CN114324156A - Stimulated radiation depletion microscope and microscopic imaging system thereof - Google Patents
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Abstract
The present disclosure provides a stimulated radiation depletion microscope and a microscopic imaging system thereof, wherein the microscopic imaging system comprises a displacement table and a phase plate, and the displacement table is used for bearing a sample; the phase plate is used for simultaneously carrying out phase modulation and convergence on a first light beam and a second light beam which are vertically incident, so that the first light beam and the second light beam respectively form a first light spot and a second light spot on a focal plane of the microscope after passing through the phase plate, and the centers of the first light spot and the second light spot are strictly coincided; wherein an overlapping area of the first beam and the second beam may cover the phase plate. Compared with the prior art, the phase plate has the characteristics of miniaturization and capability of focusing on multiple wavelengths, and the objective lens is replaced by the pure-phase modulation type phase plate, so that the loss of the objective lens to light energy is avoided, and the light intensity utilization rate is improved.
Description
Technical Field
The disclosure relates to the technical field of optical super-resolution microscopic imaging, in particular to a stimulated radiation loss microscope and a microscopic imaging system thereof.
Background
The study of microscopic imaging in life sciences today requires the use of optical microscopes for about 80%, and it can be said that advances in life sciences have been accompanied by the development of optical microscopes. However, the spatial resolution of the optical microscope is limited to about half a wavelength due to the existence of the optical diffraction limit, and such resolution seriously hinders the fine study of subcellular structures by biologists. The Stimulated radiation loss microscope (STED) uses a beam of loss light to form a loss light spot through the modulation of a phase plate, fluorescent molecules around the excitation light diffraction light spot are depleted and converted into a non-radiative state through Stimulated radiation, and the spatial resolution better than 50nm is achieved. Because of the use of all-optical setting, the image acquisition time is the same as that of the traditional confocal microscope, and no special requirements are required for sample preparation, so that the real-time imaging and dynamic tracking of the subcellular structure in the living cell can be realized.
Super-resolution STED microscopes have been widely used in biological and life medical science research since their invention. However, in practical applications, the STED microscope may have some problems, such as: the light path is complex, and miniaturization is difficult to realize; it is difficult to maintain high precision long time stable alignment (nanoscale) of the excitation and STED spots; the microscope objective has great (more than 60%) energy loss to the excitation light and the STED light, so the STED super-resolution microscope needs to select a laser with higher power; the microscope objective generates larger energy loss to the fluorescence signal, and reduces the detection sensitivity of the fluorescence signal.
Disclosure of Invention
The present disclosure provides an stimulated emission depletion microscope and a microscopic imaging system thereof, which can avoid energy loss of an objective lens to a fluorescence signal and improve detection sensitivity of the fluorescence signal.
In a first aspect, embodiments of the present disclosure provide a microscopy imaging system for a microscope, comprising:
the displacement table is used for bearing a sample;
the phase plate is used for simultaneously carrying out phase modulation and convergence on a first light beam and a second light beam which are vertically incident, so that a first light spot and a second light spot are respectively formed at the focal plane of the microscope after the first light beam and the second light beam pass through the phase plate, and the centers of the first light spot and the second light spot are strictly coincided;
wherein an overlapping area of the first beam and the second beam may cover the phase plate.
Optionally, the first light beam is 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.
Optionally, the phase plate is in millimeter scale dimensions.
Embodiments of a second aspect of the present disclosure provide a stimulated radiation depletion microscope, comprising:
an illumination system and a fluorescence detection system, and a microscopic imaging system according to the first aspect;
the illumination system emits a first light beam and a second light beam, the first light beam and the second light beam respectively pass through the phase plate and then are focused and irradiated on the surface of a sample, fluorescent substances in the sample are excited to emit fluorescence, and the excited fluorescence enters the fluorescence detection system for detection after being converged by the phase plate.
Optionally, the lighting system comprises: the laser comprises a first laser, a second laser, a first lens, a second lens, a first optical filter, a second optical filter, a first dichroic element, a second dichroic element and an 1/4 wave plate;
the first laser emits the first light beam, and the second laser emits the second light beam;
the first light beam sequentially passes through the first lens and the first optical filter and then enters the first dichroic element; the second light beam sequentially passes through the second lens and the second optical filter 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, are reflected by the second dichroic element, are incident to the 1/4 wave plate, and are then perpendicularly incident to the phase plate, so as to excite the fluorescent substances in the sample to emit fluorescence.
