low-frequency range, and solves an expanded sample frequency spectrum by utilizing multi-frame images so as to improve the image resolution. For example, the three-dimensional structured light illumination super-resolution microscopic imaging device provided in patent application with publication number CN107907981A, the structured light illumination microscope needs less number of acquired images, has high imaging speed, and is suitable for real-time living cell imaging; the required fluorescent labeling density is low, a specific fluorescent dye is not needed, but the principle is limited to only improving the resolution by one time at most, and the resolution is about 100 nm.
Disclosure of Invention
The invention provides a double-objective-lens single-molecule fluorescence microscopic imaging method and device based on three-dimensional illumination. The device combines the structured light illumination technology with the single-molecule fluorescence imaging technology, introduces the double-objective structure to improve the capability of receiving fluorescence, improves the resolution ratio by more than 3 times on the basis of the single-molecule microscopic imaging technology, realizes the ultrahigh resolution ratio superior to 5nm, and has important significance for observing the subcellular structure.
The invention is still a single molecule positioning super-resolution technology essentially, and the core of improving the resolution is to introduce the modulated illumination stripe, and the relative position of the molecule in the stripe is calculated only through the intensity change of the luminous single molecule energy in the stripe phase-shifting process without calculating in the frequency domain, so that the higher positioning precision is obtained compared with the traditional single molecule fitting positioning method, and the further improvement of the resolution is realized.
The invention adopts the following specific technical scheme:
a double-objective-lens single-molecule fluorescence microscopic imaging device based on three-dimensional illumination modulation comprises an excitation light path module and an imaging light path module;
the excitation light path module comprises the following components in sequential arrangement:
a laser for emitting a laser beam for exciting fluorescence;
the beam splitting and scanning system is used for splitting the laser beam into four linearly polarized light beams which are independently gated or cut off, and scanning the imaging position and changing the optical path difference;
the double-objective system is used for dividing the exciting light into two symmetrical groups to interfere on an image surface and collecting fluorescence;
the imaging light path module comprises the following components in sequential arrangement:
the phase modulation system is used for dividing the two paths of fluorescence into four beams of interference light according to s and p polarization and introducing appointed phase delay;
a camera for collecting said fluorescence intensity signal;
and the computer is used for controlling the beam splitting and scanning system and the camera, respectively changing the phase and the direction of the interference fringes and photographing in an accurate time sequence, and processing the acquired data to obtain a super-resolution image.
Preferably, the beam splitting and scanning system comprises:
the laser beam emitted by the laser is divided into two paths by the first polarization beam splitter after the polarization direction of the laser beam is adjusted by the first electro-optical modulator, and the first electro-optical modulator controls the polarization direction to realize gating and cutting off of the two paths of light;
the second electro-optic modulator and the second polarization beam splitter are positioned on the reflection light path of the first polarization beam splitter, and the reflection light of the first polarization beam splitter is divided into two paths by the second polarization beam splitter;
the third electro-optical modulator and the third polarization beam splitter are positioned on the transmission light path of the first polarization beam splitter; the transmission light of the first polarization beam splitter is divided into two paths by the third polarization beam splitter.
In the invention, the second electro-optical modulator and the third electro-optical modulator are used for quickly introducing appointed phase delay to incident light, and the second polarization beam splitter and the third polarization beam splitter are matched to realize quick gating and cut-off of transmission and reflection light paths.
Preferably, a reflection mirror driven by a first piezoelectric ceramic is arranged on a reflection light path or a transmission light path of the second polarization beam splitter and used for changing an optical path difference to adjust the phase of the interference fringes; a first scanning galvanometer and a second scanning galvanometer are respectively arranged on a reflection light path and a transmission light path of the second polarization beam splitter; a reflecting mirror driven by second piezoelectric ceramics is arranged on a reflection light path or a transmission light path of the third polarization beam splitter and is used for changing and changing optical path difference to adjust the phase of interference fringes; and a third scanning galvanometer and a fourth scanning galvanometer are respectively arranged on a reflection light path and a transmission light path of the third polarization beam splitter. The four scanning galvanometers are formed by vertically staggering a pair of electrically controlled swinging galvanometers, so that the accurate control of the emergent light beam within a certain angle can be realized, and the change of the period of the interference fringes is realized through light scanning.
