CN217112868U - Multi-mode multi-color fast switching structured light illumination system - Google Patents
Multi-mode multi-color fast switching structured light illumination system Download PDFInfo
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Abstract
The utility model discloses a multi-mode polychrome fast switch over structure light lighting system. The utility model adopts a variable-pitch space filter with a structured light illumination confocal microscopic clear aperture and a structured light illumination super-resolution microscopic clear aperture, which realizes multiple microscopic imaging modes in one system, including a wide-field fluorescence imaging mode, a structured light illumination super-resolution microscopic imaging mode, and a structured light illumination confocal microscopic imaging mode, and adds a fluorescence polarization microscopic imaging mode by fusion on the basis of multiple imaging functions; the fast switching of various microscopic modes and multicolor imaging are realized through adjustment, and the process of biological imaging by using a structured light illumination microscopic method is realized; according to different required structures, wide-field fluorescence microscopic imaging, optical slice imaging, fast super-resolution imaging or fluorescence polarization microscopic imaging is selected.
Description
Technical Field
The utility model relates to an optical microscopy imaging field, concretely relates to multi-mode polychrome fast switch over structure light lighting system.
Background
Wide-field Fluorescence Microscopy (WF) is the process of irradiating a fluorescently labeled sample with excitation light while collecting the fluorescent signal emitted by the fluorophore. The imaging method is an approximate dark field imaging mode, and can obtain a sample image with good sensitivity, good contrast and a large field of view.
Structured light Illumination Super Resolution Microscopy (SIM) is a Super Resolution (SR) imaging technique. According to the moire principle, a sinusoidal structured light field is irradiated on a sample, and high frequency information of the sample is shifted to a low frequency, so that the high frequency information can pass through a low pass filter formed by an optical transfer function of the optical system. The camera acquires the sample images illuminated by the structured light in different directions and phases. Reconstruction is carried out through an algorithm, and the improvement of the transverse double-time resolution can be realized. Meanwhile, the method has the advantages of high imaging speed, low phototoxicity and the like, and is suitable for living cell imaging.
Structured light illumination enables optical tomographic microscopy like Confocal microscopy (Confocal) in addition to super-resolution microscopy. When the ordinary optical microscope is used for imaging, the information of the object in focus and the information of the object out of focus can be recorded simultaneously to form a two-dimensional image. By irradiating the sample with structured light having a sufficiently high spatial frequency, only the focal plane signal will be modulated and the defocus signal will not be affected. The in-focus signal can be extracted by structured light illumination, thus achieving optical sectioning. The wide-field microscope can also have the optical sectioning capability of the confocal microscope, and the characteristics of low imaging speed, complex mechanical structure, high manufacturing cost and the like of the confocal microscope are overcome.
Fluorescence Polarization Microscopy (FPM) imaging can realize fluorescent dipole orientation analysis directly from an acquired original sample image with Polarization characteristics by utilizing the characteristic that the direction of the structured light illumination stripes on a sample surface is consistent with the Polarization direction of incident light, and endows the traditional SIM system with the characteristic of Fluorescence Polarization Microscopy imaging.
In biological imaging using structured light illumination microscopy. In order to quickly locate the structure to be observed in the sample, wide-field fluorescence microscopy imaging can be selected; in order to be able to obtain the three-dimensional structure of a thick sample, optical slice imaging (SIM-confocal) is optionally used; in order to be able to observe fine intracellular structures in thin samples, fast super-resolution imaging (SIM-SR) is an option; fluorescence polarization microscopy imaging can be used to study structures with significant dipole orientation characteristics, such as cytoskeleton.
How to apply the structured light illumination method, realize multiple microscopic modes in one system and carry out rapid switching, observe biological samples from different angles is a problem to be solved urgently.
Disclosure of Invention
To the above problem, the utility model provides a multi-mode polychrome fast switch over structure light lighting system utilizes structure light illumination principle, through becoming interval space filter, realizes multiple microscopic imaging modality in one set of system, including wide field fluorescence imaging, the microscopic imaging of structure light illumination super-resolution, the microscopic imaging of structure light illumination confocal and fluorescence polarization.
