Stimulated radiation loss optical microscope and illumination system thereof

Abstract

The invention provides a stimulated radiation loss optical microscope and an illumination system thereof. The illumination system comprises an illumination light source, a light beam adjusting assembly, a polarization splitting prism, a first 1 / 4 wave plate, a first dichroic element, a first optical delay unit, a second 1 / 4 wave plate, a second optical delay unit, a second dichroic element, a third 1 / 4 wave plate and a third dichroic element which are sequentially arranged in the light path transmission direction. Part of the first light beam is converted into a third light beam after being transmitted by the polarization splitting prism, and the other part of the first light beam is converted into a fifth light beam after being reflected by the polarization splitting prism; part of the second light beam is converted into a fourth light beam after being transmitted by the polarization splitting prism, and the other part of the second light beam is converted into a sixth light beam after being reflected by the polarization splitting prism. The illumination system can obtain a three-dimensional nanoscale spatial super-resolution image, and the imaging resolution is improved.

Description

Technical Field

[0001] The present invention relates to the field of microscopy imaging technology, and in particular to a stimulated emission depletion optical microscope and its illumination system. Background Art

[0002] Approximately 80% of the current microscopy imaging research in life sciences still uses optical microscopes. It can be said that the progress of life sciences has been accompanied by the development of optical microscopes. However, due to the existence of the optical diffraction limit, the spatial resolution of optical microscopes is limited to about half a wavelength, and such resolution seriously hinders the fine research of subcellular structures by biologists. The STED (Stimulated Emission Depletion) optical microscope uses a depletion beam modulated by a phase plate to form a depletion spot, and converts the fluorescent molecules around the excitation light diffraction spot into a non-spontaneous emission state through stimulated emission depletion, achieving a spatial resolution better than 50 nm. Due to the use of an all-optical setup, the image acquisition time is the same as that of confocal microscopes, and there are no special requirements for sample preparation. Therefore, real-time imaging and dynamic tracking of subcellular structures in living cells can be achieved.

[0003] Since the invention of the stimulated emission depletion optical microscope, it has been widely used in biological and life medical science research. For the improvement of axial resolution, stimulated emission depletion optical microscopes based on two architectures of single objective lens and dual objective lenses (4PI microscopes) have emerged. Among them, the stimulated emission depletion optical microscope micro-system with a dual objective lens architecture is complex, has extremely strict alignment requirements, great debugging difficulty, and its stability is also difficult to guarantee; in contrast, the stimulated emission depletion optical microscope with a single objective lens architecture has a smaller complexity. It realizes three-dimensional isotropic super-resolution imaging by non-coherently superimposing the intensity of a transverse doughnut spot and an axial hollow spot at the focal plane through beam shaping. However, in the actual development process, the stimulated emission depletion optical microscope with a single objective lens architecture also faces many technical problems, such as the difficulty of coaxial alignment of multiple beams, the bulky volume of the illumination system of the stimulated emission depletion optical microscope, high construction costs, poor stability, etc., which limit its wide application. Summary of the Invention

[0004] The object of the present invention is to solve at least one of the problems of difficult beam alignment, large equipment volume, and poor stability in the existing stimulated emission depletion optical microscope, and this object is achieved through the following technical solutions.

[0005] The first aspect of the embodiments of the present application provides an illumination system for a stimulated emission depletion optical microscope. The stimulated emission depletion optical microscope includes a microscope objective lens, and the illumination system includes the following components sequentially arranged along the optical path transmission direction:

[0006] A lighting light source that generates an incident light beam;

[0007] A light beam adjusting assembly, wherein the incident light beam is converted into a first light beam and a second light beam after passing through the light beam adjusting assembly;

[0008] A polarization beam splitter prism, wherein part of the first light beam is transmitted through the polarization beam splitter prism and then converted into a third light beam, and the remaining part of the first light beam is reflected by the polarization beam splitter prism and then converted into a fifth light beam; part of the second light beam is transmitted through the polarization beam splitter prism and then converted into a fourth light beam, and the remaining part of the second light beam is reflected by the polarization beam splitter prism and then converted into a sixth light beam;

[0009] A first quarter-wave plate, wherein the third light beam is converted into a first circularly polarized light after passing through the first quarter-wave plate, and the fourth light beam is converted into a second circularly polarized light after passing through the first quarter-wave plate;

[0010] A first dichroic element, wherein the first circularly polarized light is reflected by the first dichroic element and then converted into a first linearly polarized light after passing through the first quarter-wave plate again;

[0011] A first optical delay unit, wherein a first phase plate is provided at the distal end of the first optical delay unit, the second circularly polarized light is transmitted through the first dichroic element and then enters the first optical delay unit, and after being reflected by the first phase plate, it passes through the first optical delay unit, the first dichroic element, and the first quarter-wave plate in sequence and then is converted into a second linearly polarized light;