Preferably, a first beam splitter for combining the light beams is arranged on the light emitting paths of the first scanning galvanometer and the second scanning galvanometer; a second beam splitter used for beam combination is arranged on the emergent light path of the third scanning galvanometer and the fourth scanning galvanometer; emergent light of the first beam splitter and the second beam splitter is reflected and transmitted by the third beam splitter to enter the double-objective system.
The first beam splitter and the second beam splitter are used for combining the light beams separated by the second polarization beam splitter and the third polarization beam splitter, the third beam splitter is used for further dividing the light beams separated by the first polarization beam splitter into two paths with equal intensity, and the light beams finally enter the double-objective system to realize the generation of interference fringes.
Preferably, the dual objective system includes a lower objective and a lower objective disposed above and below the imaging position, and the reflected light and the transmitted light of the third beam splitter generate interference fringes for illuminating the sample through the lower objective and the lower objective, respectively.
Preferably, a drift correction system is provided, the drift correction system comprising: the monitoring laser emits 940nm monitoring laser; the detector is used for converging the monitoring laser passing through the lower objective lens and the lower objective lens on the detector; and the double-objective piezoelectric adjusting table group is used for correcting the lower objective and the lower objective posture according to the shape of the light spot on the detector.
Preferably, the phase modulation system comprises a first and a second solitaire babinet compensators respectively arranged on the fluorescence optical path of the lower and the upper objective lens for introducing a controllable phase difference in the fluorescence collected by the lower and the upper objective lens.
Preferably, a fourth beam splitter is arranged on an emergent light path of the phase modulation system, and the polarized fluorescence collected by the upper and lower objective lenses is split into upper light path s interference light, upper light path p interference light, lower light path s interference light and lower light path p interference light;
the s interference light and the p interference light of the upper light path and the s interference light and the p interference light of the lower light path enter a fourth polarization beam splitter through different reflectors, the p light of the lower light path is reflected to enter the lower light path, the s light of the lower light path is transmitted to enter the upper light path, the p light reflector of the upper light path enters the upper light path, and the s light of the upper light path is transmitted to enter the lower light path; the four beams of interference light of the upper optical path and the lower optical path are received by the camera.
The invention also provides a double-objective lens single-molecule fluorescence microscopic imaging method based on three-dimensional illumination modulation, which comprises the following steps:
1) the laser beam is divided into four linearly polarized light beams which can be independently and rapidly gated or cut off;
2) the four beams of linearly polarized light are divided into two parts of equal light intensity after passing through the beam splitter, and are interfered on a sample plane after passing through two objective lenses which are symmetrically assembled up and down to form a fringe pattern for modulating illumination;
3) fluorescent light generated by the sample under the condition of illumination modulation is received by the double objective lens, passes through the beam splitting and phase delay system, forms four images with phase difference n/2 in sequence on the plane of the detector, and is received by the camera;
4) sequentially rotating the direction of interference fringes of the structured light illumination pattern, and changing the phase of the interference fringes for multiple times in each direction to obtain multiple fluorescence intensity images in corresponding phases in each direction;
5) and collecting a plurality of fluorescence intensity images under different illumination modulations in a single-molecule luminescence period for data processing, and reconstructing to obtain a super-resolution image.
The method combines the monomolecular microscopic imaging technology with the structured light illumination technology, and calculates the relative position of the monomolecular in the interference fringes by recording the variation of the luminous intensity of the monomolecular caused by the phase variation of the interference fringes in different directions of space, thereby obviously improving the imaging resolution.
Preferably, the number of the lighting modulation images is selected according to the requirement, and the phase of the interference fringes needs to be changed three times in each direction, namely six frames of data (24 sub-images) are needed for two-dimensional resolution improvement, and nine frames of data (36 sub-images) are needed for three-dimensional resolution improvement.
The device of the invention adopts a modularized design to reserve a multifunctional application interface for the system, and can be conveniently changed into a 4pi structured light illumination microscopic imaging system and a 4pi single molecule microscopic imaging system. The device adopts a reflector driven by piezoelectric ceramics to realize phase shift of the illumination fringes by changing an optical path difference mode, adopts the combination of an electro-optical modulator and a polarization beam splitter to realize the rapid gating of the direction of the illumination fringes, and adopts a scanning galvanometer to realize the switching of the three-dimensional illumination fringes and the adjustment of the period of the interference fringes.
