Structured light illumination system based on large view field

Abstract

The invention provides a structured light illumination system based on a large view field. The structured light illumination system comprises an illumination light source, a collimating lens, a grating, a first lens, a diaphragm, a second lens, a third lens, a spectroscope and a camera, the illumination light source is used for emitting laser; the laser is collimated by the collimating lens and then enters the grating, after passing through the grating, a multi-stage interference light beam is generated and enters the first lens, and after being collimated by the first lens, the multi-stage interference light beam is focused to the diaphragm, so that a target-stage diffraction light beam passes through the second lens; the light sequentially passes through the third lens to the spectroscope; and the spectroscope reflects the received light beam to an objective lens of the camera to form interference fringes on an object plane. A required structured light period and corresponding illumination lens parameters are calculated according to system parameters, and super-resolution imaging is realized by using an image height function to control light rays.

Description

Technical Field

[0001] The present invention relates to the technical field of super-resolution microscopy, and more specifically to a structured light illumination system based on a large field of view. Background Art

[0002] In the past decade, super-resolution microscopy technology has developed rapidly. Currently, common super-resolution microscopy technologies include: Stimulated Emission Depletion Microscopy (STED), Structured Illumination Microscopy (SIM), Photo Activated Localization Microscopy (PALM), and Stochastic Optical Reconstruction Microscopy (STORM). Traditional optical manufacturers have also launched many related products based on the above technologies, such as Nikon N-SIM series, N-STORM, Leica SR GSD 3D, Zeiss Elyra 7 with Lattice SIM, etc.

[0003] Among the above-mentioned technologies, SIM has the advantages of fast imaging speed, low photobleaching, and large imaging field of view, and is very suitable for application in the gene sequencing industry.

[0004] However, most of the products currently on the market are based on high-magnification, small-field-of-view objectives, generally above 60x, with a maximum field of view of only 0.4mm. However, most of the sequencers currently on the market have a field of view of more than 1mm, and there are no relevant technical methods.

[0005] In other words, existing structured light illumination technologies are all designed based on high-magnification objectives, with a very small field of view, which does not meet the high-throughput requirements of sequencers. Summary of the invention

[0006] In view of this, in order to solve the above problems, the present invention provides a structured light illumination system based on a large field of view, and the technical solution is as follows:

[0007] A structured light illumination system based on a large field of view, the structured light illumination system comprising: an illumination light source, a collimating lens, a grating, a first lens, an aperture, a second lens, a third lens, a beam splitter and a camera;

[0008] The illumination light source is used to emit laser light;

[0009] The laser is collimated by the collimating lens and incident on the grating. After passing through the grating, a multi-level interference beam is generated and incident on the first lens. After being collimated by the first lens, it is focused on the aperture, so that the target-level diffraction beam passes through the second lens, and then passes through the third lens to the beam splitter in sequence.

[0010] The beam splitter reflects the received light beam to the objective lens of the camera, forming interference fringes on the object plane.

[0011] Preferably, in the above structured light illumination system, the first lens and the second lens are distributed in mirror-symmetric manner.

[0012] Preferably, in the above structured light illumination system, the aperture is located at the focal plane of the first lens and at the focal plane of the second lens.

[0013] Preferably, in the above structured light illumination system, the second lens and the third lens are distributed in mirror-symmetric manner.

[0014] Preferably, in the above structured light illumination system, the distance between the second lens and the third lens is twice the focal length.

[0015] Preferably, in the above structured light illumination system, the grating is mounted on a one-position translation stage, and the one-position translation stage is mounted on a rotating stage.

[0016] Preferably, in the above structured light illumination system, the first lens, the second lens or the third lens is a doublet lens.

[0017] Preferably, in the above structured light illumination system, the first lens, the second lens and the third lens are lenses of the same type.

[0018] Preferably, in the above structured light illumination system, the beam splitter is a reflector.

[0019] Preferably, in the above structured light illumination system, the illumination light source includes lasers of two wavelengths.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention provides a structured light illumination system based on a large field of view, comprising: an illumination light source, a collimating lens, a grating, a first lens, an aperture, a second lens, a third lens, a beam splitter and a camera; the illumination light source is used to emit laser light; the laser light is collimated by the collimating lens and incident on the grating, and after passing through the grating, a multi-level interference light beam is generated and incident on the first lens, and after being collimated by the first lens, it is focused on the aperture, so that the target-level diffraction light beam passes through the second lens, and then passes through the third lens to the beam splitter in turn; the beam splitter reflects the received light beam to the objective lens of the camera, and forms interference fringes on the object surface. The required structured light period and the corresponding illumination lens parameters are calculated according to the system parameters, and super-resolution imaging is achieved by controlling the light using the image height function. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0023] Figure 1 A structural diagram of a structured light illumination system based on a large field of view provided by an embodiment of the present invention;

[0024] Figure 2 A schematic diagram of a specific optical path structure of a structured light illumination system based on a large field of view provided by an embodiment of the present invention;