Optionally, the 1/4 wave plate can convert the incident first and second light beams from linearly polarized light to circularly polarized light.
Optionally, the first optical filter and the second optical filter are both neutral density optical filters, and are used for adjusting the laser intensity of a passing light beam.
Optionally, the fluorescence detection system includes a third optical filter, a third lens and a fluorescence detection element along the light beam propagation direction;
and fluorescent light beams are emitted from a sample, converged by the phase plate and incident on the second dichroic element for transmission, and then sequentially incident on the third optical filter and the third lens after transmission, and are converged and collected to the fluorescence detection element by the third lens.
Optionally, the first dichroic element and the second dichroic element are both dichroic sheets.
Optionally, the third optical filter is a fluorescent optical filter, and is capable of passing the fluorescent light beam and filtering the first light beam and the second light beam.
This disclosure compares advantage with prior art and lies in:
(1) the phase plate provided by the disclosure has the characteristic of miniaturization and capability of focusing multiple wavelengths, and is used for modulating exciting light and loss light emitted by an illuminating system of an STED microscope. The excitation light and the loss light are coupled at the dichroic element and then perpendicularly incident on the phase plate to generate a phase modulated light beam which is strictly concentric.
(2) The phase plate provided by the disclosure replaces an objective lens with a pure phase modulation type phase plate, so that the energy loss of exciting light and loss light when passing through the phase plate is greatly reduced, the energy utilization rate of laser is improved, the requirement on the power of a laser is reduced, and the cost of accessories is further reduced.
(3) According to the phase plate provided by the disclosure, the pure phase modulation type phase plate is used for replacing an objective lens, so that the energy loss of a fluorescent signal is reduced, and the detection sensitivity of the fluorescent signal is improved, thereby improving the signal-to-noise ratio of an image and improving the image quality.
(4) The phase plate provided by the disclosure is millimeter-sized, so that the excitation light beam and the loss light beam can be subjected to phase modulation and convergence under the condition of substantial coaxiality. And the excitation light spots and the loss light spots modulated and converged by the phase plate can be overlapped with each other in a stable and strict center for a long time.
(5) The phase plate that this disclosure provided, for millimeter level size, replaced objective and traditional phase plate for STED microscope's microscopic imaging system size reduces to several centimetres, can be applied to more scenes.
Drawings
Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the disclosure. Also, like reference numerals are used to refer to like parts throughout the drawings. In the drawings:
FIG. 1 shows a schematic diagram of a stimulated radiation depletion microscope provided by the present disclosure;
fig. 2 shows a height diagram of a phase plate provided by the present disclosure;
FIG. 3 shows a confocal image obtained by irradiating a fluorescent microsphere sample with a diameter of 40nm by using a conventional confocal microscope;
FIG. 4 shows an image obtained by stimulated radiation depletion microscopy imaging of a 40nm diameter fluorescent microsphere sample in the same area of FIG. 3 using the STED microscope of the present disclosure;
reference numerals:
01-a lighting system; 02-microscopic imaging system; 03-fluorescence detection system; a-a first laser;
b-a first laser; 1-a first lens; 2-a second lens; 3-a first optical filter; 4-a second optical filter;
5-a first dichroic element; 6-a second dichroic element; 7-1/4 wave plates; 8-a third optical filter;
9-a third lens; 101-a first light beam; 102-a second light beam; 103-a fluorescent light beam; a 111-phase plate; 112-displacement stage.
Detailed Description
Hereinafter, embodiments of the present disclosure will be described with reference to the accompanying drawings. It should be understood that the description is illustrative only and is not intended to limit the scope of the present disclosure. Moreover, in the following description, descriptions of well-known structures and techniques are omitted so as to not unnecessarily obscure the concepts of the present disclosure.
Various structural schematics according to embodiments of the present disclosure are shown in the figures. The figures are not drawn to scale, wherein certain details are exaggerated and possibly omitted for clarity of presentation. The shapes of various regions, layers, and relative sizes and positional relationships therebetween shown in the drawings are merely exemplary, and deviations may occur in practice due to manufacturing tolerances or technical limitations, and a person skilled in the art may additionally design regions/layers having different shapes, sizes, relative positions, as actually required.