The principle of the invention for improving the imaging resolution is as follows:
the traditional single-molecule microscopic imaging technology is that a special fluorescent dye is utilized to make a sample emit light sparsely and randomly, namely, only part of molecules of each shot image emit light and the distance between the molecules is long, so that the accurate position of the single molecule can be obtained by a fitting method, a large number of images are shot, the position coordinate of the single molecule in each image is calculated, and finally all the single-molecule coordinates are drawn in a high-resolution image to realize the reconstruction of a super-resolution image. The resolution of this technique is related to the intensity of single-molecule light emission, and the higher the intensity of light emission (illumination light intensity), the higher the resolution. The resolution of the single molecule technology is difficult to be reduced below 10nm because the excessive light intensity of the exciting light causes a series of problems such as photobleaching, phototoxicity and the like and even irreversible damage to sample cells. In view of this, in order to improve the resolution without increasing the intensity of the excitation light, the imaging method proposed by the present invention introduces a modulated illumination mechanism to excite the fluorescence.
Sinusoidal interference fringes with light and dark phases are formed through hardware control, and the higher the intensity of the fringes is, the higher the intensity of the excited fluorescence is. For any single molecule that randomly emits light, changing the phase of the three interference fringes during its emission will sequentially obtain three brightness values on the camera that vary with the illumination intensity. The relative position of the luminescent molecule in the interference fringe can be fitted through three brightness values, so that the accurate position coordinate of the luminescent molecule can be calculated. To ensure that the same single molecule can be accurately located in multiple images, its coarse position needs to be obtained before calculating its relative position in the fringe. In the transverse direction (x, y), acquiring a coarse positioning (x, y) coordinate by using a traditional single-molecule fitting positioning method; in the axial direction (z), the phases of the luminous single molecules are calculated by using four sub-images generated by a detection module phase modulation system, and the phases are settled into optical path differences so as to obtain the distance of the positions of the luminous single molecules deviated from an image plane (z is 0), thereby obtaining a coarse positioning z coordinate.
Preferably, the phase shift of the interference fringes is controlled by 0 °, 120 ° and 240 ° in each dimension. The phase shift angle is limited to be the optimal example, and theoretically, the phase shift angle can be any value, so that the phase shift angle can be different; alternatively, the two-dimensional interference fringes can be used for two-dimensional resolution enhancement, and the three-dimensional interference fringes can be used for three-dimensional resolution enhancement.
Compared with the prior art, the invention has the following beneficial technical effects:
(1) the structured light illumination technology is combined with the single molecule technology, and compared with the traditional single molecule positioning technology, the imaging resolution is improved by more than 3 times;
(2) the device can be used for the method of the patent, and is also suitable for experimental researches related to 4pi structured light illumination microscopic imaging and 4pi single-molecule microscopic imaging and other wide-field microscopic imaging;
(3) the combination of the electro-optical modulator and the polarization beam splitter is adopted to realize the rapid gating of the direction of the illumination fringes, and the scanning galvanometer is adopted to realize the switching of the three-dimensional illumination fringes and the adjustment of the period of the interference fringes, so that the imaging speed of the system is improved.
Drawings
FIG. 1 is a schematic diagram of a dual-objective monomolecular fluorescence microscopic imaging device based on three-dimensional illumination modulation according to the present invention;
FIG. 2 is a diagram of the imaging position and interference fringes of the back focal plane of the dual objective lens;
FIG. 3 is a schematic diagram of an objective attitude monitoring optical path of a dual objective system.
Detailed Description
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced in other ways than those specifically described herein, and thus the present invention is not limited to the specific embodiments disclosed below.