[0025] Figure 3 A schematic diagram of imaging of a structured light illumination stripe on a camera provided by an embodiment of the present invention;

[0026] Figure 4 A method for providing a Figure 3 Schematic diagram of the distribution of the image in the frequency domain after Fourier transform. DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Based on the technical problems pointed out in the background technology, this application proposes a structured light illumination design method based on a large field of view optical system, and realizes super-resolution imaging. First, the required structured light period and the corresponding illumination lens parameters are calculated according to the system parameters, and a corresponding software design optimization method is proposed. In traditional lens design, the default function is generally used to optimize the wave aberration of the system. However, in the design of structured light illumination, in order to ensure that the two beams of light interfere, we believe that it is simpler and more effective to use the image height function to control the light. We designed the required illumination lens through the above method and verified the accuracy of the theory through experiments.

[0029] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0030] (1) First, conduct theoretical calculation analysis:

[0031] 1. The structured light illumination system based on a large field of view includes a laser with excitation wavelengths of 532nm and 640nm.

[0032] 2. The objective lens used in the structured light illumination system based on a large field of view has a specification of NA0.8, 20x.

[0033] 3. Considering the effect of vignetting, the maximum angle we can use is about 48°. For oblique incident light, the interference period is:

[0034]

[0035] 4. The field of view of the objective lens is 1.5mm. Assuming that 110line / mm grating is selected, the grating period is 9.09um. Take three pictures in each direction, and each picture moves 3.03um.

[0036] 5. According to the grating equation:

[0037] d g *sinθ g =λ (1-2)

[0038] 6. The first-order diffraction angle of the grating can be calculated:

[0039]

[0040] 7. Diffraction angle θ at 640nm g640 =4.037°; diffraction angle θ at 532nm g532 =3.355°.

[0041] 8. Because the diffraction angle of 640nm is relatively large, the illumination magnification is calculated based on 640nm:

[0042]

[0043] 9. Back to formula (1-1), the interference period formed by 640nm is:

[0044]

[0045] 10. Substituting formula (1-2) and formula (1-3) into formula (1-1), we can get:

[0046]

[0047] It can be seen that the period of interference fringes has nothing to do with the wavelength.

[0048] The objective lens field of view is 1.5mm. Considering the difficulty of installation and adjustment, the illumination spot will be designed to be slightly larger, 1.7mm.

[0049] The spot size incident on the grating is: 1.7mm*10.55=17.9mm.

[0050] The theoretical focal length of the objective lens is 10mm, so the focal length of the illumination lens is 10*10.55=105.5mm. This value will be adjusted according to the actual focal length of the objective lens.

[0051] (2) Then design the lens:

[0052] 1. According to the above analysis and calculation, a lens with a focal length of about 105mm needs to be designed in this application. If the spot lens purchased directly from Thorlab will not match the objective lens, the optical system will have large residual aberrations, affecting the interference fringe effect.

[0053] 2. At the same time, in order to reduce production and R&D costs, the same lighting lens is used in the system.

[0054] 3. In principle, to produce interference fringes, it is necessary to ensure the overlap of the two beams of light at the + and - levels; to achieve this goal, the image height function is used for control in the design software.

[0055] 4. Figure 1 As shown, Figure 1 A structural diagram of a structured light illumination system based on a large field of view provided in an embodiment of the present invention, wherein a grating 103, a first lens 104, an aperture 105, a second lens 106, a third lens 107 and an objective lens 109, and a virtual surface exists between the second lens 106 and the third lens 107.

[0056] 5. The lens parameters in the system are as follows:

[0057]

[0058]

[0059] In this example, the first lens 104, the second lens 106, and the third lens 107 are lenses of the same type, all of which are double-cemented lenses, wherein the first row represents parameters of the initial surface, the second row represents parameters of the grating 103, 2, 3, and 4 represent parameters of the first lens 104, 5 represents parameters of the aperture 105, 6, 7, and 8 represent parameters of the second lens 106, 9 represents parameters of the virtual surface, 10, 11, and 12 represent parameters of the third lens 107, and parameters 13 and thereafter represent parameters of the objective lens 109.

[0060] 6. Optimization Function

[0061]

[0062] The main optimization functions designed here are:

[0063] REAY: Controls the height of light on the image plane.

[0064] RAED: Controls the incident angle of light on the image plane.

[0065] refer to Figure 2 , Figure 2 A schematic diagram of a specific optical path structure of a structured light illumination system based on a large field of view is provided in an embodiment of the present invention. The structured light illumination system based on a large field of view includes: an illumination light source 101, a collimating lens 102, a grating 103, a first lens 104, an aperture 105, a second lens 106, a third lens 107, a beam splitter 108 and a camera 111.

[0066] The illumination light source 101 is used to emit laser light, and the illumination light source 101 includes laser light of two wavelengths.

[0067] The laser is collimated by the collimating lens 102 and incident on the grating 103. After passing through the grating 103, a multi-level interference light beam is generated and incident on the first lens 104. After being collimated by the first lens 104, it is focused on the aperture 105, so that the target-level diffraction light beam passes through the second lens 106, and then passes through the third lens 107 to the spectrometer 108.