[0012] A second quarter-wave plate, wherein the fifth light beam is converted into a third circularly polarized light after passing through the second quarter-wave plate, and the sixth light beam is converted into a fourth circularly polarized light after passing through the second quarter-wave plate;

[0013] A second optical delay unit;

[0014] A second dichroic element, wherein a second phase plate is provided at the distal end of the second optical delay unit;

[0015] A third quarter-wave plate; and

[0016] A third dichroic element, wherein the third circularly polarized light is emitted after passing through the second optical delay unit, the second phase plate, and the second dichroic element in sequence; the fourth circularly polarized light passes through the second optical delay unit and the second phase plate in sequence, and then is reflected by the second dichroic element and then passes through the second phase plate, the second optical delay unit, and the second quarter-wave plate in sequence and then is converted into a third linearly polarized light;

[0017] The first linearly polarized light is reflected by the polarization beam splitter prism and then sequentially passes through the third quarter-wave plate and the third dichroic element, and then enters the microscope objective lens, forming a first light spot at the focal plane of the microscope objective lens;

[0018] The second linearly polarized light is reflected by the polarization beam splitter prism and then sequentially passes through the third quarter-wave plate and the third dichroic element, and then enters the microscope objective lens, forming a second light spot at the focal plane of the microscope objective lens;

[0019] The third linearly polarized light is transmitted through the polarization beam splitter prism and then sequentially passes through the third quarter-wave plate and the third dichroic element, and then enters the microscope objective lens, forming a third light spot at the focal plane of the microscope objective lens. The centers of the first light spot, the second light spot, and the third light spot coincide.

[0020] The illumination system of the stimulated emission depletion optical microscope according to the present application further has the following additional technical features.

[0021] Optionally, the beam adjustment assembly includes a filter, a linear polarizer, and a half-wave plate arranged in sequence along the optical path transmission direction, and the filter, the linear polarizer, and the half-wave plate are coaxially arranged.

[0022] Optionally, the filter is a dual-bandpass filter.

[0023] Optionally, the first light beam is an excitation light, and the second light beam is a loss light relative to the first light beam; and / or, the first light spot is a solid light spot, and the second light spot and the third light spot are hollow light spots.

[0024] Optionally, the first dichroic element is a selectively transmissive dielectric film, and the dielectric film is plated on the incident end of the first optical delay unit; and / or, the second dichroic element is a dichroic sheet.

[0025] Optionally, the first phase plate is a reflective spiral phase plate, and / or, the second phase plate is a half-wave phase plate.

[0026] Optionally, the first optical delay unit and the first phase plate are of an integral structure or a split structure, and / or, the second optical delay unit and the second phase plate are of an integral structure or a split structure.

[0027] The second aspect of the embodiments of the present application provides a stimulated emission depletion optical microscope, including the illumination system of the stimulated emission depletion optical microscope and a microscopic imaging system. The microscopic imaging system includes a microscope objective lens, and the light beam generated by the illumination system is converged to a sample through the microscope objective lens.

[0028] Optionally, the stimulated emission depletion optical microscope further includes a detection system, and the fluorescence beam generated by the sample enters the detection system.

[0029] Optionally, after being reflected by the third dichroic element, the fluorescence beam enters the detection system.

[0030] The advantages of the present invention are as follows:

[0031] The illumination system of the stimulated emission depletion optical microscope of the present application can make the centers of the first spot, the second spot, and the third spot coincide, obtain a three-dimensional nano-scale spatial super-resolution image, and improve the imaging resolution. Description of the Drawings

[0032] By reading the following detailed description of the preferred embodiments, various other advantages and benefits will become clear to those of ordinary skill in the art. The drawings are only for the purpose of showing the preferred embodiments and are not considered to be a limitation of the present invention. Moreover, throughout the drawings, the same reference numerals are used to represent the same components.

[0033] Figure 1 It is a schematic structural diagram of the illumination system of the stimulated emission depletion optical microscope provided by the embodiment of the present invention;

[0034] Figure 2 It is an image of the first spot, the second spot, the third spot, and the point spread function of the superposition of the second spot and the third spot provided by the embodiment of the present invention;

[0035] Figure 3 It is an image of the point spread function of the incoherent intensity superposition of the second spot and the third spot according to different intensity ratios provided by the embodiment of the present invention;

[0036] Figure 4 It is a confocal imaging diagram in the XY plane obtained by irradiating a 40-nm fluorescent microsphere sample with an existing confocal microscope;

[0037] Figure 5 It is for the stimulated emission depletion imaging diagram in the XY plane obtained in the same region of the 40-nm fluorescent microsphere sample irradiated by the illumination system of the stimulated emission depletion optical microscope provided by the embodiment of the present invention Figure 4 ;

[0038] Figure 6 It is a confocal imaging diagram in the XZ plane obtained by irradiating a 40-nm fluorescent microsphere sample with an existing confocal microscope;

[0039] Figure 7 It is for the stimulated emission depletion optical microscope provided by the embodiment of the present invention to irradiate a 40-nm fluorescent microsphere sample Figure 6Stimulated emission depletion imaging diagram in the XZ plane obtained from the same region.