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
In the present invention, unless otherwise expressly specified or limited, the terms "mounted," "connected," "secured," and the like are to be construed broadly and can, for example, be fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
The double-objective single-molecule fluorescence microscopic imaging device shown in figure 1 comprises: the laser comprises a laser 1, a single-mode polarization-maintaining optical fiber 2, a first electro-optical modulator 3, a first polarization beam splitter 4, a second electro-optical modulator 5, a second polarization beam splitter 6, a first half-wave plate 7, a first reflecting mirror 8, a second reflecting mirror 9, a first scanning vibration mirror 10, a first piezoelectric ceramic driven reflecting mirror 11, a third reflecting mirror 12, a second scanning vibration mirror 13, a first beam splitter 14, a second electro-optical modulator 15, a third polarization beam splitter 16, a second half-wave plate 17, a fourth reflecting mirror 18, a fifth reflecting mirror 19, a third scanning vibration mirror 20, a second piezoelectric ceramic driven reflecting mirror 21, a sixth reflecting mirror 22, a fourth scanning vibration mirror 23, a second beam splitter 24, a third beam splitter 25, a first dichroic mirror 26, a first barrel mirror 27, a lower objective lens 28, a second dichroic mirror 29, a second barrel mirror 30, an upper objective lens 31, a double-piezoelectric objective lens adjusting stage group 32, a seventh reflecting mirror 33, An eighth mirror 34, a first solitaire babinet compensator 35, a second solitaire babinet compensator 36, a fourth beam splitter 37, a ninth mirror 38, a tenth mirror 39, a fourth polarizing beam splitter 40, an eleventh mirror 41, a tenth mirror 42, a triangular reflecting prism 43, and a camera 44.
The double-objective single-molecule fluorescence microscopic imaging device mainly comprises an excitation light path module and an imaging light path module.
The excitation light path module comprises the following components in sequential arrangement: a laser 1 for emitting a laser beam for exciting fluorescence; the beam splitting and scanning system is used for splitting the laser beam into four independent linear polarized light beams which can be rapidly gated or cut off to generate interference fringes, and can change the period of the interference fringes through scanning of an imaging position and change the phase of the interference fringes through optical path difference regulation; and the double-objective system is used for dividing the exciting light into two symmetrical groups to generate interference fringes on an image surface and collecting fluorescence.
The imaging light path module comprises the following components in sequence: the phase modulation system is used for dividing the two paths of fluorescence into four groups of interference light according to s and p polarization and introducing appointed phase delay; a camera 44 for collecting said fluorescence intensity signal; and the computer is used for controlling the beam splitting and scanning system and the camera 44, respectively changing the phase and the direction of the interference fringes and the acquired data in an accurate time sequence, and processing the acquired data to obtain a super-resolution image.
In this embodiment, the beam splitting and scanning system includes:
the first electro-optical modulator 3 is used for rapidly introducing specified phase delay to incident light, and rapid gating and stopping of transmission and reflection light paths can be realized by matching the first polarization beam splitter 4; the reflection and transmission light paths of the first polarization beam splitter adopt completely symmetrical distribution;
the second electro-optical modulator 5 and the third electro-optical modulator 15, the second third polarization beam splitter 6 and the third polarization beam splitter 16 are sequentially arranged on the reflection and transmission light path of the first polarization beam splitter 4; the second electro-optical modulator and the third electro-optical modulator are used for rapidly introducing specified phase delay to incident light, and rapid gating and cut-off of transmission and reflection light paths can be realized by matching with the second polarization beam splitter and the third polarization beam splitter;
the first half-wave plate 7, the second half-wave plate 17, the first reflecting mirror 8, the second reflecting mirror 9, the fourth reflecting mirror 18, the fifth reflecting mirror 19, the first scanning galvanometer 10 and the third scanning galvanometer 13 are sequentially arranged on a transmission light path of the second polarization beam splitter 6 and the third polarization beam splitter 16; the half-wave plate is used for rotating the polarization direction of incident linearly polarized light by 90 degrees so as to achieve the effect of improving the interference contrast ratio by being the same as the polarization direction of reflected light of the second and third polarization beam splitters; the reflector is used for deflecting the light path to enable the light to be incident on the scanning galvanometer; the scanning galvanometer is formed by vertically staggering a pair of electrically controlled swinging mirrors, so that the accurate control within a certain angle of an emergent light beam can be realized, and the change of the period of interference fringes is realized through light scanning;
the first piezoelectric ceramic driven reflector 11, the second piezoelectric ceramic driven reflector 21, the third reflector 12, the sixth reflector 22, the second scanning galvanometer 13 and the fourth scanning galvanometer 23 are sequentially arranged on the reflection light paths of the second polarizing beam splitter and the third polarizing beam splitter; except one reflector added with a piezoelectric driver, the transmission light paths of the second and third polarization beam splitters are completely the same and symmetrically distributed; the reflecting mirror driven by the piezoelectric ceramics is used for deflecting a light path to enable light to be incident on the scanning galvanometer to realize light scanning, and can change optical path difference to enable interference fringes to generate phase shift;
the first beam splitter 14, the second beam splitter 24 and the third beam splitter 25 are sequentially arranged, the first beam splitter and the second beam splitter are used for combining light beams separated by the second polarization beam splitter and the third polarization beam splitter, the third beam splitter is used for further dividing the light beams separated by the first polarization beam splitter into two paths with equal intensity, and finally the light beams enter the double-objective system to realize generation of interference fringes.