The utility model discloses a multi-mode polychrome fast switch over structure light lighting system includes: the device comprises a laser coupling module, a structured light imaging main body module and a fluorescence detection module; the laser coupling module comprises N lasers, a laser beam combiner, an acousto-optic tunable filter, a first focusing lens, a collimation beam expander and a polarization-preserving single-mode fiber; the structured light imaging main body module comprises a polarization beam splitter, a liquid crystal spatial light modulator, a half-wave plate, a third focusing lens, a variable-pitch spatial filter, a subarea half-wave plate and a relay lens; the fluorescence detection module comprises an objective lens, a dichroic mirror, an emission filter, a tube lens and a camera, wherein N is a natural number not less than 2;
the variable-pitch spatial filter comprises an opaque substrate, a structured light illumination confocal microscopy clear aperture and a structured light illumination super-resolution microscopy clear aperture; the non-transparent substrate is a circular flat plate, 2M structured light illumination Confocal microscopy light-passing holes with the same shape are formed in the non-transparent substrate, M is a natural number larger than or equal to 3, the structured light illumination Confocal microscopy light-passing holes are circular, the circle centers of the 2M structured light illumination Confocal microscopy light-passing holes are centrally and symmetrically distributed on a first circle concentric with the non-transparent substrate, a line segment formed by connecting the centers of the two structured light illumination Confocal microscopy light-passing holes is located on a first circle of the non-transparent substrate, two structured light illumination Confocal microscopy light-passing holes on the same diameter are located on the first circle, the pair of structured light illumination Confocal microscopy light-passing holes correspond to the direction of a structured light illumination field, the 2M structured light illumination Confocal microscopy light-passing holes are located at the +/-1-level light position of the structured light illumination Confocal microscopy imaging, the radius of the first circle is c, and the radius c of the first circle is equal to the +/-1-level diffraction light spot interval of the Grid-Confocai light illumination Confocal microscopy imaging Half of the total weight; the non-transparent substrate is also provided with 2M structured light illumination super-resolution microscopic light-passing holes with the same shape, the structured light illumination super-resolution microscopic light-passing holes are in a round end rectangle, namely the middle of the light illumination super-resolution microscopic light-passing holes is a rectangle, the two ends of the rectangle are respectively semicircles, the semicircle close to the center of the non-transparent substrate is an inner end semicircle, the semicircle far away from the center of the non-transparent substrate is an outer end semicircle, the diameter of the semicircle is equal to the short side of the rectangle, the center of the semicircle is positioned at the center of the short side of the rectangle, a line segment formed by connecting the centers of the two structured light illumination super-resolution microscopic light-passing holes is positioned on one diameter of the non-transparent substrate, and any pair of the structured light illumination super-resolution microscopic light-passing holes and any pair of the structured light illumination confocal microscopic light-passing holes can not be on the same straight line; the circle centers of the 2M inner end semicircles are centrosymmetrically distributed on a second circle concentric with the opaque substrate, the radius b of the second circle is equal to half of the +/-1-order diffraction light spot interval of the structured light illumination super-resolution microimaging 2D-SIM, the circle centers of the 2M outer end semicircles are centrosymmetrically distributed on a third circle concentric with the opaque substrate, and the radius a of the third circle is equal to half of the +/-1-order diffraction light spot interval of the total internal reflection structured light illumination super-resolution microimaging TIRF-SIM; from the radius b of the second circle to the radius a of the third circle, half of the distance between +/-1 order diffraction light spots of three modes in the high-inclination-gradient-structure-light illumination microscopic imaging HILO-SIM is included in sequence;
the N lasers respectively emit lasers with different wavelengths; the laser coupling module couples N laser beams with different wavelengths to the same optical axis; the N coaxial laser beams with different wavelengths are transmitted to an acousto-optic tunable filter, and gating, switching and light intensity modulation of the laser beams with different wavelengths are controlled by the acousto-optic tunable filter; the laser is emitted from the acousto-optic tunable filter and then reaches a first focusing lens, the first focusing lens focuses light beams, the light beams are coupled into the polarization-maintaining single-mode fiber, the light beams are emitted from the polarization-maintaining single-mode fiber and then reach a collimation beam expander, and the collimation beam expander collimates and expands light beams and enters a polarization beam splitter of the structured light imaging main body module; loading black and white stripe images at equal intervals on the liquid crystal spatial light modulator through a polarization beam splitter to the liquid crystal spatial light modulator through a half-wave plate, wherein the function of the black and white stripe images is equivalent to that of a diffraction grating, the direction of the structured light illuminating light field is the direction of the stripes of the grating, and the phase of the structured light illuminating light field is determined by the position of the stripes of the grating; the light beam reflected from the liquid crystal spatial light modulator comprises light rays with different diffraction orders, the light rays pass through the half-wave plate again to the polarization beam splitter, the light beam passes through the half-wave plate twice, the polarization direction of the light beam is adjusted by adjusting the fast axis direction of the half-wave plate, and therefore the light intensity ratio of +/-1 order light to zero order light in diffraction light is changed; the light beams are focused by the third lens through the polarization beam splitter and are focused on the variable-pitch spatial filter; the variable-pitch spatial filter filters out diffracted light of other orders except the +/-1 order light; the +/-1-order light passes through the partition half-wave plate to adjust the polarization direction of the light beam, so that the polarization direction of a pair of +/-1-order light is vertical to the connecting line direction of diffraction light spots of the +/-1-order light and then reaches the relay lens; the relay lens conjugates the positive and negative first-order diffraction light spots on the variable-pitch spatial filter to the back focal plane of the objective lens; transmitting or reflecting the excitation light to the objective lens through the dichroic mirror; the objective lens changes a pair of positive and negative first-order diffraction light spots of a back focal plane into parallel light, and the parallel light irradiates a sample surface to generate interference to form a structured light illuminating field; fluorescent signals emitted by the structured light illuminating field irradiating the sample surface are collected by the objective lens and are reflected or transmitted by the dichroic mirror; the fluorescent signal passing through the dichroic mirror passes through an emission filter to further filter stray light and emission light of other wave bands; and focusing the image on a camera through a tube lens.