[0040] The reference numerals in the figure are as follows:

[0041] 1000, Stimulated emission depletion optical microscope;

[0042] 100, Illumination system;

[0043] 200, Microscopic imaging system;

[0044] 300, Detection system;

[0045] 1, Filter;

[0046] 2, Linear polarizer;

[0047] 3, 1 / 2 waveplate;

[0048] 4, Polarizing beam splitter prism;

[0049] 5, First 1 / 4 waveplate;

[0050] 6, First dichroic element;

[0051] 7, First optical delay unit;

[0052] 8, First phase plate;

[0053] 9, Second 1 / 4 waveplate;

[0054] 10, Second optical delay unit;

[0055] 11, Second phase plate;

[0056] 12, Second dichroic element;

[0057] 13, Third 1 / 4 waveplate;

[0058] 14, Third dichroic element;

[0059] 15, Illumination light source;

[0060] 101, First light beam;

[0061] 102, Second light beam;

[0062] 103, Third light beam;

[0063] 104, Fourth light beam;

[0064] 105, Fifth light beam;

[0065] 106, Sixth light beam;

[0066] 107, Fluorescence light beam. Detailed Implementation Modes

[0067] The exemplary implementation modes of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although the exemplary implementation modes of the present disclosure are shown in the drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the implementation modes set forth herein. On the contrary, these implementation modes are provided so that the present disclosure can be more thoroughly understood and the scope of the present disclosure can be fully conveyed to those skilled in the art.

[0068] It should be noted that in the description of the present invention, the terms "first" and "second" 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.

[0069] As Figure 1 shown, the stimulated emission depletion optical microscope 1000 includes an illumination system 100, a microscopic imaging system 200, and a detection system 300. Among them, the microscopic imaging system 200 includes a microscopic objective lens, and the number of microscopic objective lenses is one. The detection system 300 is used to detect the fluorescence beam 107 emitted by the sample, and the illumination system 100 is used to excite the sample to emit the fluorescence beam 107. Here, the sample refers to a fluorescence-labeled sample that can generate the fluorescence beam 107.

[0070] Next, Figure 1 the structure of the illumination system 100 will be introduced in detail.

[0071] The illumination system 100 includes the following components arranged in sequence along the optical path transmission direction. The components include an illumination light source 15, a beam adjustment assembly, a polarization beam splitter prism 4, a first quarter-wave plate 5, a first dichroic element 6, a first optical delay unit 7, a first phase plate 8, a second quarter-wave plate 9, a second optical delay unit 10, a second phase plate 11, a second dichroic element 12, a third quarter-wave plate 13, and a third dichroic element 14. The illumination light source 15 is used to generate an incident beam. The illumination light source 15 can be a laser light source, and the generated incident beam is a laser beam. The laser light source can emit multi-wavelength lasers or a combination of multi-wavelength lasers. The laser beam emitted by the laser light source passes through the optical path of the illumination system 100 and is then focused by the microscopic objective lens of the microscopic imaging system and irradiated onto the surface of the sample.

[0072] It should be emphasized that for the illuminating light source 15, the beam adjustment assembly, the polarization beam splitter prism 4, the first quarter-wave plate 5, the first dichroic element 6, the first optical delay unit 7, the first phase plate 8, the second quarter-wave plate 9, the second optical delay unit 10, the second phase plate 11, the second dichroic element 12, and the third quarter-wave plate 13, end-face bonding with optical glue can be used between the components to be connected to achieve stable integration of the stimulated emission depletion optical microscope 1000. Of course, an integral structure can also be used to replace two components to be connected.

[0073] The optical paths of these components will be described in more detail below.

