WO2020125518A1 - 一种基于dmd可多色激发的结构光显微系统及多色激发方法 - Google Patents

一种基于dmd可多色激发的结构光显微系统及多色激发方法 Download PDF

Info

Publication number
WO2020125518A1
WO2020125518A1 PCT/CN2019/124600 CN2019124600W WO2020125518A1 WO 2020125518 A1 WO2020125518 A1 WO 2020125518A1 CN 2019124600 W CN2019124600 W CN 2019124600W WO 2020125518 A1 WO2020125518 A1 WO 2020125518A1
Authority
WO
WIPO (PCT)
Prior art keywords
dmd
light
wavelength
color
module
Prior art date
Application number
PCT/CN2019/124600
Other languages
English (en)
French (fr)
Inventor
陈廷爱
李慧
夏先园
高玉峰
余佳
廖九零
吴婷
郑炜
Original Assignee
中国科学院深圳先进技术研究院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院深圳先进技术研究院 filed Critical 中国科学院深圳先进技术研究院
Publication of WO2020125518A1 publication Critical patent/WO2020125518A1/zh

Links

Images

Classifications

    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G02B21/361—Optical details, e.g. image relay to the camera or image sensor
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G02B21/365—Control or image processing arrangements for digital or video microscopes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/10—Beam splitting or combining systems
    • G02B27/1006—Beam splitting or combining systems for splitting or combining different wavelengths

