Stimulated emission depletion microscope and its microscopic imaging system

Abstract

The present disclosure provides a stimulated emission depletion microscope and a microscopic imaging system thereof, wherein the microscopic imaging system includes a displacement stage and a phase plate, wherein the displacement stage is used to carry a sample; the phase plate is used to simultaneously phase modulate and converge a first light beam and a second light beam incident vertically, so that after the first light beam and the second light beam pass through the phase plate, a first light spot and a second light spot are formed at the focal plane of the microscope respectively, and the centers of the first light spot and the second light spot strictly coincide; wherein the overlapping area of ​​the first light beam and the second light beam can cover the phase plate. Compared with the prior art, the phase plate disclosed in the present disclosure has the characteristics of miniaturization and multi-wavelength focusing, and a pure phase modulation phase plate is used to replace the objective lens, thereby avoiding the loss of light energy by the objective lens and improving the light intensity utilization rate.

Description

Technical Field

[0001] The present disclosure relates to the technical field of optical super-resolution microscopic imaging, and in particular to a stimulated emission depletion microscope and a microscopic imaging system thereof. Background Art

[0002] About 80% of the research on microscopic imaging in life sciences today requires the use of optical microscopes. It can be said that the progress of life sciences is 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. Such a resolution seriously hinders biologists' detailed research on subcellular structures. Stimulated Emission Depletion (STED) microscopy uses a beam of loss light to modulate the phase plate to form a loss spot, and converts the fluorescent molecules around the diffraction spot of the excitation light into a non-radiative state through stimulated emission depletion, achieving a spatial resolution better than 50nm. Due to the use of an all-optical setting, the image acquisition time is the same as that of a traditional confocal microscope, and there are no special requirements for sample preparation, so real-time imaging and dynamic tracking of subcellular structures in living cells can be achieved.

[0003] Since its invention, super-resolution STED microscopy has been widely used in biological and life science research. However, in practical applications, STED microscopy has some problems, such as: the optical path is complex and it is difficult to achieve miniaturization; it is difficult to maintain high-precision and long-term stable alignment (nanometer level) between the excitation light spot and the STED light spot; the microscope objective has a large (more than 60%) energy loss for the excitation light and the STED light, so the STED super-resolution microscope needs to use a higher-power laser; the microscope objective has a large energy loss for the fluorescence signal, which reduces the detection sensitivity of the fluorescence signal. Summary of the invention

[0004] The purpose of the present disclosure is to provide a stimulated emission depletion microscope and a microscopic imaging system thereof, which can avoid the energy loss of the objective lens to the fluorescence signal and improve the detection sensitivity of the fluorescence signal.

[0005] A first aspect of the present disclosure provides a microscopic imaging system for a microscope, comprising:

[0006] A translation stage, used to carry samples;

[0007] A phase plate is used to simultaneously phase modulate and converge a first light beam and a second light beam incident vertically, so that after the first light beam and the second light beam pass through the phase plate, a first light spot and a second light spot are formed at the focal plane of the microscope, respectively, and the centers of the first light spot and the second light spot strictly coincide with each other;

[0008] The overlapping area of ​​the first light beam and the second light beam may cover the phase plate.

[0009] Optionally, the first light beam is excitation light, and the first light spot is a solid light spot; the second light beam is loss light relative to the first light beam, and the second light spot is a hollow light spot.

[0010] Optionally, the phase plate is of millimeter size.

[0011] A second aspect of the present disclosure provides a stimulated emission depletion microscope, comprising:

[0012] An illumination system and a fluorescence detection system, and the microscopic imaging system described in the first aspect above;

[0013] The illumination system emits a first light beam and a second light beam, which are focused and irradiated onto the sample surface after passing through the phase plate respectively, thereby exciting the fluorescent material in the sample to emit fluorescence. The excited fluorescence is converged by the phase plate and enters the fluorescence detection system for detection.

