CN215181230U - Structured light filtering device and structured light illumination microscopic imaging system with same - Google Patents
Structured light filtering device and structured light illumination microscopic imaging system with same Download PDFInfo
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- CN215181230U CN215181230U CN202121130201.5U CN202121130201U CN215181230U CN 215181230 U CN215181230 U CN 215181230U CN 202121130201 U CN202121130201 U CN 202121130201U CN 215181230 U CN215181230 U CN 215181230U
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Abstract
The utility model discloses a structured light filter device and have device's micro-imaging system of structured light illumination, this structured light filter device include diffraction optical element, Fourier lens, round hole filter element, phase delay ware and first collimation lens. The structured light illumination microscopic imaging system comprises a structured light filtering device, an illumination light source module and a microscopic imaging module. The filtering device provided by the utility model can accurately control the positive and negative first-order diffraction light to be transmitted continuously through the frequency spectrum plane through the circular hole filtering element, and can improve the contrast of the illumination light of the surface structure of the subsequent sample; the utility model discloses a micro-imaging system of structured light illumination on the basis of the contrast that has improved sample surface texture illumination light, blocks the marginal part of facula through the aperture diaphragm to can obtain the better laser of intensity homogeneity, finally ensure that the structured light that generates has good homogeneity, solved the bright, the dark shortcoming in edge of formation of image picture central authorities, can reduce the fluorescence distortion degree of image.
Description
Technical Field
The utility model relates to a structured light illumination microscope field, in particular to structured light filter and have device's the microscopic imaging system of structured light illumination.
Background
The structured light fluorescence microscope is an optical microscope based on a conventional fluorescence microscope, and images with specific structured light by improving the illumination mode of the conventional fluorescence microscope, so that the diffraction limit is broken through, high-resolution sample information is obtained, and a sample microscopic image is obtained by Fourier transform. By inserting a structured light generating device (such as a grating, a spatial light modulator, or a digital micromirror array (DMD), etc.) in an illumination light path, a pattern with regularly changed brightness is formed after the illumination light is modulated, and then the pattern is projected on a sample through an objective lens, and a fluorescence signal generated by the modulated light is received by a camera. By moving and rotating the illumination pattern to cover various areas of the specimen, and combining and reconstructing the captured images with software, a super-resolution image of the specimen can be obtained. Therefore, the structured light illumination fluorescence microscope has wide application in the aspects of cell biology, neurobiology, microbial physiology and the like.
However, the existing structured light illumination fluorescence microscope has the following defects, for example: the filtering element in the existing structured light fluorescence microscope mostly adopts a rectangular hole Mask (as shown in fig. 4) to filter on the frequency spectrum surface of the light beam, except that interference is generated after positive and negative first-order diffracted lights of the light beam pass through the Mask, peripheral stray lights can pass through and the contrast of subsequent light beam interference fringes is directly reduced, so that the final imaging quality is influenced. In addition, in the conventional structured light illumination fluorescence microscope, the uniformity of the generated structured light is generally poor (generally, the center is strong, and the edge is weak), so that the phenomenon that the center of an imaging picture is bright and the edge is dark is easily caused, and the image is distorted. Therefore, there is a need to provide a more reliable solution.
SUMMERY OF THE UTILITY MODEL
The utility model aims to solve the technical problem that to the not enough among the above-mentioned prior art, provide a structured light filter and have device's the micro-imaging system of structured light illumination.
In order to solve the technical problem, the utility model discloses a technical scheme is: the filtering device for generating the structured light comprises a diffraction optical element, a Fourier lens, a round hole filtering element, a phase retarder and a first collimating lens which are sequentially arranged along an optical path;
the diffracted light emitted by the diffractive optical element is output after being focused by the Fourier lens, filtered by the circular hole filtering element, subjected to phase modulation by the phase retarder and collimated by the first collimating lens in sequence;
the circular hole filtering element is provided with a plurality of circular holes with different apertures and capable of switching into the light path to filter light.
Preferably, the circular hole filtering element comprises a MASK plate with a plurality of circular holes with different apertures arranged at intervals and a driving mechanism for driving the MASK plate to perform position movement so as to switch different circular holes into the optical path.
Preferably, only ± 1 st order light of diffracted light emitted from the diffractive optical element can pass through the circular aperture filter element.