Definitions

  • the invention relates to the technical field of microscopic optical imaging, in particular to a structured light microscopy system capable of multi-color excitation based on DMD and a multi-color excitation method.
  • Fluorescence microscopes use fluorescent molecules as probes to specifically mark the observation site of the sample, so that it has molecular specific recognition. This imaging method not only improves the image contrast of the observed sample, but also can achieve flexible sample feature labeling. Ideal imaging tool for scientific research such as biology and life medicine.
  • multi-color fluorescence microscopy allows people to not only observe the interaction between multiple proteins in the same cell at the same time. Role, you can also study the interaction process between many different cells or tissues.
  • the fluorescence structure outside its focal plane is blurred and faint. Regardless of which part of the sample the microscope focuses the excitation light on vertically, the entire thickness of the irradiated sample will be excited by fluorescence, that is, not only the fluorescence on the focal plane will be collected, but also the scattered fluorescence from above or below the focal plane Collected by objective lens. More than 90% of the fluorescence in the two-dimensional image obtained from the focal plane imaging of the sample comes from defocused light, which even completely submerges the details of the focal plane and greatly reduces the image contrast.
  • the structured light microscope is based on the traditional fluorescent microscope to change the uniform light illumination to structured light illumination, to produce a set of modulated light of different phases on the sample, and then extract from this set of image data modulated with different phases through a phase shift algorithm
  • a wide-field microscopic imaging technology developed by focal plane information, reconstructed tomographic images and three-dimensional images. It has high image quality contrast, simple system structure, fast imaging speed, and low cost. Compared with confocal laser scanning microscope and two-photon fluorescence microscope, it has great advantages in cost performance.
  • the illumination light source of the structured light microscopy system is divided into coherent light source illumination (such as laser illumination) and non-coherent light source illumination (such as high-pressure mercury lamp or LED illumination) according to whether there is coherence.
  • coherent light source illumination such as laser illumination
  • non-coherent light source illumination such as high-pressure mercury lamp or LED illumination
  • the currently announced structured light microscopy system based on Digital Micro-mirror Device (DMD) uses non-coherent light sources for illumination.
  • DMD Digital Micro-mirror Device
  • patent CN104570315A uses LED lighting
  • patent CN101655601B uses mercury lamps. illumination.
  • no matter whether it uses coherent light source illumination or incoherent light source illumination there is no proposed or published structured light microscopy system based on DMD that can be multi-color excited.
  • the DMD itself is a two-dimensional blazed grating, which diffracts incident light of different wavelengths in different directions, resulting in beams of different wavelengths no longer concentric and coaxial after being diffracted by the DMD.
  • the structured light projection shift of the different wavelengths of illumination light on the sample surface caused by the fundamental property of DMD is acceptable, but complex reconstruction of tomographic images and 3D image reconstruction algorithms is required; but for the illumination of coherent light sources, the fundamental property of DMD will directly cause the failure of structured light fringes caused by interference of illumination lights of different wavelengths on the sample surface.
  • the present invention proposes a structured light microscopy system and a multi-color excitation method based on DMD that can multi-color excitation, which can conveniently provide tomographic images and three-dimensional images under mono-color or multi-color excitation , And retain the advantages of DMD-based structured light illumination microscopy system's illumination stability, illumination space uniformity and imaging speed.
  • a structured light microscopy system capable of multi-color excitation based on DMD includes a multi-color coupling module, a multi-color declination module, a DMD, a fluorescence excitation module and a fluorescence collection module which are sequentially arranged on the optical path.
  • the multi-color coupling module is used for To couple light sources of at least two wavelengths in the same optical path, make the light beams of each wavelength concentric and coaxial, and select light sources of different wavelengths for time-sharing output; the multi-color declination module is used to couple the multi-color
  • the light beam incident on the module is pre-processed so that each beam incident on the DMD is diffracted to be concentric and coaxial;
  • the fluorescence excitation module is used to generate structured light illumination on the sample surface using the light emitted from the DMD;
  • the fluorescence collection module is used to collect and image the fluorescence signal of the sample excited by the structured light illumination.
  • the multi-color coupling module includes a plurality of lasers and a narrow-band filter, an optical processing element, and an acousto-optic filter respectively provided on the optical path of each of the lasers. After being filtered by respective narrow-band filters, processed by respective light processing elements and then coupled to a concentric coaxial axis, the acousto-optic filter is used to select and output the light beams emitted by each light processing element according to wavelength.
  • the light processing element includes a mirror and/or a dichroic mirror.
  • the light processing element includes a prism, one of the prism surfaces of the prism is an exit surface that emits light toward the acousto-optic filter, and each of the narrow-band filters is attached to the other prism surface of the prism .
  • the multi-color coupling module includes a plurality of lasers and focusing lenses respectively provided on the optical path of each of the lasers, as well as a fiber combiner and a collimating lens.
  • Each focusing lens enters the optical fiber after focusing, and the optical fiber combiner couples the light beams of each optical fiber to the same optical fiber and outputs the time-sharing according to the wavelength selection.
  • the light beam emitted from the optical fiber combiner is collimated by the collimating lens Concentric and coaxial beams.
  • the multi-color deflection angle module includes an angle compensation unit for generating a preset deflection angle of the incident light beam and a displacement compensation unit for generating a displacement of the incident light beam perpendicular to the optical axis direction .
  • the angle compensation unit is a blazed grating
  • the displacement compensation unit is an electrically controlled translation stage that can drive the blazed grating to reciprocate.
  • the grating constant and the blaze angle of the blazed grating are consistent with the DMD.
  • Another object of the present invention is to provide a multi-color excitation method, including:
  • Coupling illumination light sources of at least two wavelengths in the same optical path making the light beams of each wavelength concentric and coaxial, and selecting light sources of different wavelengths for time-sharing output;
  • the preprocessing of the light beam incident from the multicolor coupling module includes:
  • the incident light beam is displaced perpendicular to the direction of the optical axis.
  • the invention couples the illumination light sources of different wavelengths in the same optical path, makes the light beams of each wavelength concentric and coaxial, and selects the light sources of different wavelengths for time-sharing output.
  • the beams entering the DMD are diffracted to be concentric and coaxial; then, by generating structured light illumination on the sample surface, the fluorescence signals excited by the structured light illumination of the sample can be collected and imaged.
  • DMD is used to edit the structure to generate the required structured light illumination, and the structured light stripes of each wavelength can be well overlapped, retaining the advantages of illumination stability, illumination space uniformity and imaging speed of the structured light illumination microscopy system based on DMD.
  • FIG. 1 is a structural block diagram of a structured light microscope system capable of multi-color excitation according to the present invention
  • FIG. 2 is a schematic diagram of the optical path of a structured light microscope system capable of multi-color excitation according to Embodiment 1 of the present invention
  • FIG. 3 is a schematic diagram of the optical path of the multi-color coupling module according to Embodiment 1 of the present invention.
  • FIG. 4 is a schematic diagram of the optical path of the multi-color declination module according to Embodiment 1 of the present invention.
  • FIG. 5 is a schematic diagram of the optical path of the fluorescent excitation module according to Embodiment 1 of the present invention.
  • FIG. 6 is a schematic diagram of the optical path of the fluorescence collection module according to Embodiment 1 of the present invention.
  • FIG. 7 is a schematic diagram of the optical path of the multi-color coupling module according to Embodiment 2 of the present invention.
  • FIG. 8 is a schematic diagram of the optical path of the multi-color coupling module according to Embodiment 3 of the present invention.
  • the structured light microscopy system based on DMD capable of multi-color excitation of the present invention includes a multi-color coupling module 11, a multi-color declination module 12, a DMD 13, a fluorescence excitation module 14 and a fluorescence collection module 15 which are sequentially arranged on the optical path ,
  • the multi-color coupling module 11 is used to couple the light sources of at least two wavelengths in the same optical path, so that the light beams of each wavelength are concentric and coaxial, and the light sources of different wavelengths are selected for time-sharing output;
  • the multi-color declination module 12 is used to The light beam incident from the multicolor coupling module 11 is pre-processed so that each beam incident on the DMD 13 is diffracted to be concentric and coaxial;
  • the fluorescence excitation module 14 is used to generate structured light illumination on the sample surface using the light emitted from the DMD 13; fluorescence collection
  • the module 15 is used to collect and image the fluorescence signal of the sample excited by the structured light
  • the invention also correspondingly provides a method for realizing multi-color excitation, which mainly includes:
  • S02. Pre-process the light sources of each wavelength output by time-sharing, so that the beams entering the DMD13 are concentric and coaxial after being diffracted;
  • the multi-color deflection angle module 12 includes an angle compensation unit 121 for generating a preset deflection angle for the incident light beam and a displacement compensation unit 122 for generating a displacement perpendicular to the optical axis direction of the incident light beam.
  • pre-processing the light sources of each wavelength output by time-sharing includes:
  • the incident light beam is displaced perpendicular to the direction of the optical axis.
  • the beams of different wavelengths generate a preset angle offset to compensate for the diffraction of the incident light of different wavelengths after the DMD Angular deviation, meanwhile, the displacement compensation unit 122 compensates the longitudinal (vertical to the optical axis direction) displacement deviation generated by the diffracted light of different wavelengths.
  • the beams are concentric and coaxial.
  • the multi-color declination module 12 of the present invention processes the beam before it enters the DMD 13 that generates a preset declination angle and a displacement perpendicular to the direction of the optical axis.
  • the displacement deviation compensation can still maintain the multi-wavelength beams to be concentric and coaxial, and the subpole diffraction will be symmetrically distributed on both sides of the main pole diffraction light.
  • the main and sub-order diffracted beams of these different wavelengths then enter the fluorescence excitation module 14, thereby interfering on the sample surface to form structured light illumination.
  • the illumination beams of different wavelengths can not only be structured by DMD to generate the required structured light illumination on the sample surface, but also the structured light stripes of each wavelength can be well overlapped.
  • the multi-color coupling module 11 of this embodiment is used to couple illumination light sources of multiple wavelengths into the same optical path to achieve time-sharing output, including a plurality of lasers 111 and separately provided for each laser In the optical path of 111, a narrow-band filter 112, a light processing element 113, and an acousto-optic filter (AOTF) 114.
  • the beams emitted by each laser 111 are filtered by their respective narrow-band filters 112, and then pass through their respective light processing elements After the 113 processing, it is coupled to a concentric coaxial axis.
  • the acousto-optic filter 114 is used to select and output the light beams emitted by the optical processing elements 113 according to the wavelength.