[0014] Optionally, the illumination system comprises: a first laser, a second laser, a first lens, a second lens, a first filter, a second filter, a first dichroic element, a second dichroic element and a quarter wave plate;

[0015] The first laser emits the first light beam, and the second laser emits the second light beam;

[0016] The first light beam passes through the first lens and the first filter in sequence and then enters the first dichroic element; the second light beam passes through the second lens and the second filter in sequence and then enters the first dichroic element;

[0017] The first dichroic element can reflect the first light beam and transmit the second light beam, so that the first light beam and the second light beam coaxially enter the second dichroic element, after being reflected by the second dichroic element, enter the 1 / 4 wave plate, and then vertically enter the phase plate to excite the fluorescent substance in the sample to emit fluorescence.

[0018] Optionally, the quarter wave plate can convert the incident first light beam and second light beam from linearly polarized light to circularly polarized light.

[0019] Optionally, the first filter and the second filter are both neutral density filters for adjusting the intensity of the laser passing through the light beam.

[0020] Optionally, the fluorescence detection system includes a third filter, a third lens and a fluorescence detection element along the propagation direction of the light beam;

[0021] After being emitted from the sample, the fluorescent light beam is converged by the phase plate and incident on the second dichroic element for transmission. After transmission, it is incident on the third filter and the third lens in sequence, and is converged and collected by the third lens to the fluorescent detection element.

[0022] Optionally, the first dichroic element and the second dichroic element are both dichroic sheets.

[0023] Optionally, the third filter is a fluorescence filter, which can allow the fluorescence light beam to pass through and filter out the first light beam and the second light beam therein.

[0024] The advantages of the present invention compared with the prior art are:

[0025] (1) The phase plate provided by the present disclosure has the characteristics of miniaturization and multi-wavelength focus, and is used to modulate the excitation light and loss light emitted by the illumination system of the STED microscope. After the excitation light and the loss light are coupled at the dichroic element, they are vertically incident on the phase plate to generate a strictly concentric phase modulated light beam.

[0026] (2) The phase plate provided by the present invention replaces the objective lens with a pure phase modulation phase plate, which greatly reduces the energy loss of the excitation light and the loss light when passing through the phase plate, improves the energy utilization rate of the laser, reduces the power requirement of the laser, and thus reduces the cost of accessories.

[0027] (3) The phase plate provided by the present disclosure replaces the objective lens with a pure phase modulation phase plate, which reduces the energy loss of the fluorescence signal and improves the detection sensitivity of the fluorescence signal, thereby improving the signal-to-noise ratio of the image and improving the image quality.

[0028] (4) The phase plate provided by the present disclosure is of millimeter-level size, so that the excitation light beam and the loss light beam can be phase modulated and converged in a substantially coaxial state. Moreover, the excitation light spot and the loss light spot modulated and converged by the phase plate can be stably and strictly centrally overlapped for a long time.

[0029] (5) The phase plate provided by the present disclosure is of millimeter size and replaces the objective lens and the traditional phase plate, so that the size of the microscopic imaging system of the STED microscope is reduced to a few centimeters and can be applied to more scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present disclosure. Also, the same reference symbols are used throughout the accompanying drawings to represent the same components. In the accompanying drawings:

[0031] Figure 1 A schematic structural diagram of a stimulated emission depletion microscope provided by the present disclosure is shown;

[0032] Figure 2 shows a height diagram of a phase plate provided by the present disclosure;

[0033] Figure 3 The confocal image obtained by irradiating a fluorescent microsphere sample with a diameter of 40 nm using a conventional confocal microscope is shown;

[0034] Figure 4 The STED microscope of the present invention is used to observe the fluorescence microsphere sample with a diameter of 40 nm. Figure 3 The image obtained by stimulated emission depletion microscopy imaging of the same area;

[0035] Reference numerals:

[0036] 01-illumination system; 02-microscopic imaging system; 03-fluorescence detection system; A-first laser;