[0074] The incident light beam is converted into a first light beam 101 and a second light beam 102 after passing through the light beam adjustment component. A part of the first light beam 101 is transmitted through the polarization beam splitter prism 4 and then converted into a third light beam 103, and the remaining part of the first light beam 101 is reflected by the polarization beam splitter prism 4 and then converted into a fifth light beam 105. A part of the second light beam 102 is transmitted through the polarization beam splitter prism 4 and then converted into a fourth light beam 104, and the remaining part of the second light beam 102 is reflected by the polarization beam splitter prism 4 and then converted into a sixth light beam 106. Among them, the third light beam 103 is converted into a first circularly polarized light after passing through the first quarter-wave plate 5, and the fourth light beam 104 is converted into a second circularly polarized light after passing through the first quarter-wave plate 5. The first circularly polarized light is reflected by the first dichroic element 6 and then converted into a first linearly polarized light after passing through the first quarter-wave plate 5 again. A first phase plate 8 is provided at the distal end of the first optical delay unit 7. The second circularly polarized light is transmitted through the first dichroic element 6 and then enters the first optical delay unit 7. After being reflected by the first phase plate 8, it passes through the first optical delay unit 7, the first dichroic element 6, and the first quarter-wave plate 5 in sequence and then is converted into a second linearly polarized light. The fifth light beam 105 is converted into a third circularly polarized light after passing through the second quarter-wave plate 9, and the sixth light beam 106 is converted into a fourth circularly polarized light after passing through the second quarter-wave plate 9. A second phase plate 11 is provided at the distal end of the second optical delay unit 10. The third circularly polarized light is emitted after passing through the second optical delay unit 10, the second phase plate 11, and the second dichroic element 12 in sequence. The fourth circularly polarized light passes through the second optical delay unit 10 and the second phase plate 11 in sequence, and then is reflected by the second dichroic element 12 and then passes through the second phase plate 11, the second optical delay unit 10, and the second quarter-wave plate 9 in sequence and then is converted into a third linearly polarized light. The first linearly polarized light is reflected by the polarization beam splitter prism 4 and then enters the microscope objective lens after passing through the third quarter-wave plate 13 and the third dichroic element 14 in sequence, and a first light spot is formed at the focal plane of the microscope objective lens. The second linearly polarized light is reflected by the polarization beam splitter prism 4 and then enters the microscope objective lens after passing through the third quarter-wave plate 13 and the third dichroic element 14 in sequence, and a second light spot is formed at the focal plane of the microscope objective lens. The third linearly polarized light is transmitted through the polarization beam splitter prism 4 and then enters the microscope objective lens after passing through the third quarter-wave plate 13 and the third dichroic element 14 in sequence, and a third light spot is formed at the focal plane of the microscope objective lens. The centers of the first light spot, the second light spot, and the third light spot coincide.

[0075] In Figure 1 For the reason of making the figure clear, the first light beam 101, the second light beam 102, the third light beam 103, the fourth light beam 104, the fifth light beam 105, and the sixth light beam 106 are vertically offset. In actual use, the first light beam 101 and the second light beam 102 are in a coaxial state, the third light beam 103 and the fourth light beam 104 are in a coaxial state, and the fifth light beam 105 and the sixth light beam 106 are in a coaxial state.

[0076] Optionally, the beam adjustment assembly includes a filter 1, a linear polarizer 2, and a half-wave plate 3 arranged in sequence along the optical path transmission direction. The filter 1, the linear polarizer 2, and the half-wave plate 3 are coaxially arranged.

[0077] Optionally, the filter 1 is a dual-bandpass filter 1 for filtering out the first light beam 101 and the second light beam 102 with appropriate wavelengths. The filter 1 is coaxially arranged along the optical path and coincides with the optical axis of the illumination light source 15. Among them, the wavelengths of the first light beam 101 and the second light beam 102 are different.

[0078] The linear polarizer 2 here is used to filter the first light beam 101 and the second light beam 102 into linearly polarized light, and the polarization direction of the outgoing light is parallel to the transmission axis of the linear polarizer 2.

[0079] The half-wave plate 3 here is used to change the polarization directions of the linearly polarized first light beam 101 and the linearly polarized second light beam 102, and can also distribute the energy between the third light beam 103 and the fifth light beam 105, and between the fourth light beam 104 and the sixth light beam 106, realizing the free switching between the 2D-STED (2-Dimension Stimulated Emission Depletion) imaging and 3D-STED (3-Dimension Stimulated Emission Depletion) imaging modes. Among them, when the sixth light beam 106 cannot form a light spot at the focal plane of the microscope objective, 2D-STED imaging can be realized. When the fourth light beam 104, the sixth light beam 106, and the third light beam 103 all form light spots at the focal plane of the microscope objective respectively, 3D-STED imaging can be realized. At this time, the centers of the three light spots coincide.

[0080] Specifically, the polarization beam splitter prism 4 can separate the incident light into linearly polarized light with polarization directions parallel and perpendicular to the incident plane, and transmit and reflect them respectively. The first light beam 101 forms the third light beam 103 and the fifth light beam 105 after passing through the polarization beam splitter prism 4; the second light beam 102 forms the fourth light beam 104 and the sixth light beam 106 after passing through the polarization beam splitter prism 4.