  • the light processing element 113 may use various combinations of reflecting mirrors and dichroic mirrors to realize its optical processing function.
  • this embodiment takes four lasers 111 as an example for description, wherein each laser 111 is used to generate laser light sources of different wavelengths, respectively.
  • the laser 111 has a laser one 111a, a laser two 111b, a laser three 111c, and a laser four 111d, respectively generating a wavelength 1 (for example 635nm), a wavelength 2 (for example 561nm), a wavelength 3 (for example 488nm), and a wavelength 4 (for example 405nm ) Laser;
  • the narrow-band filter 112 also has four: 112a, 112b, 112c, 112d, for allowing the optical signal to pass in a specific band, while the optical signals on both sides that deviate from this band are blocked; the light processing element 113
  • the reflector 113a, dichroic mirrors 113b, 113c, and 113d are included.
  • each laser passes through a narrow-band filter 112, and then is reflected by different mirrors/dichroic mirrors to the same acousto-optic filter 114: laser one 111a outputs a wavelength of 1, and passes through the narrow-band filter 112a (E.g. 635 ⁇ 10nm) is reflected by mirror 113a (working at 45° angle) and enters acousto-optic filter 114; laser two 111b outputs wavelength 2 and passes narrowband filter 112b (e.g.
  • dichroic mirror 113b by dichroic mirror 113b Reflection (for example, reflecting laser light with a wavelength below 561 nm, transmitting laser light with a wavelength of 561 nm and above, and working at a 45° angle) enters the acousto-optic filter 114, and before the wavelength 2 enters the acousto-optic filter 114, it is coupled to a concentric coaxial with wavelength 1;
  • Laser III 111c outputs a wavelength of 3, is reflected by a dichroic mirror 113c (e.g., reflects laser light of less than 488nm wavelength, transmits laser light of greater than 488nm wavelength, and works at an angle of 45°) through a narrow-band filter 112c (e.g., 488 ⁇ 10nm).
  • Optical filter 114, wavelength 3 is coupled to wavelength 1 and wavelength 2 to be concentric and coaxial before entering the acousto-optic filter 114; laser four 111d outputs wavelength 4 and is dichroic through a narrow-band filter 112d (eg 405 ⁇ 10nm)
  • the mirror 113d (for example, reflects laser light with a wavelength of 405 nm, transmits laser light with a wavelength greater than 405 nm, and works at an angle of 45°) reflects into the acousto-optic filter 114, and wavelength 4 is coupled with wavelength 1, wavelength 2, and wavelength 3 before entering the acousto-optic filter 114. To concentric and coaxial.
  • the acousto-optic filter 114 is used to select time-division output for four wavelengths, and the light beams are concentric and coaxial when each wavelength is output from the acousto-optic filter 114.
  • the reflecting mirror 113a, the dichroic mirrors 113b, 113c, and 113d are sequentially arranged along a straight line and are parallel to each other.
  • the multi-color declination module 12 is used to generate specific angular offset and longitudinal displacement deviation compensation, so that beams of different diffraction orders of different wavelengths undergo DMD diffraction to realize multi-wavelength beams to be concentric and coaxial. It includes an angle compensation unit 121 for generating a preset deflection angle of the incident beam and a displacement compensation unit 122 for displacing the incident beam perpendicular to the optical axis direction.
  • the angle compensation unit 121 is a blazed grating
  • the displacement compensation unit 122 is an electrically controlled translation stage that can drive the blazed grating to reciprocate.
  • the multi-wavelength coaxial beams coupled by the multicolor coupling module 11 are first irradiated onto the blazed grating to diffract, so that the beams of different wavelengths are shifted by an angle in advance, and at the same time, the blazed grating is fixed on a precision electronically controlled translation stage , Directly control the movement of the blazed grating in a fixed direction (such as the direction of incident light) through the control program to compensate for the longitudinal (vertical to the optical axis) displacement deviation generated when the diffracted beams of different wavelengths propagate forward, through these two methods
  • the combination of multiple beams after being diffracted by DMD is concentric and coaxial.
  • the grating should be a specific blazed grating (such as the same DMD, or a customized reflective blazed grating, or a customized transmission blazed Grating), the grating constant d and the blaze angle ⁇ need to be consistent with the DMD in order to set a specific angular offset for the diffracted light of different wavelengths in advance.
  • the 405nm wavelength laser corresponds to a diffraction order of 10
  • the diffraction angle is 17.22 degrees
  • the 488nm wavelength laser corresponds to a diffraction order of 8
  • the diffraction angle is 16.58 degrees
  • the 561nm wavelength laser corresponds to The diffraction order is 7 and the diffraction angle is 16.68 degrees.
  • the corresponding diffraction order of the laser at a wavelength of 635 nm is 6 and the diffraction angle is 16.17 degrees.
  • the beams of different wavelengths After passing through the blazed grating, the beams of different wavelengths not only cause the angle deviation we set in advance due to the different diffraction directions, but also cause the displacement deviation in the longitudinal direction (perpendicular to the optical axis direction) due to the different diffraction orders.
  • the blazed grating of this embodiment is fixed to a precision electronically controlled translation stage (for example, with a step accuracy of 1 ⁇ m), and cooperates with the acousto-optic filter 114 in the multi-color coupling module 101, and at the same time the wavelength is selected for time-sharing output, the blazed grating is controlled by a control program Move a specific position along a fixed direction (such as the direction of incident light), different wavelengths move different positions (for example, wavelength 1 corresponds to position 1, wavelength 2 corresponds to position 2, wavelength 3 corresponds to position 3, and wavelength 4 corresponds to position 4), thereby offsetting the difference Longitudinal (perpendicular to the optical axis) displacement deviation due to wavelength.
  • a control program Move a specific position along a fixed direction (such as the direction of incident light), different wavelengths move different positions (for example, wavelength 1 corresponds to position 1, wavelength 2 corresponds to position 2, wavelength 3 corresponds to position 3, and wavelength 4 corresponds to position 4), thereby offsetting the difference Longitudinal
  • the grating 301 is a special reflective blazed grating, after exiting DMD13, the 10th order diffracted light at 405nm wavelength, the 8th order diffracted light at 488nm wavelength, the 7th order diffracted light at 561nm wavelength, the 6th order diffracted light at 635nm wavelength, When diffracted from DMD13, it can be concentric and coaxial.
  • an optical path folding module (not shown in the figure) can also be added according to the actual situation to expand the concentric coaxial beam emitted from the multicolor coupling module 11 and turn it up to Color Offset Module 12.
  • the optical path folding module includes a beam expansion group and a mirror jumping group that are sequentially arranged on the optical path.
  • the beam expansion group includes a lens Z1 and a lens Z2, and the mirror jumping group includes a mirror Z3 and a mirror Z4, and time-sharing from the acousto-optic filter 114
  • the selected concentric and coaxial beams are sequentially expanded by the beam expansion group composed of lens Z1 and lens Z2, and the expanded beams are then reflected by the jumping mirror group composed of mirrors Z3 and Z4, and the reflected beams enter the polychromatic polarization
  • the multi-wavelength concentric coaxial beam after the beam expansion and beam folding of the optical path folding module irradiates the transmissive blazed grating at the same incident angle.
  • the structured light illumination microscope uses the mutual interference between different diffraction orders of the same light beam to generate a structural pattern to generate illumination on the sample surface P.
  • the light beams of different wavelengths are concentric and coaxial (for example, 10th order diffracted light at 405nm wavelength, 8th order diffracted light at 488nm wavelength, 7th order diffracted light at 561nm wavelength, and 6th order diffracted at 635nm wavelength
  • the light when diffracted from the DMD, is concentric and coaxial).
  • the fringe pattern is loaded on the DMD13, since the fringe pattern is also a periodic grating, sub-order diffraction will be generated on the basis of the main-order diffraction of the DMD itself.
  • the fluorescence excitation module 14 of this embodiment is used to generate structured light illumination on the sample surface P to excite the sample to generate fluorescence, including the lenses 141, 142, 143 and the objective lens 144, and the DMD 13 is placed in the fluorescence excitation
  • the relay lens composed of 143 is imaged into the rear aperture of the objective lens 144 (for example, an objective lens with a large magnification and a high NA value), and the beams of each diffraction order interfere with the objective lens 144 on the sample surface P to form structured illumination light.
  • the objective lens 144 for example, an objective lens with a large magnification and a high NA value
  • the lens 141 for example, relative aperture and focal length
  • only the 0th order diffracted light with the nth order as the main pole can be allowed (for example, the 0th order diffracted light with the 10th order of the 405 nm wavelength as the main pole, 488 nm
  • the 8th order wavelength diffracted light as the main pole, the 7th order wavelength of 561nm as the main order 0th order diffracted light, the 635nm wavelength order 6 as the main order 0th order diffracted light), n -1st order diffracted light as the main pole for example, -10th order diffracted light of the 405nm wavelength as the main pole, -1st order diffracted light of the 488nm wavelength as the main pole, 561nm wavelength 7th order as the main pole of the -1st order diffracted light, 635nm wavelength 6th order as the main pole of the -1st order diffracted light), nth order as the main pole of the
  • the 6th order of the 635 nm wavelength is the +1st order sub-diffracted light of the main pole
  • a total of three sub-order diffracted lights pass through, and the diffracted light of the other sub-orders is restricted by the aperture of the lens 141 outside the optical path.
  • the DMD 13 is placed in the front focal plane of the lens 142 in the fluorescence excitation module 14.
  • the n-th order is The 0th order diffracted light focusing spot of the main pole, the -1st order diffracted light focusing spot of the nth order as the main pole, and the +1st order diffracted light focusing spot of the nth order as the main pole.
  • the fluorescence collection module 15 of this embodiment is used to collect and image the fluorescence signal of the sample excited by structured light illumination, and includes a dichroic mirror 151 which is sequentially located on the optical path of excited fluorescence , Imaging lens 152, filter 153, imaging detector 154.
  • the excited fluorescent signal on the sample surface P is collected by the objective lens 144 and enters the imaging detector 154 through the dichroic mirror 151, the imaging lens 152, and the filter 153 in sequence, and the filter 153 is used to filter background signals other than fluorescence
  • the dichroic mirror 151 may be a reflection 405nm/488nm/561nm/635nm wavelength, and a multi-band pass dichroic mirror that transmits other wavelengths
  • the filter 153 may be equipped with four 405nm long pass/488nm long pass /561nm long-pass/635nm long-pass filter wheel, or a single multi-band pass filter with 426-462nm bandpass, 502.5-544.5nm bandpass, 582-617.5nm bandpass, and 663-1200nm bandpass.
  • the main process of implementing multi-color excitation is:
  • the multi-color coupling module 11 couples the illumination light sources of different wavelengths into the same optical path, the light beams of each wavelength are concentric and coaxial, and can select the time-sharing output of lasers of different wavelengths;
  • the light sources of each wavelength output by time-sharing specifically after a laser beam of a certain wavelength is selected and output, it passes through the optical path folding module and expands and turns into the transmissive blazed grating in the multicolor declination module 12 .
  • the incident beam produce a preset declination, and at the same time, by controlling the transmission blazed grating fixed on the precision electronically controlled translation stage to move to a corresponding appropriate position along a fixed direction (such as the direction of the incident light), the incident beam A displacement perpendicular to the direction of the optical axis is generated to offset the displacement deviation of the wave length in the longitudinal direction (perpendicular to the direction of the optical axis).
  • the laser with wavelength 1 After the laser with wavelength 1 is selected for output, it passes through the optical path folding module to expand the beam, and then enters the transmissive blaze grating in the multicolor declination module 12, and the control program controls the transmissive blaze fixed on the precision electronically controlled translation stage.
  • the grating moves to position 1 in a fixed direction;