[0037] B-second laser; 1-first lens; 2-second lens; 3-first filter; 4-second filter;

[0038] 5-first dichroic element; 6-second dichroic element; 7-1 / 4 wave plate; 8-third filter;

[0039] 9-third lens; 10-fluorescence detection element; 101-first light beam; 102-second light beam; 103-fluorescence light beam; 111-phase plate;

[0040] 112-Translation stage. DETAILED DESCRIPTION

[0041] Hereinafter, embodiments of the present disclosure will be described with reference to the accompanying drawings. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of the present disclosure. In addition, in the following description, descriptions of well-known structures and technologies are omitted to avoid unnecessary confusion of the concepts of the present disclosure.

[0042] Various structural schematic diagrams according to embodiments of the present disclosure are shown in the accompanying drawings. These figures are not drawn to scale, and some details are magnified and some details may be omitted for the purpose of clear expression. The shapes of various regions and layers shown in the figures and the relative sizes and positional relationships therebetween are only exemplary, and may deviate in practice due to manufacturing tolerances or technical limitations, and those skilled in the art may further design regions / layers with different shapes, sizes, and relative positions according to actual needs.

[0043] In the context of the present disclosure, when a layer / element is referred to as being "on" another layer / element, the layer / element may be directly on the other layer / element or an intervening layer / element may exist therebetween. In addition, if a layer / element is "on" another layer / element in one orientation, the layer / element may be "below" the other layer / element when the orientation is reversed.

[0044] In order to solve the problems existing in the prior art, the embodiments of the present disclosure provide a microscopic imaging system and a method for preparing the microscopic imaging system, which are described below in conjunction with the accompanying drawings.

[0045] Figure 1 FIG. 4 shows a schematic diagram of a stimulated emission depletion microscope provided by the present disclosure, such as Figure 1 The STED microscope shown includes an illumination system 01, a microscopic imaging system 02 and a fluorescence detection system 03. The light beam emitted by the illumination system 01 is modulated by an optical element, phase-modulated and converged by a phase plate 111 in the microscopic imaging system 02, and irradiated onto the sample surface on the translation stage of the microscopic imaging system 02, which can excite the fluorescent material in the sample to emit fluorescence. The fluorescence of the sample is modulated and converged into parallel light by the phase plate 111, and finally collected to the fluorescence detection system 03.

[0046] Specifically, the microscopic imaging system 02 includes:

[0047] A translation stage 112, used for carrying a sample;

[0048] The phase plate 111 is used to simultaneously phase modulate and converge the vertically incident first light beam and the second light beam, so that after the first light beam and the second light beam pass through the phase plate, a first light spot and a second light spot are formed at the focal plane of the microscope respectively, and the centers of the first light spot and the second light spot are strictly coincident; wherein the overlapping area of ​​the first light beam and the second light beam can cover the phase plate.

[0049] Specifically, the first light beam is excitation light, and the first light spot is a solid light spot; the second light beam is loss light relative to the first light beam, and the second light spot is a hollow light spot.

[0050] The phase plate 111 provided by the present disclosure is a miniaturized focusable multi-wavelength phase plate, which can be calculated by deep learning and processed by micro-nano processing technology. Specifically, the phase plate 111 is of millimeter size, so that the excitation light and the loss light can be phase modulated and converged under the condition of millimeter coaxiality.

[0051] Figure 2The height diagram of the phase plate 111 provided by the present disclosure is shown. The phase plate simultaneously phase modulates the co-incident excitation light and loss light, and converges them into a stable coaxial, solid excitation light spot and a hollow loss light spot with strictly overlapping centers for subsequent fluorescence excitation. After being collected by the phase plate, the fluorescence enters the detection system 03.

[0052] Specifically, Figure 1 As shown, the illumination system 01 emits a first light beam 101 and a second light beam 102. The first light beam 101 and the second light beam 102 are focused and irradiated onto the sample surface after passing through the phase plate 111 respectively, so as to excite the fluorescent material in the sample to emit fluorescence. The excited fluorescence is converged by the phase plate 111 and enters the fluorescence detection system 03 for detection.