[0081] Optionally, the first light beam 101 is an excitation light beam. After passing through the polarization beam splitter prism 4, the first light beam 101 is divided into a third light beam 103 and a fifth light beam 105. Among them, the third light beam 103 is used to excite fluorescence for fluorescence imaging of the sample. The second light beam 102 is a loss light beam relative to the first light beam 101. After passing through the polarization beam splitter prism 4, the second light beam 102 is divided into a fourth light beam 104 and a sixth light beam 106. The fourth light beam 104 and the sixth light beam 106 will perform incoherent intensity superposition near the focal point of the microscope objective to suppress fluorescence, de-excite the fluorescent substances in the peripheral region of the first spot that are in the fluorescent emission state, so that the peripheral region no longer spontaneously emits fluorescence.

[0082] It should be emphasized that the functions of the first quarter-wave plate 5, the second quarter-wave plate 9, and the third quarter-wave plate 13 are the same, all of which are used to adjust the polarization state of the light beam. Among them, the first quarter-wave plate 5 can convert the incident third light beam 103 from a first linearly polarized light into a first circularly polarized light, and can also convert the incident third light beam 103 from a first circularly polarized light into a first linearly polarized light. The first quarter-wave plate 5 can convert the incident fourth light beam 104 from a second linearly polarized light into a second circularly polarized light, and can also convert the incident fourth light beam 104 from a second circularly polarized light into a second linearly polarized light. Similarly, the second quarter-wave plate 9 and the third quarter-wave plate 13 can also switch the incident light beam between linearly polarized light and circularly polarized light.

[0083] Optionally, the first spot is a solid spot, and the second spot and the third spot are hollow spots respectively. Specifically, the second spot is a transverse doughnut spot, and the third spot is an axial hollow spot, and their centers are in a coincident state.

[0084] Optionally, the first dichroic element 6 is a selectively transmissive dielectric film, and the dielectric film is plated on the incident end of the first optical delay unit 7. This dielectric film can reflect the incident third light beam 103 and transmit the fourth light beam 104. Preferably, the dielectric film of the first dichroic element 6 is plated on the surface of the incident end of the first optical delay unit 7, which can not only reduce the volume of the entire optical path system, make the arrangement of optical elements more reasonable and compact, but also reduce the influence of environmental factors such as temperature and vibration on the stimulated emission depletion microscope 1000.

[0085] Optionally, the second dichroic element 12 is a dichroic sheet, specifically a selectively transmissive dichroic sheet, which can transmit the incident fifth light beam 105 and reflect the sixth light beam 106.

[0086] Optionally, both the first optical delay unit 7 and the second optical delay unit 10 are optical glasses with strictly parallel ends, and the lengths of the first optical delay unit 7 and the second optical delay unit 10 can be designed according to actual needs, so as to generate the fourth light beam 104 and the sixth light beam 106 with appropriate delays.

[0087] Optionally, the first phase plate 8 is a reflective spiral phase plate. The reflective spiral phase plate is perpendicular to the optical axis of the first optical delay unit 7, that is, the center line of the reflective spiral phase plate coincides with the optical axis of the first optical delay unit 7, and is used to modulate the transverse loss optical wavefront and optical delay, generating a second spot in the shape of a transverse donut on the focal plane of the microscope objective.

[0088] The reflective spiral phase plate is spiral-shaped with a gradually increasing thickness. The phase difference between the light passing through the thinnest part and the thickest part is π. The round-trip phase difference of the reflection becomes 2π. After being focused by the microscope objective, they coherently superpose to form a donut-shaped spot laterally, which is the second spot mentioned above.

[0089] The second phase plate 11 includes a half-wave phase plate. The half-wave phase plate is arranged at the distal end of the second optical delay unit 10 and / or the proximal end of the second dichroic element 12, and is perpendicular to the optical axis of the second optical delay unit 10, that is, the center line of the half-wave phase plate coincides with the optical axis of the second optical delay unit 10, and is used to modulate the wavefront and optical path of the axial loss light, generating an axial hollow focal spot on the focal plane of the microscope objective. The phase plates in the embodiments of the present application perform reflective phase modulation on the loss light on the reflective spiral phase plate and the half-wave phase plate through binary optical processing technology, and can generate a phase modulation beam that is crucial in 3D-STED technology, has a certain time delay, and is strictly concentric with the excitation light.

[0090] It should be noted that there is a circular protrusion or depression in the middle of the half-wave phase plate here. The phase difference between the light passing through the inner circular part and the outer ring is π / 2. Because it is reflective and passes through twice, the total phase difference is π, and the corresponding optical path difference is half a wavelength, so it is called a half-wave phase plate. The beam modulated by the half-wave phase plate, after being focused by the microscope objective, coherently superposes to form an axially distributed hollow dark spot, that is, the third spot.