Preferably, a first reflector is further disposed between the fourier lens and the circular hole filter element, and light emitted from the fourier lens reaches the circular hole filter element after being reflected by the first reflector.
Preferably, the diffractive optical element is a spatial light modulator.
The utility model also provides a micro-imaging system of structured light illumination, it includes as above structured light filter, light source module and micro-imaging module.
Preferably, the illumination light source module includes a laser, an optical fiber, a second collimating lens, an aperture diaphragm, a second reflecting mirror, a half-wave plate and a polarizing prism, which are sequentially arranged along the optical path;
laser light emitted by the laser is transmitted to the second collimating lens through an optical fiber, then is reflected to the diffractive optical element by the polarizing prism after sequentially passing through the aperture diaphragm, the second reflecting mirror and the half-wave plate, and diffracted light generated by the diffractive optical element is transmitted to the polarizing prism and then is output to the Fourier lens of the structured light filtering device.
Preferably, the microscopic imaging module comprises a third reflecting mirror, a dichroic mirror, a camera and a microscope;
the light emitted by the first collimating lens of the structured light filtering device is reflected by the third reflector and the dichroic mirror in sequence, then is irradiated to the sample surface through the microscope objective of the microscope to generate the structured light and irradiate the sample, and the fluorescence generated by exciting the sample is collected by the microscope objective and then is transmitted through the dichroic mirror to reach the camera for imaging.
The utility model has the advantages that: the filtering device for generating the structured light, provided by the utility model, can accurately control the positive and negative first-order diffraction light to be continuously transmitted through the frequency spectrum plane through the round hole filtering element, and can improve the contrast of the illumination light of the surface structure of the subsequent sample; the utility model provides a further micro-imaging system of structured light illumination on the basis that has improved the contrast of sample surface texture illumination light, blocks the marginal part of facula through the aperture diaphragm to can obtain the better laser of intensity homogeneity, finally ensure that the structured light that generates has good homogeneity, solve the bright, the dark shortcoming in edge of formation of image picture central authorities, can reduce the fluorescence distortion degree of image.
Drawings
Fig. 1 is a schematic structural diagram of a filtering apparatus for structured light generation according to embodiment 1 of the present invention;
fig. 2 is a schematic structural diagram of a structured light illumination microscopic imaging system in embodiment 2 of the present invention;
fig. 3 is a schematic structural diagram of a circular hole filter element according to embodiment 1 of the present invention;
fig. 4 is a schematic structural view of a Mask plate with rectangular holes used in the prior art.
Description of reference numerals:
1-a laser; 2-an optical fiber; 3-a second collimating lens; 4-aperture diaphragm; 5-a second reflector; 6, a half-wave plate; 7-a polarizing prism; 8-a diffractive optical element; 9-a fourier lens; 10 — a first mirror; 11-circular hole filter element; 12-a phase retarder; 13-a first collimating lens; 14-a third mirror; 15-a dichroic mirror; 16-a camera; 17-a microscope objective; 18-sample; 110 — MASK plate; 111-round hole.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can implement the invention with reference to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
Example 1
As shown in fig. 1, a filter device for structured light generation of the present embodiment includes a diffractive optical element 8, a fourier lens 9, a first mirror 10, a circular hole filter element 11, a phase retarder 12, and a first collimating lens 13, which are arranged in this order along an optical path;
the diffracted light emitted by the diffractive optical element 8 is focused by the fourier lens 9 (light is focused to a spectrum plane), reflected by the first reflector 10, filtered by the circular hole filter element 11 (only positive and negative first-order diffracted light is allowed to pass), subjected to phase modulation by the phase retarder 12, and collimated by the first collimating lens 13, and then output. The positive and negative first-order diffracted lights output by the first collimating lens 13 are collimated and then interfere on the surface of the sample 18, so that the structured illumination light is generated to irradiate the sample 18.
The circular hole filtering element 11 has a plurality of circular holes with different apertures, which can be switched to enter the optical path for filtering. The incident light source of the filtering device can comprise lasers with various different wavelengths, and round holes corresponding to the apertures are switched for filtering aiming at the lasers with different wavelengths, so that the filtering effect can be remarkably improved.