High-power single-mode polarization maintaining optical fibers 2 are sequentially arranged between the laser 1 and the beam splitting and scanning system and used for transmitting high-power linearly polarized laser emitted by the laser 1 into the beam splitting and scanning system and ensuring the linear polarization characteristics of the high-power linearly polarized laser.
Linearly polarized light emitted by the laser 1 enters the system through the single-mode polarization-maintaining fiber 2, is divided into two paths by the first polarization beam splitter 4 after the polarization direction is adjusted by the first electro-optical modulator 3, and the first electro-optical modulator can realize gating and stopping of the two paths of light by controlling the polarization direction.
The double-objective system comprises a lower objective 28, an upper objective 31 and a double-objective piezoelectric adjusting table group 32, and is used for real-time feedback adjustment of the double objectives according to the monitoring of the postures of the double objectives to ensure the alignment accuracy of the double objectives; the dual objective system comprises a first dichroic mirror 26 and a second dichroic mirror 29 for reflecting the wavelength of the illumination laser into the objective and transmitting the fluorescence light collected from the objective into the detection module.
The phase modulation system includes: a first solitary bambinet compensator 35, a second solitary bambinet compensator 36, a fourth beam splitter 37, a ninth mirror 38, a tenth mirror 39, a fourth polarizing beam splitter 40, an eleventh mirror 41, a tenth mirror 42 and a triangular reflecting prism 43 which are arranged in this order; the first and second Sorley Babinet compensators are symmetrically arranged in the upper and lower light paths and used for introducing adjustable phase difference in s-polarized light and p-polarized light; the fourth beam splitter 37 is disposed at the intersection of the upper and lower optical paths, and is configured to divide the polarized light in the upper and lower optical paths into two parts with equal light intensity, where the transmission and reflection optical paths of the beam splitter include s and p polarized lights that are self-interfering; the ninth reflector and the tenth reflector respectively reflect the transmission light and the reflection light of the beam splitter to the polarizing beam splitter; the fourth polarization beam splitter respectively transmits and reflects s-polarized light and p-polarized light which are respectively interfered, and four interference images which are not overlapped in space and have phase delay are formed by staggering a tiny angle through the eleventh reflector and the tenth reflector; the triangular reflecting prism 43 introduces four interference images into the camera.
In this embodiment, according to the above apparatus, the specific implementation process is as follows:
linearly polarized light emitted by the laser 1 enters the system through the single-mode polarization-maintaining fiber 2, is divided into two paths by the first polarization beam splitter 4 after the polarization direction is adjusted by the first electro-optical modulator 3, and the first electro-optical modulator can realize gating and stopping of the two paths of light by controlling the polarization direction.
The reflected light of the first polarization beam splitter 4 enters the second electro-optical modulator 5 and the second polarization beam splitter 6 and then is continuously divided into two paths, and the second electro-optical modulator also plays a role in rapidly gating two paths of light. The transmission light of the second polarization beam splitter is incident to the first scanning galvanometer 10 through the first reflecting mirror 8 and the second reflecting mirror 9, the reflection light of the second polarization beam splitter is incident to the second scanning galvanometer 13 through the first piezoelectric ceramic driven reflecting mirror 11 and the third reflecting mirror 12, wherein the four reflecting mirrors are used for adjusting the direction of a light path, so that the light is ensured to be vertically incident to the scanning galvanometer, and the reflecting mirror controlled by the special piezoelectric ceramic can also realize wavelength magnitude displacement along the radial direction through driving the reflecting mirrors to change the optical path difference, thereby playing a role in adjusting the phase of interference fringes. The scanning galvanometers can rapidly and accurately control the light reflection direction within a certain angle range, the function of switching the interference fringe direction can be realized by matching with the rapid gating cut-off light path of the electro-optical modulator, and the emergent light of the two scanning galvanometers is combined by the first beam splitter 14 and is continuously transmitted to the third beam splitter 25.