Generally speaking, because the structured light illumination confocal microscopic mode only comprises one mode, only one pair of circular light through holes are needed in one direction, and the structured light illumination super-resolution microscopic mode comprises a plurality of functional modes, a plurality of pairs of circular light through holes are needed in one direction, the centers of the circular light through holes are close, so that different circular light through holes are overlapped with each other, and the circular end rectangular pattern constructed by the method is formed. In order to avoid mutual crosstalk caused by light of non-plus or minus 1-order diffraction orders of the structured light illumination super-resolution microscopic mode and the structured light illumination confocal microscopic mode irradiating the light through hole of the opposite mode. The connection between any pair of light through holes in the structured light illumination super-resolution microscopy and any pair of light through holes in the structured light illumination confocal microscopy cannot be on the same straight line. Generally, 2N light-passing controls of the structured light illumination super-resolution microscopic mode or the structured light illumination confocal microscopic mode use the center of the light blocking sheet as a rotation center, and the whole body is rotated by an angle so that any pair of light-passing holes in the two modes are not on the same straight line.
The dichroic mirror has two types of short wave pass and long wave pass; the short-wave-pass dichroic mirror can reflect light in a set short-wave range and transmit light in a set long-wave range; the long-wavelength-pass dichroic mirror can reflect light in a set long-wavelength range and transmit light in a set short-wavelength range.
The collimating beam expander uses a second focusing lens. The relay lens adopts a fourth focusing lens and a fifth focusing lens. The non-transparent substrate of the variable-pitch spatial filter is a stainless steel plate; the laser beam combiner uses a combination of a mirror and a dichroic mirror.
The width of the structured light illumination super-resolution microscopic clear hole is 0.01mm-0.2mm wider than the diameter of the +/-1 order diffraction light spot of the structured light illumination super-resolution microscopic imaging, and the diameter of the structured light illumination confocal microscopic clear hole is 0.01mm-0.2mm wider than the diameter of the +/-1 order diffraction light spot of the structured light illumination confocal microscopic imaging.
A plurality of microscopic imaging modes are realized in one set of system, including a wide-field fluorescence imaging mode, a structured light illumination super-resolution microscopic imaging mode and a structured light illumination confocal microscopic imaging mode, and the fluorescence polarization microscopic imaging mode is fused on the basis function of each imaging mode. The functions that can be achieved and the methods used for each imaging modality are described in turn below.
(1) Achieved in a wide field fluorescence microscopy (WF) modality: non-polarizing Wide-Field microscopy (Non-polarizing Wide-Field, WF), polarizing Wide-Field microscopy (PWF).
The non-polarization wide-field microscopic imaging realizes the wide-field illumination effect by collecting a plurality of structured light illumination fields with different phases in one direction, superposing a plurality of collected sample images and complementing a single-phase non-illuminated area. By irradiating structured light fields in multiple directions and averaging, non-polarized wide-field uniform illumination imaging can be realized.
By the structured light illumination field with a plurality of different phases in one direction, a wide field illumination effect can be realized. The structured light illumination fields in at least three directions are continuously illuminated and superposed for averaging, so that the orientation of the fluorescent dipole can be further analyzed while uniform illumination is completed on the basis of a wide-field image, and the polarization wide-field microscopic imaging is realized.
(2) Implemented in a structured light illuminated super-resolution microscopy (SIM-SR) modality: total Internal Reflection Structured light illuminated super-resolution Microscopy (TIRF-SIM), high oblique layer-cut Structured light illuminated Microscopy (HILO-SIM), Two-dimensional Structured light illuminated super-resolution Microscopy (2D-SIM). In each imaging mode, the imaging functions of TIRF-pSIM, HILO-pSIM and 2D-pSIM can be realized by combining Polarization imaging, illuminating and microimaging by common Structured light and expanding the imaging mode into polarized Structured light Illumination Microscopy (pSIM).