In the context of the present disclosure, when a layer/element is referred to as being "on" another layer/element, it can be directly on the other layer/element or intervening layers/elements may be present. In addition, if a layer/element is "on" another layer/element in one orientation, then that layer/element may be "under" the other layer/element when the orientation is reversed.
In order to solve the problems in the prior art, embodiments of the present disclosure provide a microscopic imaging system and a method for manufacturing the same, which are described below with reference to the accompanying drawings.
Fig. 1 shows a schematic structural diagram of an stimulated radiation depletion microscope provided by the present disclosure, such as the STED microscope shown in fig. 1, including an illumination system 01, a microscopic imaging system 02, and a fluorescence detection system 03. Light beams emitted by the illumination system 01 are modulated by an optical element, phase-modulated and converged by a phase plate 111 in the microimaging system 02, and irradiated onto the surface of a sample on a displacement table of the microimaging system 02, so that fluorescent substances in the sample can be excited to emit fluorescence, and the fluorescence of the sample is modulated and converged into parallel light by the phase plate 111 and finally collected to a fluorescence detection system 03.
Specifically, the microscopic imaging system 02 includes:
a displacement stage 112 for carrying a sample;
the phase plate 111 is used for simultaneously performing phase modulation and convergence on a first light beam and a second light beam which are vertically incident, so that the first light beam and the second light beam form a first light spot and a second light spot on a focal plane of the microscope respectively after passing through the phase plate, and the centers of the first light spot and the second light spot are strictly coincided; wherein an overlapping area of the first beam and the second beam may cover the phase plate.
Specifically, the first light beam is 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 phase plate 111 provided by the disclosure is a miniaturized multi-wavelength phase plate capable of focusing, and the phase plate 111 can be obtained by deep learning calculation and is processed by adopting a micro-nano processing technology. Specifically, the phase plate 111 has a millimeter size, so that the excitation light and the loss light can be phase-modulated and condensed on a millimeter coaxial line.
Fig. 2 shows a height view of a phase plate 111 provided by the present disclosure. The phase plate simultaneously performs phase modulation on the commonly incident exciting light and the loss light, and the commonly incident exciting light and the loss light are converged into a stable coaxial solid exciting light spot and a hollow loss light spot, wherein the centers of the solid exciting light spot and the hollow loss light spot are strictly coincident, so that the solid exciting light spot and the hollow loss light spot are used for subsequent fluorescence excitation. The fluorescence light enters the detection system 03 after being collected by the phase plate.
Specifically, as shown in fig. 1, 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 respectively pass through the phase plate 111 and then are focused to irradiate the surface of the sample, the fluorescent substance in the sample is excited to emit fluorescence, and the excited fluorescence enters the fluorescence detection system 03 after passing through the phase plate 111 and is detected.
Specifically, as shown in fig. 1, the lighting 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 an 1/4 wave plate 7.
Specifically, a first laser a emits a first light beam 101, a second laser B emits a second light beam 102, and the first light beam 101 sequentially passes through the first lens 1 and the first optical filter 3 and then enters the first dichroic element 5; the second light beam 102 sequentially passes through the second lens 2 and the second optical filter 4 and then enters the first dichroic element 5.
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 enter the second dichroic element 5 coaxially, are reflected by the second dichroic element 6, are incident on the 1/4 wave plate 7, and are then perpendicularly incident on the phase plate 111, so as to excite the fluorescent substance in the sample to emit fluorescence.
Specifically, the 1/4 wave plate 7 can convert the incident first light beam 101 and the incident second light beam 102 from linearly polarized light to circularly polarized light. The first optical filter 3 and the second optical filter 4 are both neutral density optical filters, and can adjust the laser intensity of the passing light beams.
It can be seen that the first light beam 101 and the second light beam 102 exiting 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, and forms a first light spot and a second light spot on a focal plane of the microscope, wherein centers of the first light spot and the second light spot are strictly coincident.
The first light beam 101 is excitation light, and the first light spot is a solid excitation light spot and is used for exciting fluorescence in the sample and carrying out fluorescence imaging on the sample; the second light beam 102 is a loss light, and the second light spot is a hollow loss light spot for suppressing fluorescence, so that the fluorescent substance in a fluorescence emission state on the peripheral area of the first light spot is subjected to stimulated radiation, and the peripheral area is not subjected to spontaneous fluorescence radiation any more.