  • the laser light of wavelength 1 is diffracted after passing through the transmissive blazed grating and then irradiated onto the DMD13.
  • the Littrow configuration because the grating constant of the transmission blazed grating and the DMD13 is consistent with the blaze angle design, after the beam of wavelength 1 is diffracted by the DMD13 again, its diffraction angle is restored to 0 degrees.
  • the control program controls the transmissive type fixed on the precision electronically controlled translation stage
  • the blazed grating 301 moves to position 2 in a fixed direction;
  • the laser beam of wavelength 2 is also diffracted after passing through the transmissive blazed grating and irradiated on the DMD13. After the beam of wavelength 2 is diffracted by DMD13 again, its diffraction angle is also restored to 0 degrees. Moreover, since the position of the transmissive blazed grating fixed on the precision electronically controlled translation stage is controlled in a fixed direction by the control program in advance, the displacement deviation of the wavelength 2 and the wavelength 1 in the longitudinal direction is cancelled, and the wavelength 2 and the wavelength 1 are concentric Coaxial.
  • the control program is fixed at the precision
  • the transmissive blazed grating on the controlled translation stage moves to position 3 in a fixed direction;
  • the laser light of the wavelength 3 is diffracted after passing through the transmissive blazed grating and irradiated on the DMD 13. After the beam of wavelength 3 is diffracted by DMD 13 again, its diffraction angle is also restored to 0 degrees. Like the wavelength 2 and the wavelength 1, the displacement deviation of the wavelength 3 in the longitudinal direction is also canceled, and the wavelength 3, the wavelength 2 and the wavelength 1 are concentric and coaxial.
  • the laser at wavelength 4 After the laser at wavelength 4 is selected for output, it is the same as wavelength 3, wavelength 2, and wavelength 1.
  • the control program is fixed at this time.
  • the transmissive blazed grating on the precision electronically controlled translation stage moves to position 4 in a fixed direction.
  • wavelength 2 and wavelength 1 when the incident angle is 0 degrees, the laser light of wavelength 4 is diffracted after passing through the transmissive blazed grating and irradiated on the DMD 13. After the light beam with wavelength 4 is diffracted by DMD 13 again, its diffraction angle is also restored to 0 degrees. Like Wavelength 3, Wavelength 2, and Wavelength 1, the displacement deviation of Wavelength 4 in the longitudinal direction is also cancelled. Wavelength 4, Wavelength 3, Wavelength 2, and Wavelength 1 are concentric and coaxial.
  • sub-order diffracted light for example, 635nm
  • a laser beam with a wavelength of 1 for example, 635nm wavelength
  • sub-order diffracted light for example, 635nm
  • nth order of wavelength 1 as the main pole is generated (The 6th order of the wavelength as the main pole of the 0th order, ⁇ 1st order, ⁇ 2nd order diffracted light);
  • sub-order diffracted light for example, 561 nm
  • sub-order diffracted light for example, 561 nm
  • a sub-order diffracted light (for example, 488 nm) containing the 0th order, ⁇ 1st order, ⁇ 2nd order, etc. of the nth order of wavelength 3 as the main pole is generated.
  • the 8th order of the wavelength as the main pole of the 0th order, ⁇ 1st order, ⁇ 2nd order diffracted light is generated.
  • sub-order diffracted light eg, 405 nm, including 0th order, ⁇ 1st order, ⁇ 2nd order, etc.
  • the 10th order of the wavelength serves as the 0th order, ⁇ 1st order, and ⁇ 2nd order diffracted light of the main pole.
  • the nth order of each wavelength is the main pole
  • the 0th order diffracted light is concentric and coaxial
  • the nth order of each wavelength is the main Polar diffracted light of ⁇ 1, ⁇ 2 orders will cause different diffraction directions due to different wavelengths, and will no longer be concentric.
  • these sub-order diffracted beams retain only the 0th order diffracted light as the main pole, the -1st order diffracted light as the main pole, and the +1st order diffracted as the main pole.
  • Light, diffracted light of three sub-orders in total passes, and diffracted light of other sub-orders is restricted by the aperture of the lens 141 outside the optical path.
  • the DMD 13 is placed in the front focal plane of the lens 141 in the fluorescence excitation module 14. After the diffracted beams of the main and sub-orders of different wavelengths pass through the lens 141, the n-th order of different wavelengths is formed on the back focal plane as the 0th order of the main pole Sub-diffracted light focusing spot, n-th order diffracted light focusing spot as main pole, and n-th order diffracted light focusing spot as main pole. These focusing spots are then imaged into the rear aperture of the objective lens 144 through the relay lens composed of the lens 142 and the lens 143, and the beams of each diffraction order interfere with the objective lens 144 on the sample surface P to form structured illumination light.
  • the illumination light of different wavelengths can not only be structured by DMD to generate the required structured light illumination on the sample surface, but also the structured light stripes of each wavelength can be well overlapped.
  • the multi-color coupling module 11 of this embodiment also includes a plurality of lasers 111 and a narrow-band filter 112, an optical processing element 113, and an acousto-optic filter (which are respectively provided on the optical path of each laser 111) AOTF)114.
  • the light beams emitted by the lasers 111 are respectively filtered by the respective narrow-band filters 112, and then processed by the same light processing element 113 and then coupled to the concentric Coaxial, and finally the light beam emitted by the acousto-optic filter 114 to the light processing element 113 is output in a time-selective manner according to wavelength.
  • each wavelength is also coupled to a concentric axis before entering the acousto-optic filter 114.
  • the main component of the light processing element 113 is a prism.
  • One of the prism faces of the prism is an exit face that emits light toward the acousto-optic filter 114, and each narrow-band filter 112 is attached to the other prism face of the prism.
  • each narrow-band filter 112 is attached to a different prism surface of the prism, and only one of the prism surfaces faces the acousto-optic filter 114 as the light exit surface.
  • the prism is explained by taking a pentagonal prism as an example.
  • Each prism surface of the prism is attached with a narrow-band filter 112 for filtering different wavelengths, and each narrow-band filter 112 corresponds to a laser for generating different wavelengths. 111.
  • Laser 111a outputs wavelength 1 (for example, 635nm), passes through narrow-band filter 112a (for example, 635 ⁇ 10nm, works at an angle of 72°), and enters the pentagonal prism at an incident angle of 18° to the interface normal, and then passes through the pentagonal prism After reflecting from the other two sides, it exits from the exit surface of the pentaprism and enters the acousto-optic filter 114;
  • wavelength 1 for example, 635nm
  • narrow-band filter 112a for example, 635 ⁇ 10nm, works at an angle of 72°
  • Laser II 111b outputs wavelength 2 (eg 561 nm), passes through narrow-band filter 112b (eg 561 ⁇ 10 nm, works at an angle of 72°) and enters the pentaprism at an angle of incidence of 18° from the interface normal, directly from the pentaprism After exiting, the exit surface enters the acousto-optic filter 114, and the wavelength 2 is coupled with the wavelength 1 to be concentric and coaxial before entering the acousto-optic filter 114;
  • wavelength 2 eg 561 nm
  • narrow-band filter 112b eg 561 ⁇ 10 nm, works at an angle of 72°
  • the exit surface enters the acousto-optic filter 114, and the wavelength 2 is coupled with the wavelength 1 to be concentric and coaxial before entering the acousto-optic filter 114;
  • Laser III 111c has an output wavelength of 3 (for example, 488nm), passes through a narrow-band filter 112c (for example, 488 ⁇ 10nm, works at an angle of 72°), and enters the pentaprism at an incident angle of 18° to the interface normal, and then passes through the pentaprism After being reflected from the other three sides, it exits from the exit surface of the pentaprism and enters the acousto-optic filter 114. Before the wavelength 3 enters the acousto-optic filter 114, it is coupled with the wavelength 1 and the wavelength 2 to be coaxial and coaxial;
  • the laser four 111d outputs a wavelength of 4 (for example, 405nm), passes through a narrow-band filter 112d (for example, 405 ⁇ 10nm, works at an angle of 72°), enters the pentaprism at an incident angle of 18° to the interface normal, and then passes through the pentaprism After being reflected from the other side, it exits from the pentaprism and enters the acousto-optic filter 114. Before the wavelength 4 enters the acousto-optic filter 114, it is coupled with the wavelength 1, wavelength 2, and wavelength 3 to be concentric and coaxial.
  • Each wavelength is coupled to the concentric coaxial axis from the acousto-optic filter 114 before entering, and then the acousto-optic filter 114 selects the time-sharing output for the four wavelengths, and the light beams are also concentric and coaxial when output from the acousto-optic filter 114 for each wavelength.
  • the prism may also have an empty prism surface, or the light processing element 113 of Embodiment 1 may be disposed beside the prism of this embodiment, and the light beam emitted by the light processing element 113 of Embodiment 1 is from one of the prisms. It enters the surface, couples with other beams, and exits to the acousto-optic filter 114.
  • the multi-color coupling module 11 of this embodiment includes a plurality of lasers 111 and focusing lenses 115 respectively provided on the optical path of each laser 111, and fiber combining Collimator 116, collimating lens 117, the beams emitted by each laser 111 are focused by respective focusing lenses 115 and enter the optical fiber, and the fiber combiner 116 couples the beams of each optical fiber to the same optical fiber and selects the time-sharing output according to the wavelength.
  • the beam emitted by the beam combiner 116 is collimated by the collimator lens 117 into a concentric and coaxial beam.
  • One end of the optical fiber combiner 116 couples the light beams of the optical fibers, and the other end transmits the coupled light beams to the collimator lens 117.
  • one end of the optical fiber combiner 116 is provided with an optical fiber connector 1161 corresponding to the focusing optical fiber of each focusing lens 115, including optical fiber connectors 1161a, 1161b, 1161c, 1161d, and the other end of the optical fiber combiner 116 is provided with optical fibers Connector 1162.
  • Both the focusing lens 115 and the collimating lens 117 may be single lenses, double cemented lenses, or low-power microscope objectives.
  • the fiber connectors 1161a/1161b/1161c/1161d correspond to the focusing lenses 115a/115b/115c/115d, respectively.
  • Laser 111a outputs a wavelength of 1 (for example, 635nm), is focused on the incident end face of the fiber connector 1161a through the focusing lens 115a, and is coupled into the fiber;
  • Laser II 111b outputs wavelength 2 (for example, 561 nm), focuses on the incident end surface of the fiber connector 1161b through the focusing lens 115b, and couples into the fiber. Wavelength 2 and wavelength 1 are coupled into the fiber connector 1162 at the other end through the fiber combiner 116 The incident end face enters the optical fiber;
  • Laser III 111c outputs wavelength 3 (for example, 488nm), focuses it through the focusing lens 115c to the incident end surface of the fiber connector 1161c, and couples into the fiber. Wavelengths 3, 2 and 1 are coupled through the fiber combiner 116 into the other end of the fiber connection The incident end face of the device 1162 enters the optical fiber;
  • the laser four 111d outputs a wavelength of 4 (eg 405 nm), is focused by the focusing lens 115d on the incident end face of the optical fiber connector 1161d and is coupled into the optical fiber, and the wavelength 4, wavelength 3, wavelength 2 and wavelength 1 are coupled into the other end through the optical fiber combiner 116
  • the incident end face of the optical fiber connector 1162 enters the optical fiber;
  • the optical fiber combiner 116 couples the four wavelengths into the same optical fiber connector 1162 together, and can select the time-division output of each wavelength.
  • the laser beams of wavelength 1, wavelength 2, wavelength 3, and wavelength 4 are collimated by the collimator lens 117 into a concentric coaxial beam after being emitted from the exit end face of the optical fiber connector 1162.
  • the invention couples the illumination light sources of different wavelengths in the same optical path, makes the light beams of each wavelength concentric and coaxial, and selects the light sources of different wavelengths for time-sharing output.
  • the beams entering the DMD are diffracted to be concentric and coaxial; then, by generating structured light illumination on the sample surface, the fluorescence signals excited by the structured light illumination of the sample can be collected and imaged.
  • DMD is used to edit the structure to generate the required structured light illumination, and the structured light stripes of each wavelength can be well overlapped, retaining the advantages of illumination stability, illumination space uniformity and imaging speed of the structured light illumination microscopy system based on DMD.