The structured light illumination super-resolution microscopic imaging mode generally needs to irradiate three directions on a sample, wherein each direction comprises three high-frequency sinusoidal structured light striations with different phases. By acquiring nine original pictures and carrying out algorithm reconstruction, the improvement of the resolution ratio which is two times of the transverse isotropy can be realized.
(3) Implemented in a structured light illuminated Confocal microscopy (SIM-Confocal) modality: grid Confocal optical tomography (GC), Polarization Grid Confocal optical tomography (pGC).
The structured light illumination confocal imaging mode extracts high-frequency information from one uniform illumination sample image and extracts low-frequency information from the other structured light illumination sample image. By the method of fusing high-frequency and low-frequency information, the background can be effectively eliminated, and the optical slicing capability only containing in-focus information is realized.
The utility model has the advantages that:
the utility model adopts a variable-pitch spatial filter which is provided with a structured light illumination confocal microscopic clear aperture and a structured light illumination super-resolution microscopic clear aperture to realize multiple microscopic imaging modalities in a set of system, including a wide-field fluorescence imaging modality, a structured light illumination super-resolution microscopic imaging modality, a structured light illumination confocal microscopic imaging modality, and a fluorescence polarization microscopic imaging modality is added by fusion on the basis of multiple imaging functions; the rapid switching and multicolor imaging of various microscopic modes are realized through adjustment, and the process of biological imaging by using a structured light illumination microscopic method is realized; in order to quickly locate the structure to be observed in the sample, wide-field fluorescence microscopy imaging can be selected; in order to be able to obtain the three-dimensional structure of a thick sample, optical slice imaging (SIM-confocal) is optionally used; in order to be able to observe delicate intracellular structures in thin samples, fast super-resolution imaging (SIM-SR) is an option; fluorescence polarization microscopy imaging can be used to study structures with significant dipole orientation characteristics, such as cytoskeleton.
Drawings
Fig. 1 is a schematic diagram of one embodiment of a multi-mode multi-color fast switching structured light illumination system of the present invention;
fig. 2 is a schematic diagram of a variable pitch spatial filter according to an embodiment of the multi-mode multi-color fast switching structured light illumination system of the present invention.
Detailed Description
The invention will be further elucidated by means of specific embodiments in the following with reference to the drawings.
As shown in fig. 1, the multimode multicolor fast-switching structured light illumination system of the present embodiment includes a laser coupling module LCM, a structured light imaging subject module SLISM, and a fluorescence detection module FIM; the laser coupling module LCM comprises first to fourth lasers La 1-La 4, a laser beam combiner, an acousto-optic tunable filter AOTF, a first focusing lens L1, a collimation beam expander and a polarization-maintaining single-mode fiber SMF; the structured light imaging main body module SLISM comprises a polarizing beam splitter PBS, a liquid crystal spatial light modulator SLM, a half wave plate HWP, a third focusing lens L3, a variable-pitch spatial filter M, a six-partition half wave plate PHWP and a relay lens; the fluorescence detection module FIM comprises an objective lens OB, a multiband dichroic mirror DM4, an emission filter EM, a tube lens TL and a camera CCD; the collimation beam expander adopts a second focusing lens L2; the relay lens adopts a fourth focusing lens L4 and a fifth focusing lens L5; the laser beam combiner adopts first to third dichroic mirrors DM 1-DM 3 and first to third reflecting mirrors R1-R3;
as shown in fig. 2, the variable-pitch spatial filter includes an opaque substrate, a structured-light-illuminated confocal microscopy clear aperture and a structured-light-illuminated super-resolution microscopy clear aperture; the opaque substrate is a circular flat plate, and six structured light illumination confocal lenses with the same shape are arranged on the opaque substrateThe light source comprises microscopic light-passing holes, wherein the shapes of the structured light illumination Confocal microscopic light-passing holes are circular, the circle centers of six structured light illumination Confocal microscopic light-passing holes are centrally and symmetrically distributed on a first circle concentric with a non-transparent substrate, the angle between adjacent structured light illumination Confocal microscopic light-passing holes is 60 degrees, two structured light illumination Confocal microscopic light-passing holes which are centrally positioned on the same straight line passing through the center of the non-transparent substrate are in a pair, the pair of structured light illumination Confocal microscopic light-passing holes correspond to the direction of a structured light illumination field, the six structured light illumination Confocal microscopic light-passing holes are positioned at the +/-1-level light position of the structured light illumination Confocal microscopic imaging, the radius of the first circle is c, and the radius c of the first circle is equal to half of the +/-1-level diffraction light spot interval of the Grid-common of the structured light illumination Confocal microscopic imaging; 2M structured light illumination super-resolution microscopy light-passing holes with the same shape are also formed in the opaque substrate, the structured light illumination super-resolution microscopy light-passing holes are in the shape structure that firstly, a rectangle is formed, then, the center of one short side in the rectangle is used as the center of a circle, the half of the length (width) of the short side of the rectangle is used as the radius, a semicircle is formed on the outer side of the rectangle, and the diameter of the obtained semicircle is completely coincided with the short plate of the rectangle; the other short side of the rectangle is similarly operated, a graph with two semicircular ends and a rectangle in the middle can be obtained, and the graph