In a preferred embodiment, referring to fig. 3, the circular hole filter element 11 includes a MASK plate 110 having a plurality of circular holes 111 with different apertures arranged in a circular shape and spaced apart from each other, and a driving mechanism (not shown in the figure) for driving the MASK plate 110 to perform a positional shift to switch the different circular holes 111 into the optical path.
The diffracted light from the diffractive optical element 8 includes 0 order, ± 1 order light, etc., but only ± 1 order light can pass through the circular aperture filter element 11. The circular hole filter element 11 can accurately control the positive and negative first-order diffracted lights to be transmitted continuously through a frequency spectrum plane, and the contrast ratio of the illumination light of the surface structure of the subsequent sample 18 can be improved.
In a preferred embodiment, the diffractive optical element 8 is a spatial light modulator.
Example 2
The present embodiment provides a structured light illumination microscopic imaging system, which includes the structured light filtering device, the illumination light source module, and the microscopic imaging module of embodiment 1.
The illumination light source module comprises a laser 1, an optical fiber 2, a second collimating lens 3, an aperture diaphragm 4, a second reflecting mirror 5, a half-wave plate 6 and a polarizing prism 7 which are sequentially arranged along a light path, and the microscopic imaging module comprises a third reflecting mirror 14, a dichroic mirror 15, a camera 16 and a microscope.
Laser emitted by the laser 1 is transmitted to the second collimating lens 3 through the optical fiber 2, then is reflected to the diffractive optical element 8 by the polarizing prism 7 after sequentially passing through the aperture diaphragm 4, the second reflecting mirror 5 and the half-wave plate 6, and diffracted light generated by the diffractive optical element 8 is transmitted through the polarizing prism 7 and then is output to the Fourier lens 9 of the structured light filtering device for focusing; then, the light is reflected by a first reflector 10, filtered by a circular hole filter element 11 (only positive and negative first-order diffracted lights are allowed to pass), output after phase modulation by a phase retarder 12 and collimation by a first collimating lens 13, and then is reflected by a third reflector 14 and reflected by a dichroic mirror 15 in sequence, and then is irradiated to the surface of a sample 18 through a microscope objective 17 of a microscope to generate structural light and irradiate the sample 18, and fluorescence generated by exciting the sample 18 is collected by the microscope objective 17 and then is transmitted through the dichroic mirror 15 to reach a camera 16 for imaging.
The laser 1 is a tunable laser 1 capable of emitting laser beams with different wavelengths, or includes a plurality of lasers 1 for providing laser beams with different wavelengths (such as commonly used laser beams with wavelengths of 405nm, 488nm, 561nm, and 638 nm).
The period and direction of the fringes are continuously changed by the spatial light modulator, and each period and direction of the fringes corresponds to one phase of the phase delay period, so that the phase retarder 12 continuously changes along with the spatial light modulator. For example, the spatial light modulator generates stripes in three directions of 0 °, 60 °, and 120 °, and the laser light is incident on the spatial light modulator to generate diffracted light in the corresponding direction, but the 60 ° and 120 ° diffracted light do not match with the polarization direction thereof, so the polarization direction of the diffracted light is adjusted by the phase retarder 12 to correspond to the stripe direction, so that the desired structured light distribution is obtained. It is to be understood that the spatial light modulator and phase retarder 12 is a conventional product, and the operation principle thereof is the prior art.
Wherein, the laser is collimated into a bigger parallel light spot by the second collimating lens 3, the intensity distribution of the light spot satisfies the Gaussian distribution, the light at the central position is strong, the light at the edge position is small, the light spot central part is made to penetrate the diaphragm by adjusting the size of the aperture diaphragm 4, the edge part is shielded, the light penetrating the aperture diaphragm 4 is incident to the polarizing prism 7 after passing through the half-wave plate 6, the half-wave plate 6 can rotate along the optical axis to adjust the direction of the polarization state of the laser, ensuring that the light entering the polarizing prism 7 is reflected to the spatial light modulator at the inclined plane of the polarizing prism 7, the spatial light modulator generates Moire fringe pattern which is equivalent to the function of a reflection type grating to diffract the light out, the diffracted light is output to the Fourier lens 9 of the structured light filtering device after transmitting the polarizing prism 7, and then is reflected to the circular hole filtering element 11 by the first reflecting mirror 10, the diffracted light comprises 0-order light, +/-1-order light and the like, wherein only +/-1-order light can be transmitted continuously through the circular hole filtering element 11, interference occurs on the surface of the sample 18 after the light passes through the subsequent optical elements, structured light is generated, the +/-1-order light is interfered with each other to form moire fringes with better contrast, and the contrast of the structured illumination light can be improved.