[0053] Specifically, Figure 1 As shown, the illumination system 01 includes: a first laser A, a second laser B, a first lens 1 , a second lens 2 , a first filter 3 , a second filter 4 , a first dichroic element 5 , a second dichroic element 6 and a quarter wave plate 7 .

[0054] Specifically, the first laser A emits a first light beam 101, and the second laser B emits a second light beam 102. The first light beam 101 passes through the first lens 1 and the first filter 3 in sequence and then enters the first dichroic element 5; the second light beam 102 passes through the second lens 2 and the second filter 4 in sequence and then enters the first dichroic element 5.

[0055] The first dichroic element 5 can reflect the first light beam 101 and transmit the second light beam 102, so that the first light beam 101 and the second light beam 102 coaxially enter the second dichroic element 6, and after being reflected by the second dichroic element 6, enter the 1 / 4 wave plate 7, and then vertically enter the phase plate 111 to excite the fluorescent substance in the sample to emit fluorescence.

[0056] Specifically, the 1 / 4 wave plate 7 can convert the incident first light beam 101 and second light beam 102 from linear polarized light to circular polarized light. The first filter 3 and the second filter 4 are both neutral density filters, which can adjust the laser intensity of the light beam.

[0057] It can be seen that the first light beam 101 and the second light beam 102 emitted from the illumination system 01 can coaxially pass through the phase plate 111 of the microscopic imaging system 02. The phase plate 111 simultaneously performs phase modulation and convergence on the first light beam 101 and the second light beam 102, forming a first light spot and a second light spot at the focal plane of the microscope, and the centers of the first light spot and the second light spot strictly coincide.

[0058] The first light beam 101 is excitation light, and the first light spot is a solid excitation light spot, which is used to excite fluorescence in the sample and perform fluorescence imaging on the sample; the second light beam 102 is loss light, and the second light spot is a hollow loss light spot, which is used to suppress fluorescence, so that the fluorescent material in the fluorescence emission state in the peripheral area of ​​the first light spot undergoes stimulated radiation, so that the peripheral area no longer spontaneously radiates fluorescence.

[0059] The detection system 03 includes: a third filter 8, a third lens 9 and a fluorescence detection element 10 along the propagation direction of the light beam. After being emitted from the sample, the fluorescence light beam 103 passes through the phase plate 111, converges and is incident on the second dichroic element 6, and after transmission, it is incident on the third filter 8 and the third lens 9 in sequence, and is converged and collected to the fluorescence detection element 10 through the third lens 9.

[0060] Specifically, the third filter 8 is a fluorescence filter, which can allow the fluorescent light beam of the sample to pass through and filter out the first light beam 101 and the second light beam 102 therein.

[0061] Specifically, the first dichroic element 5 and the second dichroic element 6 are both dichroic sheets.

[0062] It is worth mentioning that Figure 1 Several total reflection mirrors 0 are also used to change the propagation direction of the light beam.

[0063] For ease of understanding, the following Figure 1 The working principle of the STED microscope provided in the present disclosure is introduced as follows:

[0064] In the illumination optical path of the illumination system 01, the first laser A is the light source of the first light beam 101, the first light beam 101 is the excitation light, and the first light spot is a solid light spot. The second laser B is the light source of the second light beam 102, the second light beam 102 is the loss light relative to the first light beam 101, and the second light spot is a hollow light spot.

[0065] The first light beam 101 and the second light beam 102 emitted by the first laser A and the second laser B are expanded into parallel light after passing through the first lens 1 and the second lens 2 respectively. The first filter 3 and the second filter 4 are neutral density filters, which are used to adjust the laser intensity of the first light beam 101 and the second light beam 102 respectively.