is called as a round-end rectangle; the two structured light illumination super-resolution microscopic light-passing holes on the same diameter are in a pair, the pair of structured light illumination super-resolution microscopic light-passing holes and the pair of structured light illumination confocal microscopic light-passing holes are not in the same direction, the centers of the six inner semi-circles are centrosymmetrically distributed on a second circle concentric with the opaque substrate, the radius b of the second circle is equal to half of the +/-1-order diffraction light spot interval of the 2D-SIM under the structured light illumination super-resolution microscopic imaging mode, the centers of the six outer semi-circles are centrosymmetrically distributed on a third circle concentric with the opaque substrate, and the radius a of the third circle is equal to half of the +/-1-order diffraction light spot interval of the TIRF-SIM under the total internal reflection structured light illumination super-resolution microscopic imaging mode; the length from the radius a of the third circle to the radius b of the second circle comprises +/-1 order derivatives of three modes in HILO-SIM under a high-inclination laminar-cut structured light illumination microscopic imaging modeHalf of the spot pitch;
the width of the structured light illumination super-resolution microscopic light-passing hole is slightly larger than the diameter of a +/-1 order diffraction light spot of the structured light illumination super-resolution microscopic imaging,
the diameter of the structured light illumination confocal microscopy clear hole is slightly larger than the diameter of the +/-1 st order diffraction light spot of the structured light illumination confocal microscopy image, the width of the +/-1 st order diffraction light spot of the embodiment is 0.5mm,
the diameter of the opaque substrate is 25.4 mm;
the first to fourth lasers respectively emit laser with the wavelengths of 405nm, 488nm, 561nm and 640 nm; the laser with 405nm wavelength emitted by the first laser is sequentially transmitted by first to third dichroic mirrors DM 1-DM 3, the laser with 488nm wavelength emitted by the second laser is firstly reflected by a first reflecting mirror R1, then reflected by a first dichroic mirror DM1, then sequentially transmitted by second and third dichroic mirrors DM2 and DM3, the laser with 561nm wavelength emitted by the third laser is firstly reflected by a second reflecting mirror R2, then reflected by a second dichroic mirror DM2, then transmitted by a third dichroic mirror DM3, the laser with 640nm wavelength emitted by the fourth laser is firstly reflected by a third reflecting mirror R3, then reflected by a third dichroic mirror DM3, and the lasers with four different wavelengths are coupled to the same optical axis; the four coaxial lasers with different wavelengths are reflected by the fourth reflector R4 and the fifth reflector R5 and then transmitted to the acousto-optic tunable filter, and gating, switching and light intensity modulation of the lasers with different wavelengths are controlled by the acousto-optic tunable filter; the laser is emitted from the acousto-optic tunable filter and then reaches a first focusing lens, and the first focusing lens focuses the light beam and couples the light beam into the polarization-maintaining single-mode fiber; the light is collimated and expanded by the second focusing lens after being emitted from the polarization-maintaining single-mode fiber, and is reflected by a seventh reflector R7, an eighth reflector R8 and a ninth reflector R9 in sequence and then is transmitted to the polarization beam splitter; reflecting the back half wave plate to the liquid crystal spatial light modulator through the polarization beam splitter, loading black and white stripe images with equal intervals on the liquid crystal spatial light modulator, wherein the black and white stripe images have the function of being equivalent to a diffraction grating, the direction of the structured light illuminating light field is the direction of the stripes of the grating, and the phase of the structured light illuminating light field is determined by the position of the stripes of the grating; the light beam reflected from the liquid crystal spatial light modulator comprises light rays with different diffraction orders, the light rays pass through the half-wave plate again to the polarization beam splitter, the light beam passes through the half-wave plate twice, the polarization direction of the light beam is adjusted by adjusting the fast axis direction of the half-wave plate, and therefore the light intensity ratio of +/-1 order light to zero order light in the diffracted light is changed; the light beams are transmitted to a third focusing lens through the polarization beam splitter and focused on a variable-pitch spatial filter through the third focusing lens; the variable-pitch spatial filter filters out diffracted light of other orders except the +/-1 order light; the +/-1-order light passes through the six-subarea half-wave plate to adjust the polarization direction of the light beam, so that the polarization direction of a pair of +/-1-order light is vertical to the connecting line direction of diffraction light spots of the +/-1-order light and then reaches a fourth focusing lens and a fifth focusing lens; the fourth and fifth focusing lenses conjugate the positive and negative first-order diffraction light spots on the variable-pitch spatial filter to the back focal plane of the objective lens; the multi-waveband dichroic mirror selectively transmits excitation light with different wavebands to the objective lens after being reflected by the tenth and eleventh reflecting mirrors R10 and R11; the objective lens changes a pair of positive and negative first-order diffraction light spots of a back focal plane into parallel light, and the parallel light irradiates a sample surface S to interfere to form a sinusoidal structured light illuminating field; the structured light illuminating light field irradiates a sample surface to generate a fluorescence signal, the fluorescence signal is collected by an objective lens, and the fluorescence signals of different wave bands are selectively reflected through a multiband dichroic mirror; the fluorescent signal after passing through the multiband dichroic mirror passes through an emission filter to further filter stray light and sample emission light of other wave bands; and focusing the image on a camera through a tube lens to form an image.