The transmitted light from the first polarization beam splitter 4 is also split into two paths after entering the third electro-optical modulator 15 and the third polarization beam splitter 16. The transmission light path and the reflection light path of the first polarization beam splitter 4 are completely symmetrically distributed, and the emergent light of the third scanning galvanometer 20 and the fourth scanning galvanometer 23 is combined by the second beam splitter 24 and then continuously transmitted to the third beam splitter 25 and the light of the first scanning galvanometer and the second scanning galvanometer to be combined into one beam and divided into two beams by the third beam splitter 25.
The reflected light and the transmitted light of the third beam splitter 25 include the outgoing light of the four scanning galvanometers, and are respectively converged on the back focal planes of the lower objective lens 28 and the upper objective lens 31 through the first dichroic mirror 26, the first barrel mirror 27, the second dichroic mirror 29 and the second barrel mirror 30. The dichroscope is used for reflecting light with the illumination wavelength in the illumination module into the objective lens and transmitting light with the fluorescence wavelength collected by the objective lens into the detection module. The light transmitted by the dichroic mirror enters the detection module through a seventh mirror 33 and an eighth mirror 34, respectively.
The lower objective fluorescence and the upper objective fluorescence are respectively led into controllable phase difference by a first solitaire Babinet compensator 35 and a second solitaire Babinet compensator 36
And
then passes through a fourth beam splitter 37, the s and p polarized fluorescence of the upper and lower objective lenses in the upper and lower optical paths respectively generate interference and are recorded as s interference light of the upper optical path and p interference light of the upper optical pathThe interference light, the lower light path s interference light and the lower light path p interference light respectively pass through the ninth reflector 38 and the tenth reflector 39 and then enter the fourth polarization beam splitter 40, the lower light path p light is reflected to enter the lower light path, the lower light path s light is transmitted to enter the upper light path, the upper light path p light reflector enters the upper wide light path, and the upper light path s light is transmitted to enter the lower light path. After passing through the eleventh reflecting mirror 41, the tenth reflecting mirror 42, and the triangular reflecting prism 43, the four interference lights are received by the camera.
Before the system works, the imaging position of the rear focal plane of the objective lens corresponding to the emergent angle of the scanning galvanometer needs to be calibrated in advance, the schematic diagram of interference fringes corresponding to the imaging position of the rear focal plane of the objective lens is shown in fig. 2, when imaging light spots of the objective lens are longitudinally arranged, transverse interference fringes are generated, when imaging light spots of the objective lens are transversely arranged, longitudinal interference fringes are generated, and when imaging light spots of the double objective lenses are positioned in the center of the rear focal plane, axial interference fringes are generated. The interference fringe period can be adjusted by changing the distance between the imaging light spots, and preferably, the closer the imaging light spots are to the edge of the back focal plane of the objective lens, the smaller the interference fringe interval is, and the larger the imaging resolution is improved. After the calibration of the scanning galvanometer is completed, the scanning galvanometer and the electro-optical modulation are matched to realize the quick switching of the interference fringes in different directions.
When the system works, the scanning galvanometer and the electro-optical modulator are controlled to generate interference fringes in a certain direction, the piezoelectric ceramic drives the reflecting mirror to move to carry out three-step phase shifting, and three images (each image comprises four sub-images) are shot in the period; the scanning galvanometer and the electro-optical modulator control and switch the direction of interference fringes, the piezoelectric ceramic drives the reflector to move to carry out three-step phase shifting, and three images are shot in the period; the operation is repeated in this way, and a large number of images under illumination modulation are acquired. Optionally, the system has high resolution and high requirement on the alignment precision of the objective lens, and the attitude drift of the objective lens has a large influence on the image recovery precision during long-time shooting, so that a drift correction system shown in fig. 3 is introduced. The seventh reflecting mirror 33 and the eighth reflecting mirror 34 in the original system are replaced by a third dichroic mirror 47 and a fourth dichroic mirror 46, which still function as reflecting mirrors for fluorescence and can transmit 940nm monitoring laser. The laser 45 emits 940nm laser, which passes through four dichroic mirrors, a double objective lens and two cylindrical mirrors and then is converged on the detector 50 through the imaging lens 48 and the cylindrical lens 49. When the posture of the double-objective lens is changed, the light spots shot by the detector 50 are changed, and the light spot change conditions corresponding to different types of posture drifts can be solved through a related algorithm so as to guide the posture correction of the objective lens. During the operation of the system, the shape of the light spot on the detector 50 is monitored in real time, and the calculated objective lens offset is input to the piezoelectric adjusting stage group 32 to correct the attitude of the objective lens in real time.