Landscapes

  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Multimedia (AREA)
  • Computer Vision & Pattern Recognition (AREA)
  • Microscoopes, Condenser (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

一种基于DMD可多色激发的结构光显微系统及多色激发方法。结构光显微系统包括依次设于光路上的多色耦合模块(11)、多色偏角模块(12)、DMD(13)、荧光激发模块(14)和荧光采集模块(15)。多色耦合模块(11)将至少两种波长的光源耦合于同一光路中，使各波长的光束共心共轴，并选择不同波长的光源分时输出。多色偏角模块(12)对自多色耦合模块(11)射入的光束进行预处理，使射入DMD(13)的各光束经衍射后共心共轴。荧光激发模块(14)利用DMD(13)射出的光在样品面产生结构光照明，荧光采集模块(15)将样品被结构光照明激发出的荧光信号进行收集成像。由此，结构光照明显微系统具有照明稳定性、照明空间均匀性和成像速度的优势。

Description

一种基于DMD可多色激发的结构光显微系统及多色激发方法 技术领域
本发明涉及显微光学成像技术领域，尤其涉及一种基于DMD可多色激发的结构光显微系统及多色激发方法。
背景技术
荧光显微镜利用荧光分子作为探针对样品观察部位予以特殊标记，使之具有分子特异性识别，这种成像方式不仅提升了所观察样品的图像对比度，还可以实现灵活的样品特征标记，目前已经成为生物学与生命医学等科学研究理想的成像工具。同时随着各种新型荧光分子探针的出现以及可在同一样品上进行多种不同目标标记的荧光标记技术发展，多色荧光显微镜使得人们不仅可以同时观测同一细胞中多种蛋白质之间的相互作用，还可以研究多种不同细胞或组织之间的相互作用过程。
当需要对较厚的样品成像时，传统的荧光显微镜的其中一个难以克服的缺点就会显现出：其焦平面以外的荧光结构模糊、发虚。无论显微镜将激发光垂直聚焦到样品的哪一部分，被照射到的整个厚度的样品都会被激发出荧光，即不仅焦平面上的荧光会被收集，而且来自焦平面上方或下方的散射荧光也被物镜所收集。针对样品焦平面成像所获取的二维图像中超过90％的荧光都来自离焦光线，这些光线甚至于完全淹没了焦平面应有的细节，并极大地降低了图像对比度。
结构光显微镜是在传统荧光显微镜基础上变更均匀光照明为结构光照明、以产生一组不同相位的调制结构光照射在样品上，再通过相移算法从这组不同相位调制的图像数据中提取焦平面信息、重建出层析图像和三维图像所发展的一种宽场显微成像技术。其图像质量对比度高、系统结构简单、成像速度快、成本低，与共聚焦激光扫描显微镜与双光子荧光显微镜相比，其在性价比方面具有很大的优势。
结构光显微系统的照明光源根据其是否存在相干性分为相干光源照明(如 激光照明)与非相干光源照明(如高压汞灯或者LED照明)。目前公布的基于数字微反射镜阵列(Digital Micro-mirror Device，简称DMD)的结构光显微系统，全是采用非相干光源照明，如专利CN104570315A采用的是LED照明，专利CN101655601B采用的是汞灯照明。然而在基于DMD的结构光显微系统中无论是采用相干光源照明还是非相干光源照明目前都没有提出或者公布基于DMD可多色激发的结构光显微系统。这主要是因为DMD本身是一个二维的闪耀光栅，会对不同波长的入射光进行不同方向的衍射，导致经DMD衍射后不同波长的光束不再共心共轴。对于非相干光源照明情况，DMD这一根本属性引起的不同波长照明光在样品面产生的结构光投影偏移尚可接受，但需要对层析图像以及三维图像的重建算法进行复杂的修改；但对于相干光源照明，DMD这一根本属性则会直接造成不同波长照明光在样品面因干涉产生的结构光条纹的失败。
因此，基于现有基于DMD的结构光显微系统存在的问题，不同波长照明光在样品面难以形成重合的结构光条纹，如果将其应用来实现多色激发尚存在一定的障碍。
发明内容
鉴于现有技术存在的不足，本发明提出了一种基于DMD可多色激发的结构光显微系统及多色激发方法，可以方便地提供单色或多色激发下的层析图像和三维图像，并保留基于DMD的结构光照明显微系统的照明稳定性、照明空间均匀性和成像速度的优势。
为了实现上述的目的，本发明采用了如下的技术方案：
一种基于DMD可多色激发的结构光显微系统，包括依次设于光路上的多色耦合模块、多色偏角模块、DMD、荧光激发模块和荧光采集模块，所述多色耦合模块用于将至少两种波长的光源耦合于同一光路中，使各波长的光束共心共轴，并选择不同波长的光源分时输出；所述多色偏角模块用于对自所述多色耦合模块射入的光束进行预处理，使射入所述DMD的各光束经衍射后共心共轴；所述荧光激发模块用于利用所述DMD射出的光在样品面产生结构光照明；所述荧光采集模块用于将样品被结构光照明激发出的荧光信号进行收集成像。
作为其中一种实施方式，所述多色耦合模块包括多个激光器和分别设于每个所述激光器的光路上的窄带滤光片、光处理元件，以及声光滤波器，各激光器发出的光束分别由各自的窄带滤光片过滤后，经各自的光处理元件处理后耦合至共心共轴，所述声光滤波器用于对各光处理元件发出的光束按照波长选择分时输出。
作为其中一种实施方式，所述光处理元件包括反射镜和/或二向色镜。
或者，所述光处理元件包括棱镜，所述棱镜的其中一个棱镜面为朝声光滤波器出光的出射面，每个所述窄带滤光片均贴合在所述棱镜的一个其他棱镜面上。
作为其中一种实施方式，所述多色耦合模块包括多个激光器和分别设于每个所述激光器的光路上的聚焦透镜，以及光纤合束器、准直透镜，各激光器发出的光束分别由各自的聚焦透镜聚焦后进入光纤，所述光纤合束器将各光纤的光束耦合至同一光纤并按照波长选择分时输出，自所述光纤合束器射出的光束由所述准直透镜准直成共心共轴光束。
作为其中一种实施方式，所述多色偏角模块包括用于使射入的光束产生预置偏角的角度补偿单元和用于使射入的光束产生垂直于光轴方向位移的位移补偿单元。
作为其中一种实施方式，所述角度补偿单元为闪耀光栅，所述位移补偿单元为可驱动所述闪耀光栅往复移动的电控平移台。
作为其中一种实施方式，所述闪耀光栅的光栅常数、闪耀角与所述DMD一致。
本发明的另一目的在于提供一种多色激发方法，包括：
将至少两种波长的照明光源耦合于同一光路中，使各波长的光束共心共轴，并选择不同波长的光源分时输出；
对分时输出的各波长的光源预处理，使射入DMD的各光束经衍射后共心共轴；
利用DMD射出的光在样品面产生结构光照明。
作为其中一种实施方式，所述对自所述多色耦合模块射入的光束进行预处理，包括：
使射入的光束产生预置偏角；
使射入的光束产生垂直于光轴方向的位移。
本发明通过将不同波长的照明光源耦合于同一光路中，使各波长的光束共心共轴，并选择不同波长的光源分时输出，通过对分时输出的各波长的光源预处理，使射入DMD的各光束经衍射后共心共轴；随后即可通过在样品面产生结构光照明，将样品被结构光照明激发出的荧光信号进行收集成像，不同波长的照明光在样品面不仅可以通过DMD进行结构编辑生成需要的结构光照明，而且各波长的结构光条纹可以良好重合，保留基于DMD的结构光照明显微系统的照明稳定性、照明空间均匀性和成像速度的优势。
附图说明
图1为本发明的一种可多色激发的结构光显微系统的结构框图；
图2为本发明实施例1的一种可多色激发的结构光显微系统的光路原理示意图；
图3为本发明实施例1的多色耦合模块的光路原理示意图；
图4为本发明实施例1的多色偏角模块的光路原理示意图；
图5为本发明实施例1的荧光激发模块的光路原理示意图；
图6为本发明实施例1的荧光采集模块的光路原理示意图；
图7为本发明实施例2的多色耦合模块的光路原理示意图；
图8为本发明实施例3的多色耦合模块的光路原理示意图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
参阅图1，本发明的基于DMD可多色激发的结构光显微系统包括依次设于光路上的多色耦合模块11、多色偏角模块12、DMD13、荧光激发模块14和荧光采集模块15，多色耦合模块11用于将至少两种波长的光源耦合于同一光路中，使各波长的光束共心共轴，并选择不同波长的光源分时输出；多色偏角模块12用于对自多色耦合模块11射入的光束进行预处理，使射入DMD13的各光束经衍射后共心共轴；荧光激发模块14用于利用DMD13射出的光在样品面产生结构光照明；荧光采集模块15用于将样品被结构光照明激发出的荧光信号进行收集成像。
根据该可多色激发的结构光显微系统，本发明还相应地提供了一种实现多色激发方法，主要包括：
S01、将至少两种波长的照明光源耦合于同一光路中，使各波长的光束共心共轴，并选择不同波长的光源分时输出；
S02、对分时输出的各波长的光源预处理，使射入DMD13的各光束经衍射后共心共轴；
S03、利用DMD13射出的光在样品面产生结构光照明。
在样品面产生结构光照明后，只需要在随后将样品被结构光照明激发出的荧光信号进行收集成像即可。
优选地，多色偏角模块12包括用于使射入的光束产生预置偏角的角度补偿单元121和用于使射入的光束产生垂直于光轴方向位移的位移补偿单元122。
相应地，对分时输出的各波长的光源预处理，包括：
使射入的光束产生预置偏角；以及
使射入的光束产生垂直于光轴方向的位移。
根据DMD的光学参数，通过利用角度补偿单元121合理地设计预置偏角，让不同波长的光束生成一个预先设定的角度偏移，以补偿稍后不同波长的入射光经DMD后产生的衍射角度偏移，同时，通过位移补偿单元122对不同波长的衍射光产生的纵向(垂直于光束的光轴方向)位移偏差进行补偿，通过这两 种方式的结合，实现经过DMD衍射后的多波长光束共心共轴。
由于从多色偏角模块12出射的衍射光照射到已经加载一定条纹图案的DMD 13上时，除了因为DMD 13本身发生主极衍射外，还会因加载的条纹图案发生子级衍射。但正是由于本发明的多色偏角模块12在光束进入DMD 13前对光束进行处理产生了预置偏角和垂直于光轴方向的位移，主极衍射光会因为角度预先偏移与纵向位移偏差补偿而可以仍然维持多波长光束共心共轴，子极衍射会对称分布于主极衍射光两边。这些不同波长的主级与子级衍射光束随后进入荧光激发模块14，从而在样品面干涉形成结构光照明。最终，不同波长的照明光束在样品面不仅可以通过DMD进行结构编辑生成需要的结构光照明，而且各波长的结构光条纹可以良好地重合。
下面结合几种具体的实施例对本发明的基于DMD可多色激发的结构光显微系统及多色激发方法的实现过程进行进一步说明。
实施例1
结合图2和图3所示，本实施例的多色耦合模块11用于将多种波长的照明光源耦合进同一光路中，实现分时输出，包括多个激光器111和分别设于每个激光器111的光路上的窄带滤光片112、光处理元件113，以及声光滤波器(AOTF)114，各激光器111发出的光束分别由各自的窄带滤光片112过滤后，经各自的光处理元件113处理后耦合至共心共轴，声光滤波器114用于对各光处理元件113发出的光束按照波长选择分时输出。光处理元件113可以采用反射镜、二向色镜的各种组合实现其光学处理功能。
为方便理解，本实施例以四个激光器111为例进行说明，其中，每个激光器111分别用于产生不同波长的激光光源。具体地，激光器111具有激光器一111a、激光器二111b、激光器三111c、激光器四111d，分别产生波长1(例如635nm)、波长2(例如561nm)、波长3(例如488nm)、波长4(例如405nm)的激光；窄带滤光片112也具有四个：112a、112b、112c、112d，用于在特定的波段允许光信号通过，而偏离这个波段以外的两侧光信号被阻止；光处理元件113包括反射镜113a、二向色镜113b、113c、113d。
每个激光器发出的光分别穿过一个窄带滤光片112后，由不同的反射镜/二向色镜反射至同一个声光滤波器114：激光器一111a输出波长1，经过窄带 滤光片112a(例如635±10nm)被反射镜113a反射(工作于45°角)进入声光滤波器114；激光器二111b输出波长2，经过窄带滤光片112b(例如561±10nm)被二向色镜113b反射(例如反射561nm以下波长的激光，透射561nm波长以及以上的激光，工作于45°角)进入声光滤波器114，波长2进入声光滤波器114前与波长1耦合至共心共轴；激光器三111c输出波长3，经过窄带滤光片112c(例如488±10nm)被二向色镜113c(例如反射小于488nm波长的激光，透射大于488nm波长的激光，工作于45°角)反射进入声光滤波器114，波长3进入声光滤波器114前与波长1、波长2耦合至共心共轴；激光器四111d输出波长4，经过窄带滤光片112d(例如405±10nm)被二向色镜113d(例如反射405nm波长的激光，透射大于405nm波长的激光，工作于45°角)反射进入声光滤波器114，波长4进入声光滤波器114前与波长1、波长2、波长3耦合至共心共轴。声光滤波器114用于对四种波长选择分时输出，各波长从声光滤波器114输出时光束共心共轴。如图3所示，反射镜113a、二向色镜113b、113c、113d依次沿直线布置且相互平行。
如图2和图4所示，多色偏角模块12用以产生特定的角度偏移与纵向位移偏差补偿，使得不同波长的不同衍射级光束经过DMD衍射后实现多波长光束共心共轴，包括用于使射入的光束产生预置偏角的角度补偿单元121和用于使射入的光束产生垂直于光轴方向位移的位移补偿单元122。
作为一种优选的实施方式，这里，角度补偿单元121为闪耀光栅，位移补偿单元122为可驱动闪耀光栅往复移动的电控平移台。经多色耦合模块11耦合成多波长共心共轴的光束首先照射到闪耀光栅上衍射，让不同波长的光束预先有一个角度偏移，与此同时，闪耀光栅固定在精密电控平移台上，通过控制程序直接控制闪耀光栅沿固定方向(例如入射光方向)移动，用以补偿不同波长的衍射光束向前传播时产生的纵向(垂直于光轴方向)的位移偏差，通过这两种方式的结合，实现经过DMD衍射后的多波长光束共心共轴。
根据DMD的光学参数(例如德州仪器D4100，光栅常数13.68微米，闪耀角12度)，光栅应为特定闪耀光栅(例如可以为同款DMD，或定制的反射式闪耀光栅，或定制的透射式闪耀光栅)，其光栅常数d、闪耀角γ需要与DMD保持一致，以便对各不同波长的衍射光预先设置特定的角度偏移。再依据光栅方程：mλ＝d(sin θ m+sinθi)＝2d sin γ，当从多色耦合模块11出射的多波长共心共轴光束以相同的入射角照射到闪耀光栅上时，由于波长不同，其衍射方向不同， 而且衍射级次也不同。例如，当入射角为0度时，405nm波长的激光对应的衍射级为10，衍射角为17.22度，488nm波长的激光对应的衍射级为8，衍射角为16.58度，561nm波长的激光对应的衍射级为7，衍射角为16.68度，635nm波长的激光对应的衍射级为6，衍射角为16.17度。不同波长的光束在经过闪耀光栅后，不仅因为衍射方向不同产生了我们预先设置的角度偏移，还因为衍射级次不同会产生了多余的纵向(垂直于光轴方向)的位移偏差。