Only + -1 st-order diffracted light is needed to interfere in a light field of a sample illumination sine structure, and diffracted light beams of other orders except the + -1 st-order light need to be filtered. For this reason, a pair of circular light through holes need to be designed in one direction;
in order to realize isotropic super-resolution imaging and complete dipole analysis, linear polarization illumination in at least three directions is required; for this purpose, a pair of circular light through holes is needed to be designed in each of the three directions;
the pitches of the + -1 st order diffraction light are different among a plurality of modes, and prototype light through holes with different pitches need to be designed for the purpose.
The system realizes the functions that the + -1-order diffraction light spots pass through under SIM-SR modes, the Grid-common mode under SIM-common mode and the + -1-order diffraction light spots pass through under corresponding polarization function, and the light of other orders is filtered out by a variable-spacing spatial filter (shown in figure 2). Therefore, sinusoidal fringe illumination fields with different frequencies, different directions and different phases are obtained on the sample surface.
(1) Wide field microscopic mode
The principle is as follows:
the acquisition of wide-field fluorescence microscopic image is realized in a structured light illumination system, and n phase intervals are irradiated in one direction
The structured light field of the light source is overlapped with the structured light fields of a plurality of phases, so that complementation among irradiation areas is realized, and a wide field illumination is formed. In m directions (at least three directions) in succession, each rotation
The same operation is carried out, each wide field image is superposed, and the non-polarization wide field imaging function under uniform illumination can be realized. The obtained uniform wide-field microscopic image is actually illuminated by the structured light from multiple directions, so that the method can be used for analyzing the orientation of the fluorescent dipole and realizing the polarized wide-field microscopic imaging.
The specific design method comprises the following steps:
in wide-field microscopic imaging, a variable-pitch spatial filter needs to design +/-1-order diffraction light-passing holes in three directions, and SIM-SR or SIM-Confocal light-passing holes are used in actual design.
(2) Structured light super-resolution microscopic modality
The principle is as follows:
structured light illumination super-resolution microscopic imaging is through the sinusoidal structure light field of shining spatial coding on the sample, in carrying the high frequency information of sample to the low frequency, can realize the resolution ratio promotion of twice.
In practical biological imaging applications, the required imaging depth and range will vary from subject to subject. On the basis of a structured light illumination super-resolution microscopic imaging mode, the functions of TIRF-SIM, HILO,2D-SIM and the like are further refined and realized, wherein the HILO function comprises imaging modes of HILO1-SIM, HILO2-SIM, HILO3-SIM and the like.
The <1> TIRF-SIM is that the light beams with the incidence angle exceeding the critical angle are interfered, the illumination exists only in the range of about 100nm, the 2-time expansion frequency is realized, the required light through hole interval is maximum when the light spot is diffracted in the +/-1 order on the space filter with the variable spacing.
<2> HILO-SIM extends the imaging depth to 1um using interference from highly oblique incident light slightly less than the critical angle for total reflection. The HILO1-SIM, the HILO2-SIM and the HILO3-SIM respectively realize 1.97 times, 1.95 times and 1.93 times of spreading frequency so as to adapt to the observation ranges and depths of different samples. The required clear aperture spacing for + -1 order diffraction spots on a variable pitch spatial filter is generally smaller than for TIRF-SIM and in turn decreases.