[0091] Optionally, the first optical delay unit 7 and the first phase plate 8 are of an integral structure or a split structure, and / or the second optical delay unit 10 and the second phase plate 11 are of an integral structure or a split structure. Among them, when a split structure is adopted, they can be connected by bonding. Through the integrated design of end-face bonding in the embodiments of the present application, the arrangement of optical elements is made more compact, and at the same time, the influence of external environments such as temperature and vibration on the optical path can be reduced, improving the reliability of the optical system.

[0092] A beam expander for expanding and shaping the light beam emitted by the illumination light source 15 is further provided between the illumination light source 15 and the polarizer, and the widths of the first light beam 101 and the second light beam 102 are adjusted.

[0093] The following will combine Figure 1 to introduce the propagation direction of the light beam in more detail.

[0094] The illumination light source 15 generates an incident light beam, and the incident light beam includes a first light beam 101 and a second light beam 102. Among them, after the first light beam 101 passes through the filter 1, the linear polarizer 2 and the 1 / 2 wave plate 3 in sequence, a part of the first light beam 101 is transmitted through the polarization beam splitter prism 4 and then converted into a third light beam 103. The third light beam 103 is converted into a first circularly polarized light through the first 1 / 4 wave plate 5. The first circularly polarized light is reflected by the first dichroic element 6 and then converted into a first linearly polarized light through the first 1 / 4 wave plate 5 again. The first linearly polarized light is reflected by the polarization beam splitter prism 4 and then passes through the third 1 / 4 wave plate 13 and the third dichroic element 14 in sequence and then enters the microscope objective lens, and a first light spot is formed at the focal plane of the microscope objective lens.

[0095] The remaining part of the first light beam 101 is reflected by the polarization beam splitter prism 4 and then converted into a fifth light beam 105. Among them, the fifth light beam 105 is converted into a third circularly polarized light through the second 1 / 4 wave plate 9. The third circularly polarized light passes through the second optical delay unit 10, the second phase plate 11 and the second dichroic element 12 in sequence and then exits.

[0096] A part of the second light beam 102 is transmitted through the polarization beam splitter prism 4 and then converted into a fourth light beam 104. The fourth light beam 104 is converted into a second circularly polarized light through the first 1 / 4 wave plate 5. The second circularly polarized light is transmitted through the first dichroic element 6 and enters the first optical delay unit 7. After being reflected by the first phase plate 8, it passes through the first optical delay unit 7, the first dichroic element 6, and the first 1 / 4 wave plate 5 in sequence and then is converted into a second linearly polarized light. The second linearly polarized light is reflected by the polarization beam splitter prism 4 and then passes through the third 1 / 4 wave plate 13 and the third dichroic element 14 in sequence and then enters the microscope objective lens, and a second light spot is formed at the focal plane of the microscope objective lens. The second light spot is a transverse doughnut light spot.

[0097] The remaining part of the second light beam 102 is converted into a sixth light beam 106 after being reflected by the polarization beam splitter prism 4. The sixth light beam 106 is converted into a fourth circularly polarized light after passing through the second quarter-wave plate 9. The fourth circularly polarized light sequentially passes through the second optical delay unit 10 and the second phase plate 11, and then is reflected by the second dichroic element 12 and sequentially passes through the second phase plate 11, the second optical delay unit 10, and the second quarter-wave plate 9 and is converted into a third linearly polarized light. The third linearly polarized light is transmitted through the polarization beam splitter prism 4 and sequentially passes through the third quarter-wave plate 13 and the third dichroic element 14 and then enters the microscope objective lens, and a third light spot is formed at the focal plane of the microscope objective lens. Among them, the third light spot is an axially hollow light spot.