After the system works, nine frame data (36 sub-images) in three directions are used as a group, a group of unimolecular data with improved resolution is calculated, and a super-resolution image with the resolution improved by more than 3 times can be recovered through superposition of a large amount of data.
In another embodiment, a method for dual-objective monomolecular fluorescence microscopic imaging based on three-dimensional illumination modulation is provided, which comprises the following steps:
1) the laser beam is divided into four linearly polarized light beams which can be independently and rapidly gated or cut off;
2) the four beams of linearly polarized light are divided into two parts of equal light intensity after passing through the beam splitter, and are interfered on a sample plane after passing through two objective lenses which are symmetrically assembled up and down to form a fringe pattern for modulating illumination;
3) fluorescent light generated by the sample under the condition of illumination modulation is received by the double objective lens, passes through the beam splitting and phase delay system, forms four images with phase difference n/2 in sequence on the plane of the detector, and is received by the camera;
4) sequentially rotating the direction of interference fringes of the structured light illumination pattern, and changing the phase of the interference fringes for multiple times in each direction to obtain multiple fluorescence intensity images in corresponding phases in each direction;
5) and collecting a plurality of fluorescence intensity images under different illumination modulations in a single-molecule luminescence period for data processing, and reconstructing to obtain a super-resolution image.
The method in this embodiment may be implemented according to the above-described embodiment of the apparatus, or may be implemented in other systems based on variations or modifications of the above-described embodiment of the apparatus.
The above description is only exemplary of the preferred embodiments of the present invention, and is not intended to limit the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The utility model provides a two objective monomolecular fluorescence microscopic imaging device based on three-dimensional illumination modulation, includes excitation light path module and imaging light path module, its characterized in that:
the excitation light path module comprises the following components in sequential arrangement:
a laser for emitting a laser beam for exciting fluorescence;
the beam splitting and scanning system is used for splitting the laser beam into four linearly polarized light beams which are independently gated or cut off, and scanning the imaging position and changing the optical path difference;
the double-objective system is used for dividing the exciting light into two symmetrical groups to interfere on an image surface and collecting fluorescence;
the imaging light path module comprises the following components in sequential arrangement:
the phase modulation system is used for dividing the two paths of fluorescence into four beams of interference light according to s and p polarization and introducing appointed phase delay;
a camera for collecting said fluorescence intensity signal;
and the computer is used for controlling the beam splitting and scanning system and the camera, respectively changing the phase and the direction of the interference fringes and photographing in an accurate time sequence, and processing the acquired data to obtain a super-resolution image.
2. The dual objective single molecule fluorescence microscopy imaging device based on three dimensional illumination modulation as claimed in claim 1, wherein the beam splitting and scanning system comprises:
the laser beam emitted by the laser is divided into two paths by the first polarization beam splitter after the polarization direction of the laser beam is adjusted by the first electro-optical modulator, and the first electro-optical modulator controls the polarization direction to realize gating and cutting off of the two paths of light;
the second electro-optic modulator and the second polarization beam splitter are positioned on the reflection light path of the first polarization beam splitter, and the reflection light of the first polarization beam splitter is divided into two paths by the second polarization beam splitter;
the third electro-optical modulator and the third polarization beam splitter are positioned on the transmission light path of the first polarization beam splitter; the transmission light of the first polarization beam splitter is divided into two paths by the third polarization beam splitter.