The incident light angle of the <3>2D-SIM is further reduced, enabling a larger imaging depth and 1.9 times the extension frequency compared to the first two modalities. The required light-passing hole interval of the +/-1 st order diffraction light spots on the variable-spacing spatial filter is smaller than that of the HILO-SIM.
The method comprises the following specific design:
the SIM-SR mode comprises functions of TIRF-SIM, HILO1-SIM, HILO2-SIM, HILO3-SIM, 2D-SIM and the like, and theoretically, a plurality of pairs of light-passing holes need to be designed on the variable-spacing spatial filter. In engineering, the light-transmitting holes have certain physical sizes, and the difference of the expansion frequencies corresponding to different functions is small. Therefore, a plurality of light-transmitting holes are overlapped together to form a round-end rectangle.
In actual design, the diameter of a small hole is used as the width, the TIRF-SIM +/-1 order diffraction light-passing hole is used as the outer end, and the 2D-SIM +/-1 order diffraction light-passing hole is used as the inner end. The length of the distance between the pair of light through holes in the TIRF-SIM +/-1 order diffraction minus the distance between the pair of light through holes in the 2D-SIM +/-1 order diffraction is used as the length, and a round-end rectangular light through hole can be drawn.
(3) Optical layer cutting microscopic mode
The principle is as follows:
the invention is based on the principle of structured light illumination confocal imaging, extracts high-frequency information by collecting one uniform illumination image and extracts low-frequency information by collecting the other structured light illumination image. After the frequency domain information of the two pictures is processed, the optical layer cutting microscopic imaging can be realized by splicing and fusing. SIM-Confocal was able to achieve slicing capability and 1.5 times spreading frequency for the samples.
The method comprises the following specific design:
the + -1 order diffraction spot spacing on the SIM-Confocal mode on the spatial filter is much smaller than the + -1 order diffraction spot spacing on the SIM-SR mode. In practical design, the light through hole of the SIM-SR and the light through hole of the SIM-Confocal on the spatial filter are designed in different directions, so that mutual crosstalk of different orders of diffraction light spots among different imaging modalities is avoided. The requirements of different interference fringe frequencies of the SIM-SR and the SIM-common on the sample surface are met.
Finally, it is noted that the disclosed embodiments are intended to aid in further understanding of the invention, but will be understood by those skilled in the art that: various substitutions and modifications are possible without departing from the spirit and scope of this disclosure and the appended claims. Therefore, the present invention should not be limited to the embodiments disclosed, and the scope of the present invention is defined by the appended claims.
Claims (5)
1. A multi-modal multi-color fast-switching structured light illumination system, comprising: the device comprises a laser coupling module, a structured light imaging main body module and a fluorescence detection module; the device comprises a laser coupling module, a structured light imaging main body module and a fluorescence detection module; the laser coupling module comprises N lasers, a laser beam combiner, an acousto-optic tunable filter, a first focusing lens, a collimation beam expander and a polarization-preserving single-mode fiber; the structured light imaging main body module comprises a polarization beam splitter, a liquid crystal spatial light modulator, a half-wave plate, a third focusing lens, a variable-pitch spatial filter, a subarea half-wave plate and a relay lens; the fluorescence detection module comprises an objective lens, a dichroic mirror, an emission filter, a tube lens and a camera, wherein N is a natural number not less than 2;
the variable-pitch spatial filter comprises an opaque substrate, a structured light illumination confocal microscopy clear aperture and a structured light illumination super-resolution microscopy clear aperture; the non-transparent substrate is a circular flat plate, 2M structured light illumination confocal microscopy light-passing holes with the same shape are formed in the non-transparent substrate, M is a natural number larger than or equal to 3, the structured light illumination confocal microscopy light-passing holes are circular, the circle centers of the 2M structured light illumination confocal microscopy light-passing holes are distributed on a first circle concentric with the non-transparent substrate in a central symmetry mode, a line segment formed by connecting the centers of the two structured light illumination confocal microscopy light-passing holes is located on a first circle of the non-transparent substrate, the two structured light illumination confocal microscopy light-passing holes on one diameter are located in a pair, the pair of structured light illumination confocal microscopy light-passing holes correspond to the direction of a structured light illumination field, the 2M structured light illumination confocal microscopy light-passing holes are located at +/-1-level light positions of the structured light illumination confocal microscopy imaging, the radius of the first circle is c, and the radius c of the first circle is equal to half of the +/-1-level diffraction light spot interval of the structured light illumination confocal microscopy imaging; the non-transparent substrate is also provided with 2M structured light illumination super-resolution microscopic light-passing holes with the same shape, the structured light illumination super-resolution microscopic light-passing holes are in a round end rectangle, namely the middle of the light illumination super-resolution microscopic light-passing holes is a rectangle, the