The detection system 03 comprises, along the direction of propagation of the light beam: a third filter 8, a third lens 9 and a fluorescence detecting element 10. The fluorescent light beam 103 is emitted from the sample, passes through the phase plate 111, is converged and incident on the second dichroic element 6, is transmitted, sequentially enters the third optical filter 8 and the third lens 9, is converged and collected to the fluorescence detection element 10 through the third lens 9.
Specifically, the third filter 8 is a fluorescent filter, and is capable of passing a fluorescent light beam of the sample and filtering out the first light beam 101 and the second light beam 102 therein.
Specifically, the first dichroic element 5 and the second dichroic element 6 are both dichroic sheets.
It is worth mentioning that several total reflection mirrors 0 are also used in fig. 1 for changing the propagation direction of the light beam.
For ease of understanding, the working principle of the STED microscope provided by the present disclosure is described below with reference to fig. 1 as follows:
in the illumination optical path of the illumination system 01, the first laser a is a light source of the first light beam 101, the first light beam 101 is excitation light, and the first light spot is a solid light spot. The second laser B is a light source of the second light beam 102, the second light beam 102 is a loss light relative to the first light beam 101, and the second light spot is a hollow light spot.
First light beams 101 and second light beams 102 emitted by the first laser A and the second laser B respectively pass through the first lens 1 and the second lens 2, and then are expanded into parallel light. The first filter 3 and the second filter 4 are neutral density filters, and are used for adjusting the laser intensity of the first light beam 101 and the laser intensity of the second light beam 102, respectively.
The first dichroic element 5 is a dichroic sheet, and the first dichroic element 5 enables the first light beam 101 to be reflected and the second light beam 102 to be transmitted, so that the first light beam 101 and the second light beam 102 are coaxial. The second dichroic element 6 is a dichroic sheet that reflects the first light beam 101, the second light beam 102, and the fluorescent light beam 103 that transmits the sample. After passing through the second dichroic element 6, the first light beam 101 and the second light beam 102 enter 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.
In the microscopic imaging system 02, the phase plate 111 is a miniaturized multi-wavelength phase modulation phase plate capable of focusing, and the phase plate 111 simultaneously performs phase modulation and convergence on the first light beam 101 and the second light beam 102, so as to obtain a first light spot and a second light spot with strictly coincident centers. Fluorescent materials in the sample emit fluorescence, and the fluorescent light beams are converged by the phase plate 111, and then enter the second dichroic element 6 to be transmitted, and then enter the fluorescence detection system 03. Then enters the third optical filter 8, further filters the first light beam 101 and the second light beam 102, makes the fluorescent light beam purer, finally enters the third lens 9 for convergence, and enters the fluorescent light collecting element 10.
It should be noted that the first light beam 101 and the second light beam 102 incident on the phase plate 111 should be kept substantially coaxial and incident perpendicularly to the phase plate 111.
Fig. 3 shows an image obtained by performing a confocal imaging test on a fluorescent microsphere sample with the diameter of 40nm by using a conventional confocal microscope, and fig. 4 shows an image obtained by performing a stimulated radiation depletion (STED) microscopic imaging test on the fluorescent microsphere sample with the diameter of 40nm in the same area of fig. 3 by using the STED microscope. It can be seen from the figure that conventional confocal imaging cannot resolve the nanospheres within the dashed box in fig. 3, whereas STED super-resolution imaging of the present disclosure clearly identifies 3 fluorescent microspheres within the dashed box in fig. 3 (as shown within the dashed box in fig. 4), measured as full widths at half maximum of less than 50nm for microscopic imaging of the fluorescent microspheres. By the contrast, the STED microscope provided by the disclosure can greatly improve the imaging resolution and obtain the super-resolution imaging effect.
This disclosure compares advantage with prior art and lies in:
(1) the phase plate provided by the disclosure has the characteristic of miniaturization and capability of focusing multiple wavelengths, and is used for modulating exciting light and loss light emitted by an illuminating system of an STED microscope. The excitation light and the loss light are coupled at the dichroic element and then vertically enter the phase plate to generate a strictly concentric phase modulation 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 light path of the illumination system is optimized, and optical elements are reduced. The physical adjustment of the mutual geometric relationship of the unit devices and the inherent temperature and vibration instability of a mechanical adjusting mechanism are avoided, and the phenomena of exciting light spots, loss of light spot deviation and the like are avoided. The STED microscope can work reliably for a long time under various environments.