本实施例的闪耀光栅固定于精密电控平移台(例如步进精度1μm)，配合多色耦合模块101中的声光滤波器114，在波长选择分时输出的同时，通过控制程序让闪耀光栅沿固定方向(例如入射光方向)移动特定位置，不同波长其移动位置不同(例如波长1对应位置1，波长2对应位置2，波长3对应位置3，波长4对应位置4)，进而抵消了不同波长产生的纵向(垂直于光轴方向)位移偏差。当预先有一定偏角的不同波长光束再照射到DMD13衍射并出射时便可以实现共心共轴。例如，光栅301为特制反射式闪耀光栅时，经过DMD13出射后，405nm波长的10级衍射光，488nm波长的8级衍射光，561nm波长的7级衍射光，635nm波长的6级衍射光，在从DMD13衍射出射时便能共心共轴。
如图2所示，在有的实施方式中，还可以根据实际情况增加光路折叠模块(图未标)，用以将从多色耦合模块11出射的共心共轴光束扩束，并转折至多色偏角模块12。这里，光路折叠模块包括依次设于光路上的扩束组和跳镜组，扩束组包括透镜Z1、透镜Z2，跳镜组包括反射镜Z3、反射镜Z4，从声光滤波器114分时选择输出的共心共轴光束依次经过透镜Z1和透镜Z2组成的扩束组扩束，扩束后的光束再经过反射镜Z3与Z4组成的跳镜组反射，反射后的光束进入多色偏角模块12中的透射式闪耀光栅。经光路折叠模块扩束、转折后的多波长共心共轴光束以相同的入射角照射到透射式闪耀光栅上。
结构光照明显微镜利用同一光束的不同衍射级之间的相互干涉产生结构性图案在样品面P产生照明。经多色偏角模块12后，不同波长的光束共心共轴(例如，405nm波长的10级衍射光，488nm波长的8级衍射光，561nm波长的7级衍射光，635nm波长的6级衍射光，在从DMD衍射出射时共心共轴)。当在DMD13上加载条纹图案时，由于条纹图案也是一种周期性光栅，会在DMD本身主级衍射的基础上产生子级衍射。
如图2和图5所示，本实施例的荧光激发模块14用于在样品面P产生结构光照明，激发样品产生荧光，包括透镜141、142、143和物镜144，DMD 13置于荧光激发模块14中透镜141的前焦面位置，不同波长的主级与子级衍射光束通过透镜141后，在其后焦面形成不同波长的子衍射光聚焦斑，这些聚焦斑再经过透镜142与透镜143组成的中继透镜成像至物镜144(例如大放大倍率、高NA值的物镜)的后倍孔径内，各衍射级次的光束再经物镜144在样品面P干涉形成结构化照明光。
具体地，通过合理选择透镜141(例如相对孔径、焦距)可以只允许第n级作为主极的0级子衍射光(例如，405nm波长的第10级作为主极的0级子衍射光，488nm波长的第8级作为主极的0级子衍射光，561nm波长的第7级作为主极的0级子衍射光，635nm波长的第6级作为主极的0级子衍射光)、第n级作为主极的-1级子衍射光(例如，405nm波长的第10级作为主极的-1级子衍射光，488nm波长的第8级作为主极的-1级子衍射光，561nm波长的第7级作为主极的-1级子衍射光，635nm波长的第6级作为主极的-1级子衍射光)、第n级作为主极的+1级子衍射光(例如，405nm波长的第10级作为主极的+1级子衍射光，488nm波长的第8级作为主极的+1级子衍射光，561nm波长的第7级作为主极的+1级子衍射光，635nm波长的第6级作为主极的+1级子衍射光)，共计三个子级衍射光通过，而其他子级的衍射光被透镜141的口径限制在光路以外。DMD13置于荧光激发模块14中透镜142的前焦面位置，不同波长的主级与子级衍射光束通过透镜141后，在其后焦面形成不同波长的子衍射光聚焦斑：第n级作为主极的0级子衍射光聚焦斑、第n级作为主极的-1级子衍射光聚焦斑、第n级作为主极的+1级子衍射光聚焦斑。
如图2、图5和图6所示，本实施例的荧光采集模块15用于将样品被结构光照明激发的荧光信号进行收集成像，包括依次位于激发荧光的光路上的二向色镜151、成像透镜152、滤光片153、成像探测器154。
样品面P受激发的荧光信号被物镜144收集后依次经二向色镜151、成像透镜152、滤光片153进入到成像探测器154中，滤光片153用以滤除荧光以外的背景信号，例如，二向色镜151可以是反射405nm/488nm/561nm/635nm波长，而透射其他波长的多带通二向色镜，滤光片153可以是装有四片405nm长通/488nm长通/561nm长通/635nm长通的滤光片轮，或者单片426～462nm带通、502.5～544.5nm带通、582～617.5nm带通以及663～1200nm带 通的多带通滤光片。
结合图2所示，根据上述的基于DMD可多色激发的结构光显微系统，实现多色激发的方法主要过程是：
(1)多色耦合模块11将不同波长的照明光源耦合进同一光路，各波长的光束共心共轴，并可以选择不同波波长的激光分时输出；
(2)对分时输出的各波长的光源预处理，具体是当某一波长的激光被选择输出后，经过光路折叠模块扩束、转折后进入多色偏角模块12中的透射式闪耀光栅，使射入的光束产生预置偏角，同时，通过控制固定于精密电控平移台上的透射式闪耀光栅沿固定方向(例如入射光方向)移动到相应的合适位置，使射入的光束产生垂直于光轴方向的位移，以抵消波该长在纵向(垂直于光轴方向)产生的位移偏差。
当波长1的激光被选择输出后，经过光路折叠模块扩束、转折后进入多色偏角模块12中的透射式闪耀光栅，此时控制程序控制固定于精密电控平移台上的透射式闪耀光栅沿固定方向移动到位置1；
当入射角为0度时，由于光栅的衍射效应，波长1的激光经过透射式闪耀光栅后发生衍射后照射到DMD13上。当采用利特洛配置(Littrow configuration)时，由于透射式闪耀光栅与DMD13的光栅常数与闪耀角设计一致，波长1的光束再被DMD13衍射后，其衍射角重新恢复为0度。
当波长2的激光被选择输出后，经过光路折叠模块扩束、转折后同样进入多色偏角模块12中的透射式闪耀光栅，此时控制程序控制固定于精密电控平移台上的透射式闪耀光栅301沿固定方向移动到位置2；
当入射角为0度时，波长2的激光同样经过透射式闪耀光栅后发生衍射，照射到DMD13上。波长2的光束再被DMD13衍射后，其衍射角同样重新恢复为0度。而且由于预先通过控制程序控制固定于精密电控平移台上的透射式闪耀光栅沿固定方向调整了位置，波长2与波长1在纵向产生的位移偏差被抵消，波长2与波长1实现了共心共轴。
当波长3的激光被选择输出后，与波长2、波长1一样，经过光路折叠模块扩束、转折后进入多色偏角模块12中的透射式闪耀光栅，此时控制程序控 制固定于精密电控平移台上的透射式闪耀光栅沿固定方向移动到位置3；
与波长2、波长1一样，当入射角为0度时，波长3的激光经过透射式闪耀光栅后发生衍射，照射到DMD 13上。波长3的光束再被DMD 13衍射后，其衍射角同样重新恢复为0度。与波长2、波长1一样，波长3在纵向产生的位移偏差也被抵消，波长3、波长2与波长1实现了共心共轴。
当波长4的激光被选择输出后，与波长3、波长2、波长1一样，经过光路折叠模块扩束、转折后进入多色偏角模块12中的透射式闪耀光栅，此时控制程序控制固定于精密电控平移台上的透射式闪耀光栅沿固定方向移动到位置4。
与波长3、波长2、波长1一样，当入射角为0度时，波长4的激光经过透射式闪耀光栅后发生衍射，照射到DMD 13上。波长4的光束再被DMD 13衍射后，其衍射角同样重新恢复为0度。与波长3、波长2、波长1一样，波长4在纵向产生的位移偏差也被抵消，波长4、波长3、波长2与波长1实现了共心共轴。
(3)从透射式闪耀光栅衍射出的光束照射到DMD 13的同时，在DMD 13上加载条纹图案，DMD13射出的光在样品面产生结构光照明。
由于条纹图案也是一种周期性光栅，会在DMD 13本身主级衍射的基础上产生子级衍射。
当波长1(例如，635nm波长)的激光束从DMD衍射出射后，会产生波长1的第n级作为主极的包含0级、±1级、±2级等的子级衍射光(例如635nm波长的第6级作为主极的0级、±1级、±2级子级衍射光)；
当波长2(例如，561nm波长)的激光束从DMD衍射出射后，会产生波长2的第n级作为主极的包含0级、±1级、±2级等的子级衍射光(例如561nm波长的第7级作为主极的0级、±1级、±2级子级衍射光)；
当波长3(例如，488nm波长)的激光束从DMD衍射出射后，会产生波长3的第n级作为主极的包含0级、±1级、±2级等的子级衍射光(例如488nm波长的第8级作为主极的0级、±1级、±2级子级衍射光)；
当波长4(例如，405nm波长)的激光束从DMD衍射出射后，会产生波 长4的第n级作为主极的包含0级、±1级、±2级等的子级衍射光(例如405nm波长的第10级作为主极的0级、±1级、±2级子级衍射光)。
由于多色偏角模块12对各不同波长的预先角度偏移与纵向位移偏差补偿，各波长的第n级作为主极的0级子衍射光共心共轴，各波长的第n级作为主极的±1、±2级子衍射光会因波长的不同，导致衍射方向不同，不再共心共轴。这些子级衍射光束经过透镜141后只保留第n级作为主极的0级子衍射光、第n级作为主极的-1级子衍射光、第n级作为主极的+1级子衍射光，共计三个子级的衍射光通过，而其他子级的衍射光被透镜141的口径限制在光路以外。
DMD 13置于荧光激发模块14中透镜141的前焦面位置，不同波长的主级与子级衍射光束通过透镜141后，在其后焦面形成不同波长的第n级作为主极的0级子衍射光聚焦斑、第n级作为主极的-1级子衍射光聚焦斑、第n级作为主极的+1级子衍射光聚焦斑。这些聚焦斑再经过透镜142与透镜143组成的中继透镜成像至物镜144的后倍孔径内，各衍射级次的光束再经物镜144在样品面P干涉形成结构化照明光。
最终，不同波长照明光在样品面不仅可以通过DMD进行结构编辑生成需要的结构光照明，而且各波长的结构光条纹可以良好重合。
实施例2
如图7所示，本实施例的多色耦合模块11同样包括多个激光器111和分别设于每个激光器111的光路上的窄带滤光片112、光处理元件113，以及声光滤波器(AOTF)114。与实施例1不同的是，本实施例的多色耦合模块11中，各激光器111发出的光束分别由各自的窄带滤光片112过滤后，经同一个光处理元件113处理后耦合至共心共轴，最后由声光滤波器114对光处理元件113发出的光束按照波长选择分时输出。
本实施例中，各波长进入声光滤波器114前也耦合至共心共轴。光处理元件113的主要组成是棱镜，棱镜的其中一个棱镜面为朝声光滤波器114出光的出射面，每个窄带滤光片112均贴合在棱镜的一个其他棱镜面上。
优选地，每一个窄带滤光片112贴合在棱镜的一个不同的棱镜面上，仅其中一个棱镜面朝向声光滤波器114作为光的出射面。这里，棱镜以五棱镜为例进行说明，棱镜的每一棱镜面均贴合有一用于过滤不同波长的窄带滤光片112， 每个窄带滤光片112分别对应一个用于产生不同波长的激光器111。
激光器一111a输出波长1(例如635nm)，经过窄带滤光片112a(例如635±10nm，工作于72°角)以与界面法线呈18°角的入射角透射进入五棱镜，后经五棱镜其他两面反射后从五棱镜的出射面出射进入声光滤波器114；
激光器二111b输出波长2(例如561nm)，经过窄带滤光片112b(例如561±10nm，工作于72°角)以与界面法线呈18°角的入射角透射进入五棱镜，直接从五棱镜的出射面出射后进入声光滤波器114，波长2进入声光滤波器114前与波长1耦合至共心共轴；
激光器三111c输出波长3(例如488nm)，经过窄带滤光片112c(例如488±10nm，工作于72°角)以与界面法线呈18°角的入射角透射进入五棱镜，后经五棱镜其他三面反射后从五棱镜的出射面出射后进入声光滤波器114，波长3进入声光滤波器114前与波长1、波长2耦合至共心共轴；
激光器四111d输出波长4(例如405nm)，经过窄带滤光片112d(例如405±10nm，工作于72°角)以与界面法线呈18°角的入射角透射进入五棱镜，后经五棱镜另一面反射后从五棱镜出射进入声光滤波器114，波长4进入声光滤波器114前与波长1、波长2、波长3耦合至共心共轴。
各波长从声光滤波器114进入前耦合至共心共轴，随后，声光滤波器114对四种波长选择分时输出，各波长从声光滤波器114输出时光束也共心共轴。
可以理解的是，棱镜也可以具有空置的棱镜面，或者也可以将实施例1的光处理元件113设置在本实施例的棱镜旁，实施例1的光处理元件113发出的光束从其中一个棱镜面射入，与其他光束耦合后射出到声光滤波器114。
实施例3
如图8所示，与实施例1和实施例2不同，本实施例的多色耦合模块11包括多个激光器111和分别设于每个激光器111的光路上的聚焦透镜115，以及光纤合束器116、准直透镜117，各激光器111发出的光束分别由各自的聚焦透镜115聚焦后进入光纤，光纤合束器116将各光纤的光束耦合至同一光纤并按照波长选择分时输出，自光纤合束器116射出的光束由准直透镜117准直成共心共轴光束。
光纤合束器116的一端将各光纤的光束耦合，另一端将耦合后的光束射入准直透镜117。具体地，光纤合束器116的一端设有对应每个聚焦透镜115的聚焦光纤的光纤连接器1161，包括光纤连接器1161a、1161b、1161c、1161d，光纤合束器116的另一端设有光纤连接器1162。聚焦透镜115、准直透镜117均可以是单透镜、双胶合透镜或低倍显微物镜，光纤连接器1161a/1161b/1161c/1161d分别对应聚焦透镜115a/115b/115c/115d。
激光器一111a输出波长1(例如635nm)，经过聚焦透镜115a聚焦于光纤连接器1161a的入射端面耦合进入光纤；
激光器二111b输出波长2(例如561nm)，经过聚焦透镜115b聚焦于光纤连接器1161b的入射端面耦合进入光纤，波长2与波长1通过光纤合束器116一起耦合进入另一端的光纤连接器1162的入射端面进入光纤；
激光器三111c输出波长3(例如488nm)，经过聚焦透镜115c聚焦于光纤连接器1161c的入射端面耦合进入光纤，波长3、波长2与波长1通过光纤合束器116一起耦合进入另一端的光纤连接器1162的入射端面进入光纤；
激光器四111d输出波长4(例如405nm)，经过聚焦透镜115d聚焦于光纤连接器1161d的入射端面耦合进入光纤，波长4、波长3、波长2与波长1通过光纤合束器116一起耦合进入另一端的光纤连接器1162的入射端面进入光纤；
光纤合束器116将四种波长一起耦合进入同一光纤连接器1162，并能选择分时输出各波长。波长1、波长2、波长3和波长4的激光束在从光纤连接器1162的出射端面出射后被准直透镜117准直成共心共轴光束。
本发明通过将不同波长的照明光源耦合于同一光路中，使各波长的光束共心共轴，并选择不同波长的光源分时输出，通过对分时输出的各波长的光源预处理，使射入DMD的各光束经衍射后共心共轴；随后即可通过在样品面产生结构光照明，将样品被结构光照明激发出的荧光信号进行收集成像，不同波长的照明光在样品面不仅可以通过DMD进行结构编辑生成需要的结构光照明，而且各波长的结构光条纹可以良好重合，保留基于DMD的结构光照明显微系统的照明稳定性、照明空间均匀性和成像速度的优势。
以上仅是本申请的具体实施方式，应当指出，对于本技术领域的普通技术 人员来说，在不脱离本申请原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本申请的保护范围。