[0068] The beam splitter 108 reflects the received light beam to the objective lens 109 of the camera 111 , forming interference fringes on the object plane 110 .

[0069] Specifically, in the embodiment of the present invention Figure 1 Reference numeral 100 indicates a component part for emitting a target light beam, light rays are indicated by solid lines, and the direction of arrows indicates the direction in which the light rays travel.

[0070] The illumination light source 101 is a 2W laser, and the illumination light source 101 includes two wavelengths, 532nm and 640nm.

[0071] After being emitted from the illumination light source 101 , the laser is collimated by the collimating lens 102 and incident on the grating 103 .

[0072] The number of line pairs of grating 103 is adjusted according to the design requirements. Here, the number of line pairs is 110 / mm, that is, the grating period d g =1 / 110mm.

[0073] The grating 103 is mounted on a one-dimensional translation stage, and the translation stage is mounted on a rotation stage, and can achieve translation and rotation according to system requirements.

[0074] After the light beam passes through the grating 103 , a multi-level interference light beam of 0, +-1, +-2, etc. is generated. The light beam is collimated by the first lens 104 and then focused on the aperture 105 .

[0075] The aperture 105 is located at the focal plane of the first lens 104 and allows only +-1 order diffraction beams to pass through and filters out beams of other energy levels to avoid generating structured light of other frequencies.

[0076] The second lens 106 is installed in a mirror-symmetrical manner to the first lens 104 , and the aperture 105 is also placed at the focal plane of the second lens 106 .

[0077] The third lens 107 and the second lens 106 are also installed in a mirror-symmetrical manner, and the distance between the two lenses is twice the focal length.

[0078] The beam splitter 108 reflects the illumination beam to the objective lens 109 of the camera 111, and finally forms interference fringes on the object plane 110. Specifically, the beam splitter 108 is a dichroic mirror.

[0079] like Figure 3 and Figure 4 As shown, Figure 3 A schematic diagram of imaging of a structured light illumination stripe on a camera provided by an embodiment of the present invention; Figure 4 A method for providing a Figure 3 The distribution diagram of the image in the frequency domain after Fourier transform is shown. Figure 4 The peak value of the spectrum shown in the middle circle is 4.02 pixels. The pixel size of the camera 111 used in the structured light illumination system based on a large field of view is 3.2um, and the magnification of the imaging system is 29.3. It can be calculated that the corresponding object-side structured light period is 4.02*3.2 / 29.3=0.439um, which is consistent with the theoretical design.

[0080] The above is a detailed introduction to a structured light illumination system based on a large field of view provided by the present invention. Specific examples are used in this article to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, for those skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as limiting the present invention.

[0081] It should be noted that each embodiment in this specification is described in a progressive manner, and each embodiment focuses on the differences from other embodiments, and the same or similar parts between the embodiments can be referred to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant parts can be referred to the method part description.

[0082] It should also be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that the process, method, article or device that includes a series of elements is inherent to the elements, or also includes elements inherent to these processes, methods, articles or devices. In the absence of further restrictions, the elements defined by the sentence "including a..." do not exclude the presence of other identical elements in the process, method, article or device that includes the elements.

[0083] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A structured light illumination system based on a large field of view, characterized in that: The structured light illumination system comprises: an illumination light source, a collimating lens, a grating, a first lens, an aperture, a second lens, a third lens, a beam splitter and a camera; The illumination light source is used to emit laser light; The laser is collimated by the collimating lens and incident on the grating. After passing through the grating, a multi-level interference beam is generated and incident on the first lens. After being collimated by the first lens, it is focused on the aperture, so that the target-level diffraction beam passes through the second lens, and then passes through the third lens to the beam splitter in sequence. The beam splitter reflects the received light beam to the objective lens of the camera, forming interference fringes on the object plane.

2. The structured light illumination system according to claim 1, characterized in that: The first lens and the second lens are distributed in mirror-symmetric manner.

3. The structured light illumination system according to claim 1, characterized in that: The aperture stop is located at a focal plane of the first lens and at a focal plane of the second lens.

4. The structured light illumination system according to claim 1, characterized in that: The second lens and the third lens are distributed in mirror-symmetric manner.

5. The structured light illumination system according to claim 4, characterized in that: The distance between the second lens and the third lens is twice the focal length.

6. The structured light illumination system according to claim 1, characterized in that: The grating is mounted on a one-position translation stage, and the one-position translation stage is mounted on a rotating stage.

7. The structured light illumination system according to claim 1, characterized in that: The first lens, the second lens or the third lens is a doublet lens.

8. The structured light illumination system according to claim 1, characterized in that: The first lens, the second lens and the third lens are lenses of the same type.

9. The structured light illumination system according to claim 1, characterized in that: The beam splitter is a dichroic mirror.

10. The structured light illumination system according to claim 1, characterized in that: The illumination light source includes lasers of two wavelengths.

Images