(2) The phase plate provided by the disclosure replaces an objective lens with a pure phase modulation type phase plate, so that the energy loss of exciting light and loss light when passing through the phase plate is greatly reduced, the energy utilization rate of laser is improved, the requirement on the power of a laser is reduced, and the cost of accessories is further reduced.
(3) According to the phase plate provided by the disclosure, the pure phase modulation type phase plate is used for replacing an objective lens, so that the energy loss of a fluorescent signal is reduced, and the detection sensitivity of the fluorescent signal is improved, thereby improving the signal-to-noise ratio of an image and improving the image quality.
(4) The phase plate provided by the disclosure is millimeter-sized, so that the excitation light beam and the loss light beam can be subjected to phase modulation and convergence under the condition of substantial coaxiality (the overlapped area of the two light beams can cover the phase plate). And the excitation light spots and the loss light spots modulated and converged by the phase plate are in central coincidence stably and strictly for a long time.
(5) The phase plate that this disclosure provided, for millimeter level size, replaced objective and traditional phase plate for STED microscope's microscopic imaging system size reduces to several centimetres, can be applied to more scenes.
One skilled in the art can also devise methods that are not exactly the same as those described above in order to form the same structure. In addition, although the embodiments are described separately above, this does not mean that the measures in the embodiments cannot be used in advantageous combination.
The embodiments of the present disclosure have been described above. However, these examples are for illustrative purposes only and are not intended to limit the scope of the present disclosure. The scope of the disclosure is defined by the appended claims and equivalents thereof. Various alternatives and modifications can be devised by those skilled in the art without departing from the scope of the present disclosure, and such alternatives and modifications are intended to be within the scope of the present disclosure.
Claims (10)
1. A microscopic imaging system for a microscope, comprising:
the displacement table is used for bearing a sample;
the phase plate is used for simultaneously carrying out phase modulation and convergence on a first light beam and a second light beam which are vertically incident, so that a first light spot and a second light spot are respectively formed at the focal plane of the microscope after the first light beam and the second light beam pass through the phase plate, and the centers of the first light spot and the second light spot are strictly coincided;
wherein an overlapping area of the first beam and the second beam may cover the phase plate.
2. The microscopy imaging system of claim 1, wherein the first light beam is 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.
3. The microscopic imaging system according to claim 1, wherein the phase plate is in millimeter scale dimensions.
4. A stimulated radiation depletion microscope, comprising: an illumination system and a fluorescence detection system, and the microscopic imaging system of any one of claims 1 to 3;
the illumination system emits a first light beam and a second light beam, the first light beam and the second light beam respectively pass through the phase plate and then are focused and irradiated on the surface of a sample, fluorescent substances in the sample are excited to emit fluorescence, and the excited fluorescence enters the fluorescence detection system for detection after being converged by the phase plate.
5. The stimulated radiation depletion microscope of claim 4, wherein the illumination system comprises: the laser comprises a first laser, a second laser, a first lens, a second lens, a first optical filter, a second optical filter, a first dichroic element, a second dichroic element and an 1/4 wave plate;
the first laser emits the first light beam, and the second laser emits the second light beam;
the first light beam sequentially passes through the first lens and the first optical filter and then enters the first dichroic element; the second light beam sequentially passes through the second lens and the second optical filter 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, are reflected by the second dichroic element, are incident to the 1/4 wave plate, and are then perpendicularly incident to the phase plate, so as to excite the fluorescent substances in the sample to emit fluorescence.
6. The stimulated radiation loss microscope of claim 5, wherein the 1/4 wave plate is capable of converting the incident first and second light beams from linearly polarized light to circularly polarized light.
7. The stimulated radiation loss microscope of claim 5, wherein the first filter and the second filter are each neutral density filters for adjusting the laser intensity of the passing beam.
8. The stimulated emission depletion microscope of claim 5, wherein the fluorescence detection system comprises a third filter, a third lens, and a fluorescence detection element along the direction of beam propagation;
and fluorescent light beams are emitted from a sample, converged by the phase plate and incident on the second dichroic element for transmission, and then sequentially incident on the third optical filter and the third lens after transmission, and are converged and collected to the fluorescence detection element by the third lens.
9. The stimulated radiation loss microscope of claim 8, wherein the first dichroic element and the second dichroic element are both dichroic plates.
10. The stimulated emission depletion microscope of claim 8, wherein the third filter is a fluorescence filter capable of passing the fluorescence beam and filtering out the first and second beams therein.
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