In the embodiment, the spectrum surface of the incident beam can be accurately matched with the wavelength of the incident beam and the period of the diffraction element, diffracted stray light can be effectively blocked, only positive and negative first-order diffracted light can be continuously transmitted through the spectrum surface, and structured light interference fringes with high contrast can be generated on the rear focal plane of the lens group.
In the embodiment, the edge part of the light spot is blocked by the aperture diaphragm 4, so that laser with better intensity uniformity can be obtained, the generated structured light is ensured to have good uniformity, and the defects of bright center and dark edge of an imaging picture are overcome; and the more uniform structured light illumination can reduce misjudgment on the relative brightness of the fluorescence of different areas of the picture and reduce the fluorescence distortion degree of the image.
While the embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields where the invention is suitable, and further modifications may readily be made by those skilled in the art, and the invention is therefore not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.
Claims (8)
1. A filtering device for generating structured light is characterized by comprising a diffraction optical element, a Fourier lens, a circular hole filtering element, a phase retarder and a first collimating lens which are sequentially arranged along an optical path;
the diffracted light emitted by the diffractive optical element is output after being focused by the Fourier lens, filtered by the circular hole filtering element, subjected to phase modulation by the phase retarder and collimated by the first collimating lens in sequence;
the circular hole filtering element is provided with a plurality of circular holes with different apertures and capable of switching into the light path to filter light.
2. The filtering apparatus for structured light generation according to claim 1, wherein the circular hole filtering element comprises a MASK plate with a plurality of circular holes with different apertures arranged at intervals and a driving mechanism for driving the MASK plate to perform position movement to switch different circular holes into the optical path.
3. The filter apparatus for structured light generation of claim 2, wherein only ± 1 st order light of the diffracted light emitted by the diffractive optical element is transmitted through the circular aperture filter element.
4. The filtering device for structured light generation according to claim 1, wherein a first reflector is further disposed between the fourier lens and the circular aperture filtering element, and light emitted from the fourier lens reaches the circular aperture filtering element after being reflected by the first reflector.
5. A filtering arrangement for structured light generation according to claim 1, wherein the diffractive optical element is a spatial light modulator.
6. A structured light illuminated microscopy imaging system comprising a structured light filtering device according to any one of claims 1 to 5, an illumination source module and a microscopy imaging module.
7. The structured light illuminated microimaging system of claim 6, wherein the illumination source module comprises a laser, an optical fiber, a second collimating lens, an aperture stop, a second reflecting mirror, a half-wave plate and a polarizing prism arranged in sequence along the optical path;
laser light emitted by the laser is transmitted to the second collimating lens through an optical fiber, then is reflected to the diffractive optical element by the polarizing prism after sequentially passing through the aperture diaphragm, the second reflecting mirror and the half-wave plate, and diffracted light generated by the diffractive optical element is transmitted to the polarizing prism and then is output to the Fourier lens of the structured light filtering device.
8. The structured light illuminated microscopy imaging system of claim 7, wherein the microscopy imaging module comprises a third mirror, a dichroic mirror, a camera, and a microscope;
the light emitted by the first collimating lens of the structured light filtering device is reflected by the third reflector and the dichroic mirror in sequence, then is irradiated to the sample surface through the microscope objective of the microscope to generate the structured light and irradiate the sample, and the fluorescence generated by exciting the sample is collected by the microscope objective and then is transmitted through the dichroic mirror to reach the camera for imaging.
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| CN113238389A (en) * | 2021-05-25 | 2021-08-10 | 中国科学院苏州生物医学工程技术研究所 | Structured light filtering device and structured light illumination microscopic imaging system with same |
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| CN113238389A (en) * | 2021-05-25 | 2021-08-10 | 中国科学院苏州生物医学工程技术研究所 | Structured light filtering device and structured light illumination microscopic imaging system with same |
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