Claims (20)

  1. 一种基于DMD(数字微反射镜阵列)可多色激发的结构光显微系统，其中，所述结构光显微系统包括依次设于光路上的多色耦合模块(11)、多色偏角模块(12)、DMD(13)、荧光激发模块(14)和荧光采集模块(15)，所述多色耦合模块(11)用于将至少两种波长的光源耦合于同一光路中，使各波长的光束共心共轴，并选择不同波长的光源分时输出；所述多色偏角模块(12)用于对自所述多色耦合模块(11)射入的光束进行预处理，使射入所述DMD(13)的各光束经衍射后共心共轴；所述荧光激发模块(14)用于利用所述DMD(13)射出的光在样品面产生结构光照明；所述荧光采集模块(15)用于将样品被结构光照明激发出的荧光信号进行收集成像。
  2. 根据权利要求1所述的基于DMD可多色激发的结构光显微系统，其中，所述多色耦合模块(11)包括多个激光器(111)和分别设于每个所述激光器(111)的光路上的窄带滤光片(112)、光处理元件(113)，以及声光滤波器(114)，各激光器(111)发出的光束分别由各自的窄带滤光片(112)过滤后，经各自的光处理元件(113)处理后耦合至共心共轴，所述声光滤波器(114)用于对各光处理元件(113)发出的光束按照波长选择分时输出。
  3. 根据权利要求2所述的基于DMD可多色激发的结构光显微系统，其中，所述光处理元件(113)包括反射镜和/或二向色镜。
  4. 根据权利要求2所述的基于DMD可多色激发的结构光显微系统，其中，所述光处理元件(113)包括棱镜，所述棱镜的其中一个棱镜面为朝声光滤波器(114)出光的出射面，每个所述窄带滤光片(112)均贴合在所述棱镜的一个其他棱镜面上。
  5. 根据权利要求1所述的基于DMD可多色激发的结构光显微系统，其中，所述多色耦合模块(11)包括多个激光器(111)和分别设于每个所述激光器(111)的光路上的聚焦透镜(115)，以及光纤合束器(116)、准直透镜(117)，各激光器(111)发出的光束分别由各自的聚焦透镜(115)聚焦后进入光纤，所述光纤合束器(116)将各光纤的光束耦合至同一光纤并按照波长选择分时输出，自所述光纤合束器(116)射出的光束由所述准直透镜(117)准直成共心共轴光束。
  6. 根据权利要求1所述的基于DMD可多色激发的结构光显微系统，其中，所述多色偏角模块(12)包括用于使射入的光束产生预置偏角的角度补偿单元(121)和用于使射入的光束产生垂直于光轴方向位移的位移补偿单元(122)。
  7. 根据权利要求2所述的基于DMD可多色激发的结构光显微系统，其中，所述多色偏角模块(12)包括用于使射入的光束产生预置偏角的角度补偿单元(121)和用于使射入的光束产生垂直于光轴方向位移的位移补偿单元(122)。
  8. 根据权利要求3所述的基于DMD可多色激发的结构光显微系统，其中，所述多色偏角模块(12)包括用于使射入的光束产生预置偏角的角度补偿单元(121)和用于使射入的光束产生垂直于光轴方向位移的位移补偿单元(122)。
  9. 根据权利要求4所述的基于DMD可多色激发的结构光显微系统，其中，所述多色偏角模块(12)包括用于使射入的光束产生预置偏角的角度补偿单元(121)和用于使射入的光束产生垂直于光轴方向位移的位移补偿单元(122)。
  10. 根据权利要求5所述的基于DMD可多色激发的结构光显微系统，其中，所述多色偏角模块(12)包括用于使射入的光束产生预置偏角的角度补偿单元(121)和用于使射入的光束产生垂直于光轴方向位移的位移补偿单元(122)。
  11. 根据权利要求6所述的基于DMD可多色激发的结构光显微系统，其中，所述角度补偿单元(121)为闪耀光栅，所述位移补偿单元(122)为可驱动所述闪耀光栅往复移动的电控平移台。
  12. 根据权利要求7所述的基于DMD可多色激发的结构光显微系统，其中，所述角度补偿单元(121)为闪耀光栅，所述位移补偿单元(122)为可驱动所述闪耀光栅往复移动的电控平移台。
  13. 根据权利要求9所述的基于DMD可多色激发的结构光显微系统，其中，所述角度补偿单元(121)为闪耀光栅，所述位移补偿单元(122)为可驱动所述闪耀光栅往复移动的电控平移台。
  14. 根据权利要求10所述的基于DMD可多色激发的结构光显微系统，其中，所述角度补偿单元(121)为闪耀光栅，所述位移补偿单元(122)为可驱动所述闪耀光栅往复移动的电控平移台。
  15. 根据权利要求11所述的基于DMD可多色激发的结构光显微系统，其 中，所述闪耀光栅的光栅常数、闪耀角与所述DMD(13)一致。
  16. 根据权利要求12所述的基于DMD可多色激发的结构光显微系统，其中，所述闪耀光栅的光栅常数、闪耀角与所述DMD(13)一致。
  17. 根据权利要求13所述的基于DMD可多色激发的结构光显微系统，其中，所述闪耀光栅的光栅常数、闪耀角与所述DMD(13)一致。
  18. 根据权利要求14所述的基于DMD可多色激发的结构光显微系统，其中，所述闪耀光栅的光栅常数、闪耀角与所述DMD(13)一致。
  19. 一种多色激发方法，其中，包括：
    将至少两种波长的照明光源耦合于同一光路中，使各波长的光束共心共轴，并选择不同波长的光源分时输出；
    对分时输出的各波长的光源预处理，使射入DMD(13)的各光束经衍射后共心共轴；
    利用DMD(13)射出的光在样品面产生结构光照明。
  20. 根据权利要求19所述的多色激发方法，其中，所述对分时输出的各波长的光源预处理，包括：
    使射入的光束产生预置偏角；
    使射入的光束产生垂直于光轴方向的位移。
PCT/CN2019/124600 2018-12-18 2019-12-11 一种基于dmd可多色激发的结构光显微系统及多色激发方法 WO2020125518A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201811546262.2 2018-12-18
CN201811546262.2A CN109407295B (zh) 2018-12-18 2018-12-18 一种基于dmd可多色激发的结构光显微系统及多色激发方法