[0098] Since the third light beam 103 does not pass through the first optical delay unit 7 and the second optical delay unit 10; the fourth light beam 104 passes through the first optical delay unit 7, and the sixth light beam 106 will pass through the second optical delay unit 10, a fixed pulse time delay will be generated among the three, and the recombined third light beam 103, fourth light beam 104, and sixth light beam 106 are coaxial. The recombined third light beam 103, fourth light beam 104, and sixth light beam 106 respectively pass through the third quarter-wave plate 13 to convert linearly polarized light into circularly polarized light, then pass through the third dichroic element 14, and then enter the microscopic imaging system 200, and are converged by the microscope objective lens and irradiated onto the sample to form concentric light spots. The third light beam 103 is converged and irradiated onto the sample to form a first light spot, that is, a solid light spot; the fourth light beam 104 is converged and irradiated onto the sample to form a second light spot, that is, a transverse doughnut light spot; the sixth light beam 106 is converged and irradiated onto the sample to form a third light spot, that is, an axially hollow light spot. The fourth light beam 104 and the sixth light beam 106 have the same wavelength, while the wavelength of the third light beam 103 is different from the wavelengths of the fourth light beam 104 and the sixth light beam 106. The third light beam 103 excites fluorescence on the surface of the sample after passing through the coaxial transmission of the illumination optical path. The above solid light spot overlaps with the above doughnut light spot and the above axially hollow light spot. The solid light spot excites the fluorescent substance on the sample to emit fluorescence, while the doughnut light spot and the axially hollow light spot suppress the fluorescence emitted by the periphery of the fluorescent substance in three-dimensional space. In this way, only a point smaller than the diffraction limit in the center emits fluorescence and is observed. The excited fluorescence beam 107 is reflected by the third dichroic element 14 and received by the detection system 300 for detection. Among them, the detection system 300 is a fluorescence detection system.

[0099] The illumination system 100 of the stimulated emission depletion optical microscope 1000 of the present application can achieve the coincidence of the centers of the first light spot, the centers of the second light spot, and the centers of the third light spot, obtain a three-dimensional nanoscale spatial super-resolution image, and improve the resolution of imaging.

[0100] The illumination system 100 of the stimulated emission depletion optical microscope 1000 provided by the present invention adopts an integrated design and multiplexes the optical path by utilizing the polarization characteristics of the light beam, realizing the stable coaxial input and output of the excitation light, the transverse and axial STED lights, avoiding the physical adjustment of the geometric relationship between each unit device and the temperature and vibration instabilities inherent in the mechanical adjustment mechanism, enabling the developed stimulated emission depletion optical microscope 1000 to work reliably for a long time and obtaining three-dimensional nano-scale spatial super-resolution images.

[0101] By adding excitation lights of different wavelengths in the embodiments of the present application, three-dimensional super-resolution imaging of two colors or even multiple colors can also be realized without building an additional illumination optical path.

[0102] In the embodiments of the present application, by rotating the half-wave plate 3 to adjust the polarization direction of the light beam, the free switching between two-dimensional and three-dimensional super-resolution imaging modes can be realized, and the ratio adjustment of different transverse resolutions and axial resolutions can be achieved.

[0103] The special optical path design of the illumination system 100 provided in the embodiments of the present application reduces redundant optical devices. By optically bonding them into a simple integrated module, the stability of the stimulated emission depletion optical microscope 1000 can be greatly improved and it can be miniaturized, providing an ideal choice for the combination of super-resolution imaging technology and other technical devices.

[0104] Continue to refer to Figures 2 to 7 as shown in Figure 2 are images of the point spread functions of the first spot, the second spot, the third spot, and the superposition of the second spot and the third spot provided by the embodiment of the present invention. Among them, the images in the first row are the point spread functions in the XY plane, and the images in the second row are the point spread functions in the XZ plane. The centers of the point spread functions of the first spot, the second spot, and the third spot coincide, and can reach the accuracy of the nanometer level. Figure 3 are images of the point spread functions of the incoherent intensity superposition of the second spot and the third spot according to different intensity ratios provided by the embodiment of the present invention. Different energy ratios can be achieved by adjusting the half-wave plate 3 to meet the requirements of different imaging resolutions. It can be seen in Figure 4 that the nano-microspheres that cannot be resolved by the existing confocal microscope can be clearly identified in Figure 5 , significantly improving the imaging effect. The full width at half maximum of the stimulated emission depletion optical microscope 1000 of the present application is less than 50 nanometers. It can be seen from the comparison between Figure 4 and Figure 5 that by adopting the illumination system 100 of the stimulated emission depletion optical microscope 1000 provided by the present application, the transverse imaging resolution can be greatly improved, and the super-resolution imaging effect can be obtained. Continue to refer to Figure 6 and Figure 7 as shown in Figure 6It can be seen that the full width at half maximum (FWHM) of the fluorescence microsphere in the axial direction obtained by traditional confocal imaging is 780 nm, while the FWHM of the fluorescence microsphere in the axial direction obtained by the super-resolution imaging of the stimulated emission depletion optical microscope 1000 of the present application is 130 nm. From the above comparison, it can be known that the illumination system 100 of the stimulated emission depletion optical microscope 1000 provided by the present invention can significantly improve the axial imaging resolution and obtain super-resolution images.