[0066] The first dichroic element 5 is a dichroic plate, which can reflect the first light beam 101 and transmit the second light beam 102, so as to realize the coaxiality of the first light beam 101 and the second light beam 102. The second dichroic element 6 is a dichroic plate, which can reflect the first light beam 101, the second light beam 102 and the fluorescent light beam 103 transmitted through the sample. After passing through the second dichroic element 6, the first light beam 101 and the second light beam 102 are incident on the 1 / 4 wave plate 7, and the first light beam 101 and the second light beam 102 are converted from linearly polarized light to circularly polarized light.

[0067] In the microscopic imaging system 02, the phase plate 111 is a miniaturized focusable multi-wavelength phase modulation phase plate, which simultaneously phase modulates and converges the first light beam 101 and the second light beam 102, thereby obtaining the first light spot and the second light spot with strictly coincident centers. The fluorescent substance in the sample emits fluorescence, and the fluorescent light beam is then converged by the phase plate 111, incident on the second dichroic element 6 for transmission, and enters the fluorescence detection system 03. Then, it is incident on the third filter 8, and the first light beam 101 and the second light beam 102 are further filtered out, making the fluorescent light beam purer, and finally incident on the third lens 9 for convergence, and enters the fluorescence collection element 10.

[0068] It is worth mentioning that the first light beam 101 and the second light beam 102 incident on the phase plate 111 need to remain substantially coaxial and vertically incident on the phase plate 111 .

[0069] Figure 3 The image obtained by using a conventional confocal microscope to perform confocal imaging on a fluorescent microsphere sample with a diameter of 40 nm is shown. Figure 4 The STED microscope is used to observe the fluorescence microsphere sample with a diameter of 40 nm. Figure 3 The image is obtained by performing stimulated emission depletion (STED) microscopy experiments on the same area. It can be seen from the figure that traditional confocal imaging cannot distinguish Figure 3 The nanospheres in the dashed box can be clearly identified by the STED super-resolution imaging of the present invention. Figure 3 The three fluorescent microspheres in the dashed box (such as Figure 4 The full width at half maximum of the fluorescent microsphere microscopic imaging is measured to be less than 50 nm. From the above comparison, it can be seen that the STED microscope provided by the present disclosure can greatly improve the resolution of imaging and obtain the effect of super-resolution imaging.

[0070] The advantages of the present invention compared with the prior art are:

[0071] (1) The phase plate provided by the present invention has the characteristics of miniaturization and multi-wavelength focus, and is used to modulate the excitation light and loss light emitted by the illumination system of the STED microscope. After the excitation light and the loss light are coupled at the dichroic element, they are vertically incident on the phase plate to produce a strictly concentric phase-modulated light beam (the excitation light is modulated and converged by the phase plate to obtain a solid circular spot, and the loss light is modulated and converged by the phase plate to form a hollow spot). The optical path of the illumination system is optimized and the optical components are reduced. The physical adjustment of the geometric relationship between the unit devices and the temperature and vibration instability inherent in the mechanical adjustment mechanism are avoided, and the phenomenon of excitation light spot and loss light spot offset is avoided. The STED microscope can work reliably for a long time in various environments.

[0072] (2) The phase plate provided by the present invention replaces the objective lens with a pure phase modulation phase plate, which greatly reduces the energy loss of the excitation light and the loss light when passing through the phase plate, improves the energy utilization rate of the laser, reduces the power requirement of the laser, and thus reduces the cost of accessories.

[0073] (3) The phase plate provided by the present disclosure replaces the objective lens with a pure phase modulation phase plate, which reduces the energy loss of the fluorescence signal and improves the detection sensitivity of the fluorescence signal, thereby improving the signal-to-noise ratio of the image and improving the image quality.

[0074] (4) The phase plate provided by the present disclosure is of millimeter-level size, so that the excitation light beam and the loss light beam can be phase modulated and converged in a substantially coaxial state (the overlapping area of ​​the two light beams can cover the phase plate). Moreover, the excitation light spot and the loss light spot modulated and converged by the phase plate can be stably and strictly centrally overlapped for a long time.