3. The dual-objective monomolecular fluorescence microscopic imaging device based on three-dimensional illumination modulation according to claim 2, wherein a first piezoceramic driven reflector is arranged on the reflection light path or the transmission light path of the second polarization beam splitter and is used for changing the optical path difference to adjust the phase of the interference fringes;
a first scanning galvanometer and a second scanning galvanometer are respectively arranged on a reflection light path and a transmission light path of the second polarization beam splitter;
a reflecting mirror driven by second piezoelectric ceramics is arranged on a reflection light path or a transmission light path of the third polarization beam splitter and is used for changing and changing optical path difference to adjust the phase of interference fringes;
and a third scanning galvanometer and a fourth scanning galvanometer are respectively arranged on a reflection light path and a transmission light path of the third polarization beam splitter.
4. The dual-objective-lens single-molecule fluorescence microscopic imaging device based on three-dimensional illumination modulation according to claim 3, wherein a first beam splitter for beam combination is arranged on the light outgoing paths of the first scanning galvanometer and the second scanning galvanometer;
a second beam splitter used for beam combination is arranged on the emergent light path of the third scanning galvanometer and the fourth scanning galvanometer;
emergent light of the first beam splitter and the second beam splitter is reflected and transmitted by the third beam splitter to enter the double-objective system.
5. The dual-objective single-molecule fluorescence microscopy imaging device based on three-dimensional illumination modulation of claim 4, wherein the dual-objective system comprises a lower objective and a lower objective disposed above and below the imaging position, and the reflected light and the transmitted light of the third beam splitter generate interference fringes for illuminating the sample through the lower objective and the lower objective, respectively.
6. The dual-objective single-molecule fluorescence microscopic imaging device based on three-dimensional illumination modulation according to claim 5, wherein a drift correction system is provided, the drift correction system comprising: the monitoring laser emits 940nm monitoring laser; the detector is used for converging the monitoring laser passing through the lower objective lens and the lower objective lens on the detector; and the double-objective piezoelectric adjusting table group is used for correcting the lower objective and the lower objective posture according to the shape of the light spot on the detector.
7. The dual-objective unimolecular fluorescence microscopy imaging device based on three-dimensional illumination modulation as claimed in claim 6, wherein the phase modulation system comprises a first and a second solipedrail compensator respectively disposed on the lower and upper objective fluorescence optical paths for introducing a controllable phase difference in the fluorescence collected by the lower and upper objectives.
8. The dual-objective single-molecule fluorescence microscopic imaging device based on three-dimensional illumination modulation of claim 1, wherein a fourth beam splitter is arranged on an outgoing light path of the phase modulation system, and polarized fluorescence collected by the upper and lower objective lenses is split into upper light path s interference light, upper light path p interference light, lower light path s interference light and lower light path p interference light;
the s interference light and the p interference light of the upper light path and the s interference light and the p interference light of the lower light path enter a fourth polarization beam splitter through different reflectors, the p light of the lower light path is reflected to enter the lower light path, the s light of the lower light path is transmitted to enter the upper light path, the p light reflector of the upper light path enters the upper light path, and the s light of the upper light path is transmitted to enter the lower light path; the four beams of interference light of the upper optical path and the lower optical path are received by the camera.
9. A double-objective lens single-molecule fluorescence microscopic imaging method based on three-dimensional illumination modulation is characterized by comprising the following steps:
1) the laser beam is divided into four linearly polarized light beams which can be independently and rapidly gated or cut off;
2) the four beams of linearly polarized light are divided into two parts of equal light intensity after passing through the beam splitter, and are interfered on a sample plane after passing through two objective lenses which are symmetrically assembled up and down to form a fringe pattern for modulating illumination;
3) fluorescent light generated by the sample under the condition of illumination modulation is received by the double objective lens, passes through the beam splitting and phase delay system, forms four images with phase difference n/2 in sequence on the plane of the detector, and is received by the camera;
4) sequentially rotating the direction of interference fringes of the structured light illumination pattern, and changing the phase of the interference fringes for multiple times in each direction to obtain multiple fluorescence intensity images in corresponding phases in each direction;
5) and collecting a plurality of fluorescence intensity images under different illumination modulations in a single-molecule luminescence period for data processing, and reconstructing to obtain a super-resolution image.
10. The method for two-objective single-molecule fluorescence microscopic imaging based on three-dimensional illumination modulation according to claim 1, wherein in step 4), the number of illumination modulation images to be shot is selected according to requirements, and the phase of the interference fringes needs to be changed three times in each direction, namely six frames of data are needed for two-dimensional resolution improvement and nine frames of data are needed for three-dimensional resolution improvement.
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