two ends of the rectangle are respectively semicircles, the semicircle close to the center of the non-transparent substrate is an inner end semicircle, the semicircle far away from the center of the non-transparent substrate is an outer end semicircle, the diameter of the semicircle is equal to the short side of the rectangle, the center of the semicircle is positioned at the center of the short side of the rectangle, a line segment formed by connecting the centers of the two structured light illumination super-resolution microscopic light-passing holes is positioned on one diameter of the non-transparent substrate, and any pair of the structured light illumination super-resolution microscopic light-passing holes and any pair of the structured light illumination confocal microscopic light-passing holes can not be on the same straight line; the circle centers of the 2M inner end semicircles are centrosymmetrically distributed on a second circle concentric with the opaque substrate, the radius b of the second circle is equal to one half of the distance between +/-1-level diffraction light spots of the structured light illumination super-resolution microscopic imaging, the circle centers of the 2M outer end semicircles are centrosymmetrically distributed on a third circle concentric with the opaque substrate, and the radius a of the third circle is equal to one half of the distance between +/-1-level diffraction light spots of the total internal reflection structured light illumination super-resolution microscopic imaging; from the radius b of the second circle to the radius a of the third circle, half of the distance between +/-1-order diffraction light spots of three modes in the high-inclination laminar-cut structured light illumination microscopic imaging is included in sequence;
the N lasers respectively emit lasers with different wavelengths; the laser coupling module couples N laser beams with different wavelengths to the same optical axis; the N coaxial laser beams with different wavelengths are transmitted to an acousto-optic tunable filter, and gating, switching and light intensity modulation of the laser beams with different wavelengths are controlled by the acousto-optic tunable filter; the laser is emitted from the acousto-optic tunable filter and then reaches a first focusing lens, the first focusing lens focuses light beams, couples the light beams into the polarization-maintaining single-mode fiber, emits the light beams from the polarization-maintaining single-mode fiber and then reaches a collimation beam expander, and the collimation beam expander collimates and expands light rays and enters a polarization beam splitter of the structured light imaging main body module; loading black and white stripe images at equal intervals on the liquid crystal spatial light modulator through a polarization beam splitter to the liquid crystal spatial light modulator through a half-wave plate, wherein the function of the black and white stripe images is equivalent to that of a diffraction grating, the direction of the structured light illuminating light field is the direction of the stripes of the grating, and the phase of the structured light illuminating light field is determined by the position of the stripes of the grating; the light beam reflected from the liquid crystal spatial light modulator comprises light rays with different diffraction orders, the light rays pass through the half-wave plate again to the polarization beam splitter, the light beam passes through the half-wave plate twice, the polarization direction of the light beam is adjusted by adjusting the fast axis direction of the half-wave plate, and therefore the light intensity ratio of +/-1 order light to zero order light in diffraction light is changed; the light beams are focused by the third lens through the polarization beam splitter and are focused on the variable-pitch spatial filter; the variable-pitch spatial filter filters out diffracted light of other orders except the +/-1 order light; the +/-1-order light passes through the partition half-wave plate to adjust the polarization direction of the light beam, so that the polarization direction of a pair of +/-1-order light is vertical to the connecting line direction of diffraction light spots of the +/-1-order light and then reaches the relay lens; the relay lens conjugates the positive and negative first-order diffraction light spots on the variable-pitch spatial filter to the back focal plane of the objective lens; transmitting or reflecting the excitation light to the objective lens through the dichroic mirror; the objective lens changes a pair of positive and negative first-order diffraction light spots of a back focal plane into parallel light, and the parallel light irradiates a sample surface to generate interference to form a structured light illuminating field; fluorescent signals emitted by the structured light illuminating field irradiating the sample surface are collected by the objective lens and are reflected or transmitted by the dichroic mirror; the fluorescent signal passing through the dichroic mirror passes through an emission filter to further filter stray light and emission light of other wave bands; and focusing the image on a camera through a tube lens.
2. The multi-modal multi-color fast switching structured light illumination system of claim 1, wherein the collimating beam expander employs a second focusing lens.
3. The multi-modal multi-color fast switching structured light illumination system of claim 1, wherein the relay lens employs a fourth focusing lens and a fifth focusing lens.
4. The multi-modal multi-color fast-switching structured light illumination system of claim 1, wherein the opaque substrate of the variable pitch spatial filter comprises a stainless steel plate.
5. The multi-modal multi-color fast switching structured light illumination system of claim 1, wherein the laser beam combiner employs a combination of mirrors and dichroic mirrors.
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