[0075] (5) The phase plate provided by the present disclosure is of millimeter size and replaces the objective lens and the traditional phase plate, so that the size of the microscopic imaging system of the STED microscope is reduced to a few centimeters and can be applied to more scenarios.

[0076] In order to form the same structure, those skilled in the art may also design a method that is not completely the same as the method described above. In addition, although the various embodiments are described above, this does not mean that the measures in the various embodiments cannot be used in combination advantageously.

[0077] The embodiments of the present disclosure are described above. However, these embodiments are for illustrative purposes only and are not intended to limit the scope of the present disclosure. The scope of the present disclosure is defined by the appended claims and their equivalents. Without departing from the scope of the present disclosure, a person skilled in the art may make a variety of substitutions and modifications, which should all fall within the scope of the present disclosure.

Claims

1. A stimulated emission depletion microscope, characterized in that: include: Illumination system and fluorescence detection system, as well as microscopic imaging system; The microscopic imaging system comprises: A translation stage, used to carry samples; A phase plate is used to simultaneously phase modulate and converge a first light beam and a second light beam incident vertically, so that after the first light beam and the second light beam pass through the phase plate, a first light spot and a second light spot are formed at the focal plane of the microscope, respectively, and the centers of the first light spot and the second light spot strictly coincide with each other; The overlapping area of ​​the first light beam and the second light beam can cover the phase plate; the phase plate is of millimeter size and is a miniaturized pure phase modulation phase plate that can focus multiple wavelengths, and the height distribution on it is calculated by deep learning; the first light beam is the excitation light, and the first light spot is a solid light spot; the second light beam is the loss light relative to the first light beam, and the second light spot is a hollow light spot; The illumination system emits a first light beam and a second light beam, which are respectively converged into a stable coaxial solid excitation light spot and a hollow loss light spot with strictly overlapping centers after passing through the phase plate, and are focused and irradiated onto the sample surface to excite the fluorescent material in the sample to emit fluorescence, and the excited fluorescence is converged by the phase plate and enters the fluorescence detection system for detection; The illumination system comprises: a first laser, a second laser, a first lens, a second lens, a first filter, a second filter, a first dichroic element, a second dichroic element and a quarter wave plate; the first filter and the second filter are both neutral density filters for adjusting the laser intensity of a light beam passing through; The first laser emits the first light beam, and the second laser emits the second light beam; The first light beam passes through the first lens and the first filter in sequence and then enters the first dichroic element; the second light beam passes through the second lens and the second filter in sequence and then enters the first dichroic element; The first dichroic element can reflect the first light beam and transmit the second light beam, so that the first light beam and the second light beam coaxially enter the second dichroic element, after being reflected by the second dichroic element, enter the 1 / 4 wave plate, and then vertically enter the phase plate to excite the fluorescent substance in the sample to emit fluorescence.

2. The stimulated emission depletion microscope according to claim 1, characterized in that: The 1 / 4 wave plate can convert the incident first light beam and second light beam from linearly polarized light to circularly polarized light.

3. The stimulated emission depletion microscope according to claim 1, characterized in that: The fluorescence detection system includes a third filter, a third lens and a fluorescence detection element along the propagation direction of the light beam; After being emitted from the sample, the fluorescent light beam is converged by the phase plate and incident on the second dichroic element for transmission. After transmission, it is incident on the third filter and the third lens in sequence, and is converged and collected by the third lens to the fluorescent detection element.

4. The stimulated emission depletion microscope according to claim 3, characterized in that: The first dichroic element and the second dichroic element are both dichroic sheets.

5. The stimulated emission depletion microscope according to claim 3, characterized in that: The third filter is a fluorescent filter, which can allow the fluorescent light beam to pass through and filter out the first light beam and the second light beam therein.

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