[0105] As described above, the above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. An illumination system for a stimulated emission depletion optical microscope, the stimulated emission depletion optical microscope including a microscope objective, characterized in that, The illumination system includes the following components arranged in sequence along the optical path transmission direction: An illumination light source that generates an incident light beam; A beam adjustment assembly, after passing through which the incident light beam is converted into a first light beam and a second light beam; A polarization beam splitter prism, a part of the first light beam is transmitted through the polarization beam splitter prism and converted into a third light beam, and the remaining part of the first light beam is reflected by the polarization beam splitter prism and converted into a fifth light beam; a part of the second light beam is transmitted through the polarization beam splitter prism and converted into a fourth light beam, and the remaining part of the second light beam is reflected by the polarization beam splitter prism and converted into a sixth light beam; A first quarter-wave plate, the third light beam is converted into a first circularly polarized light after passing through the first quarter-wave plate, and the fourth light beam is converted into a second circularly polarized light after passing through the first quarter-wave plate; A first dichroic element, after the first circularly polarized light is reflected by the first dichroic element and then passes through the first quarter-wave plate, it is converted into a first linearly polarized light; A first optical delay unit, a first phase plate is arranged at the distal end of the first optical delay unit, the second circularly polarized light is transmitted through the first dichroic element and enters the first optical delay unit, and after being reflected by the first phase plate, it passes through the first optical delay unit, the first dichroic element, and the first quarter-wave plate in sequence and is converted into a second linearly polarized light; A second quarter-wave plate, the fifth light beam is converted into a third circularly polarized light after passing through the second quarter-wave plate, and the sixth light beam is converted into a fourth circularly polarized light after passing through the second quarter-wave plate; A second optical delay unit; A second dichroic element, a second phase plate is arranged at the distal end of the second optical delay unit; A third quarter-wave plate; And A third dichroic element, the third circularly polarized light is emitted after passing through the second optical delay unit, the second phase plate, and the second dichroic element in sequence; the fourth circularly polarized light passes through the second optical delay unit and the second phase plate in sequence, and then is reflected by the second dichroic element and passes through the second phase plate, the second optical delay unit, and the second quarter-wave plate in sequence and is converted into a third linearly polarized light; The first linearly polarized light is reflected by the polarization beam splitter prism and then passes through the third quarter-wave plate and the third dichroic element in sequence and enters the microscope objective lens, and a first light spot is formed at the focal plane of the microscope objective lens; The second linearly polarized light is reflected by the polarization beam splitter prism and then passes through the third quarter-wave plate and the third dichroic element in sequence and enters the microscope objective lens, and a second light spot is formed at the focal plane of the microscope objective lens; The third linearly polarized light is transmitted through the polarization beam splitter prism and then passes through the third quarter-wave plate and the third dichroic element in sequence and enters the microscope objective lens, and a third light spot is formed at the focal plane of the microscope objective lens, and the centers of the first light spot, the second light spot, and the third light spot coincide.

2. The illumination system of the stimulated emission depletion optical microscope according to claim 1, wherein The beam adjustment assembly includes a filter, a linear polarizer, and a half-wave plate arranged in sequence along the optical path transmission direction, and the filter, the linear polarizer, and the half-wave plate are coaxially arranged.

3. The illumination system of the stimulated emission depletion optical microscope according to claim 2, characterized in that The filter is a dual-bandpass filter.

4. The illumination system of a stimulated emission depletion optical microscope according to any one of claims 1 to 3, characterized in that, The first light beam is an excitation light beam, and the second light beam is a loss light beam relative to the first light beam; and / or, the first light spot is a solid light spot, and the second and third light spots are hollow light spots.

5. The illumination system of a stimulated emission depletion optical microscope according to any one of claims 1 to 3, characterized in that, The first dichroic element is a selectively transmissive dielectric film, and the dielectric film is coated on the incident end of the first optical delay unit; and / or, the second dichroic element is a dichroic sheet.

6. The illumination system of a stimulated emission depletion optical microscope according to any one of claims 1 to 3, characterized in that The first phase plate is a reflective spiral phase plate, and / or, the second phase plate is a half-wave phase plate.

7. The illumination system of a stimulated emission depletion optical microscope according to any one of claims 1 to 3, characterized in that The first optical delay unit and the first phase plate are of an integral structure or a split structure, and / or, the second optical delay unit and the second phase plate are of an integral structure or a split structure.

8. A stimulated emission depletion optical microscope, characterized in that Comprising: The illumination system of the stimulated emission depletion optical microscope according to any one of claims 1 to 7; And A microscopic imaging system, the microscopic imaging system includes a microscopic objective lens, and the light beam generated by the illumination system is converged to a sample through the microscopic objective lens.

9. The stimulated emission depletion optical microscope according to claim 8, wherein The stimulated emission depletion optical microscope further includes a detection system, and the fluorescence light beam generated by the sample enters the detection system.

10. The stimulated emission depletion optical microscope according to claim 9, characterized in that, The fluorescence light beam enters the detection system after being reflected by the third dichroic element.

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