Publications (1)

Publication Number Publication Date
WO2020125518A1 true WO2020125518A1 (zh) 2020-06-25

Family

ID=65459878

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/124600 WO2020125518A1 (zh) 2018-12-18 2019-12-11 一种基于dmd可多色激发的结构光显微系统及多色激发方法

Country Status (2)

Country Link
CN (1) CN109407295B (zh)
WO (1) WO2020125518A1 (zh)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109407295B (zh) * 2018-12-18 2020-07-24 中国科学院深圳先进技术研究院 一种基于dmd可多色激发的结构光显微系统及多色激发方法
CN110161671B (zh) * 2019-04-30 2021-04-02 上海理工大学 暗场、明场、相衬、荧光多模式同步成像显微成像装置
CN110501319A (zh) * 2019-08-29 2019-11-26 中国科学院长春应用化学研究所 多通路结构光照明的拉曼超分辨显微成像方法
CN110955107B (zh) * 2019-11-18 2022-11-15 长春理工大学 一种基于反射成像技术的超高速时间分辨摄像装置和方法
CN111650739B (zh) * 2020-05-21 2022-06-03 中国科学院苏州生物医学工程技术研究所 基于dmd的单帧曝光快速三维荧光成像系统及方法
CN111589478B (zh) * 2020-06-05 2021-06-29 珠海市尚维高科生物技术有限公司 一种双通道实时荧光定量pcr仪光路系统及检测方法
CN112859315A (zh) * 2021-01-11 2021-05-28 北京大学 一种多色双模式结构光照明显微成像系统及其成像方法
CN114509911A (zh) * 2021-12-28 2022-05-17 中国空气动力研究与发展中心超高速空气动力研究所 一种同轴超高速多序列激光阴影成像装置
CN114460731B (zh) * 2022-01-24 2023-03-17 浙江大学 一种基于dmd的多色结构光照明超分辨显微成像方法和装置

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130010098A1 (en) * 2009-12-22 2013-01-10 Kalkbrenner Thomas High-Resolution Microscope and Method for Determining the Two- or Three-Dimensional Positions of Objects
WO2015162921A1 (ja) * 2014-04-25 2015-10-29 株式会社ニコン 構造化照明顕微鏡装置及び構造化照明観察方法
CN106500589A (zh) * 2016-12-05 2017-03-15 苏州大学 一种多波长可调谐显微干涉的测量方法及其装置
CN107167929A (zh) * 2017-06-12 2017-09-15 华南师范大学 基于dmd的双模式光学超分辨显微成像装置及方法
CN107389631A (zh) * 2017-04-28 2017-11-24 中国科学院生物物理研究所 高速多色多模态结构光照明超分辨显微成像系统及其方法
CN107430240A (zh) * 2015-03-16 2017-12-01 镭亚股份有限公司 采用角度选择性反射层的基于单向光栅的背光
CN107728314A (zh) * 2017-08-17 2018-02-23 中国科学院光电技术研究所 一种基于旋转双闪耀光栅的阵列光束偏转的方法
CN109407295A (zh) * 2018-12-18 2019-03-01 中国科学院深圳先进技术研究院 一种基于dmd可多色激发的结构光显微系统及多色激发方法

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106872034B (zh) * 2017-01-13 2018-11-13 清华大学 快速三维多光谱显微成像系统
CN107917680B (zh) * 2017-11-07 2019-10-25 南京航空航天大学 基于闪耀光栅的微小角度快速识别方法
CN108169887B (zh) * 2017-12-27 2020-04-24 清华大学 3d多焦面结构光快速显微成像系统及方法
CN108333160B (zh) * 2018-02-02 2020-11-06 北京工业大学 面向空间应用的一种微型结构光产生装置及超分辨荧光显微系统
CN207946356U (zh) * 2018-03-06 2018-10-09 华南师范大学 基于上转换纳米材料的多焦点结构光照明显微成像装置

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130010098A1 (en) * 2009-12-22 2013-01-10 Kalkbrenner Thomas High-Resolution Microscope and Method for Determining the Two- or Three-Dimensional Positions of Objects
WO2015162921A1 (ja) * 2014-04-25 2015-10-29 株式会社ニコン 構造化照明顕微鏡装置及び構造化照明観察方法
CN107430240A (zh) * 2015-03-16 2017-12-01 镭亚股份有限公司 采用角度选择性反射层的基于单向光栅的背光
CN106500589A (zh) * 2016-12-05 2017-03-15 苏州大学 一种多波长可调谐显微干涉的测量方法及其装置
CN107389631A (zh) * 2017-04-28 2017-11-24 中国科学院生物物理研究所 高速多色多模态结构光照明超分辨显微成像系统及其方法
CN107167929A (zh) * 2017-06-12 2017-09-15 华南师范大学 基于dmd的双模式光学超分辨显微成像装置及方法
CN107728314A (zh) * 2017-08-17 2018-02-23 中国科学院光电技术研究所 一种基于旋转双闪耀光栅的阵列光束偏转的方法
CN109407295A (zh) * 2018-12-18 2019-03-01 中国科学院深圳先进技术研究院 一种基于dmd可多色激发的结构光显微系统及多色激发方法

Also Published As

Publication number Publication date
CN109407295A (zh) 2019-03-01
CN109407295B (zh) 2020-07-24

Similar Documents

Publication Publication Date Title
WO2020125518A1 (zh) 一种基于dmd可多色激发的结构光显微系统及多色激发方法
US10642015B2 (en) Light sheet microscope with a phase-selective element for illumination
US9897790B2 (en) Structured illumination device and structured illumination microscope device
JP7073523B2 (ja) Ｓｔｅｄ光学顕微鏡に用いる照明システム及びｓｔｅｄ光学顕微鏡
JP4723806B2 (ja) 共焦点顕微鏡
JP6033798B2 (ja) 蛍光顕微鏡検査法における照明位相制御のためのシステムおよび方法
JP4899408B2 (ja) 顕微鏡装置
US11921045B2 (en) Holographic three-dimensional multi-spot light stimulation device and method
JP2008058003A (ja) 顕微鏡
JPH10510054A (ja) 移相回折干渉計
US11555991B2 (en) Method for illuminating samples in microscopic imaging methods
US20060214106A1 (en) Point scanning laser scanning microscope and methods for adjustment of a microscope
US20040227101A1 (en) Microscope
CN114460731B (zh) 一种基于dmd的多色结构光照明超分辨显微成像方法和装置
ITFI20070260A1 (it) Dispositivo per illuminare un oggetto con una sorgente di luce multispettrale e rivelare lo spettro della luce emessa.
JP7130628B2 (ja) 光学顕微鏡
JP2617175B2 (ja) 光学相関メモリ処理システム
CN211086783U (zh) 一种便于阵列应用的显微镜
JP5278522B2 (ja) 顕微鏡装置
US6903869B2 (en) Illumination system for microscopy and observation or measuring method using the same
WO2023221400A1 (zh) 超分辨单物镜光片显微成像光学系统及其成像系统
CN218974142U (zh) 一种多波段结构光显微成像系统
CN107941777B (zh) 一种抗漂白单分子定位三维超分辨显微系统
US20070081214A1 (en) Image scanning apparatus
JP2010128473A (ja) 分散素子及び分散素子を備える光学機器

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19900618

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19900618

Country of ref document: EP

Kind code of ref document: A1