CN113624682A - Annular pupil confocal Brillouin microscope system - Google Patents
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- CN113624682A CN113624682A CN202110756824.1A CN202110756824A CN113624682A CN 113624682 A CN113624682 A CN 113624682A CN 202110756824 A CN202110756824 A CN 202110756824A CN 113624682 A CN113624682 A CN 113624682A
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Abstract
The invention provides an annular pupil confocal Brillouin microscope system, which solves the problems of complex calculation and poor calculation precision of the conventional confocal Brillouin spectrum measurement system. The system, comprising: the device comprises an illumination module, a light splitting plain film, an annular diaphragm, a measuring objective lens and a spectrum detection module; the illumination module is used for generating linearly polarized laser with selectable polarization direction as incident light; the incident light is reflected by the light splitting flat sheet to enter the annular diaphragm, and is focused on a sample to be measured through the measuring objective lens after being shaped by the annular diaphragm; brillouin scattered light generated by a sample to be detected after excitation is collected by the measuring objective lens, enters the annular diaphragm, is modulated by the annular diaphragm and then is transmitted to the spectrum detection module through the light splitting flat sheet; the spectrum detection module is used for collecting and detecting the Brillouin scattering light spectrum of the sample to be detected. The invention can realize a high-spatial resolution and high-spectral resolution annular pupil confocal Brillouin microscope system.
Description
Technical Field
The invention relates to the technical field of micro-spectral measurement, in particular to an annular pupil confocal Brillouin microscopic system.
Background
The confocal Brillouin spectrum detection system introduces confocal microscopic technology with microscopic characterization capability on the basis of Brillouin scattering, can realize high-spatial-resolution micro-area mechanical performance parameter measurement, and is applied to the aspects of biological tissue detection, cell imaging, material characterization and the like. How to effectively take account of the spatial resolution and the spectral resolution of the confocal Brillouin spectrum detection system and further ensure the spectral measurement accuracy of the confocal Brillouin spectrum detection system is always a hotspot problem in the research of the field of Brillouin spectral microscopic imaging. The existing spectral analysis method has the defects that a theoretical model needs to be established according to parameters such as numerical apertures of a sample and an objective lens, the calculation process is complex, and accurate calculation cannot be carried out on an unknown sample; the existing confocal Brillouin spectrum detection system is complex in light path, devices with different numerical apertures need to be redesigned and calculated in large quantities, and the devices generally cannot realize high-spatial-resolution Brillouin spectrum microscopic imaging.
Disclosure of Invention
The invention provides an annular pupil confocal Brillouin microscope system, which solves the problems of complex calculation and poor calculation precision of the conventional confocal Brillouin spectrum measurement system.
In order to solve the problems, the invention is realized as follows:
the embodiment of the invention provides an annular pupil confocal Brillouin microscope system, which comprises: the device comprises an illumination module, a light splitting plain film, an annular diaphragm, a measuring objective lens and a spectrum detection module; the illumination module is used for generating linearly polarized laser with selectable polarization direction as incident light; the incident light is reflected by the light splitting flat sheet to enter the annular diaphragm, and is focused on a sample to be measured through the measuring objective lens after being shaped by the annular diaphragm; brillouin scattered light generated by a sample to be detected after excitation is collected by the measuring objective lens, enters the annular diaphragm, is modulated by the annular diaphragm and then is transmitted to the spectrum detection module through the light splitting flat sheet; the spectrum detection module is used for collecting and detecting the Brillouin scattering light spectrum of the sample to be detected.
Preferably, the annular diaphragm is realized by at least one of the following modes: a digital micro-mirror array, a spatial light modulator, or a light barrier.
Preferably, the adjustment error of the annular diaphragm is less than or equal to 1% of the outer circle radius of the annular diaphragm.
Preferably, the pupil coefficient of the annular diaphragm is less than 0.9.
Preferably, the normalized pinhole radius of the confocal pinhole is 2.5 ± 0.2.
Further, the lighting module includes: the device comprises a continuous laser, a beam expanding system, a half wave plate and a polarizer; the laser emitted by the continuous laser passes through the beam expanding system to expand the beam, the half wave plate and the polarizer to obtain the linearly polarized laser with the optional polarization direction.
Further, the spectrum detection module comprises: the device comprises an analyzer, a collecting lens, a confocal pinhole and a Brillouin spectrometer; brillouin scattered light generated by the sample to be tested after excitation is collected by the measuring objective lens, enters the annular diaphragm, is modulated by the annular diaphragm, then passes through the beam splitter, the analyzer and the collecting lens, then is converged in the confocal pinhole, and then is collected and detected by the Brillouin spectrometer.
Further, the system further comprises: and the three-dimensional translation table is used for placing a sample to be detected and performing three-dimensional movement.
The beneficial effects of the invention include: the invention provides an annular pupil confocal Brillouin microscopic system, which can perform Brillouin spectral imaging with high spatial resolution and high spectral resolution on a sample by using a simple optical path without establishing a theoretical model according to instrument parameters, and realizes high-precision mechanical performance imaging. The system can realize annular light beam illumination and annular light beam collection on the basis of the light path of the traditional confocal Brillouin microscope system by adding the annular pupil at the superposed light path part of the illumination light path and the collection light path, and has obvious advantages in the aspects of cost and instrument complexity.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 is an embodiment of an annular pupil confocal Brillouin microscopy system;
FIG. 2 is an embodiment of an annular pupil confocal Brillouin microscopy system incorporating a Brillouin spectrometer;
FIG. 3(a) is a schematic diagram of an illumination beam and a collection beam of a conventional confocal Brillouin microscope system according to an embodiment of the beams;
FIG. 3(b) is a schematic diagram of an illumination beam and a collection beam of an annular pupil confocal Brillouin microscope system of the beam embodiment;
FIG. 4 is a schematic diagram of an embodiment of the transverse point spread function distribution and the spectral distribution at different annular pupil parameters.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the specific embodiments of the present invention and the accompanying drawings. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The brillouin scattering is used as inelastic scattering caused by interaction of photons and thermophonons, and parameters such as pressure, temperature, elasticity and the like of a sample can be directly obtained in a non-contact mode, so that the brillouin scattering is used as an extremely important optical probe and is widely applied to the fields of physical chemistry, material science, biomedicine and the like. The confocal Brillouin spectrum detection system introduces confocal microscopic technology with microscopic characterization capability on the basis of Brillouin scattering, can realize high-spatial-resolution micro-area mechanical performance parameter measurement, and is applied to the aspects of biological tissue detection, cell imaging, material characterization and the like.
Although the research heat of the present confocal brillouin spectral detection system still keeps a very strong trend, a very important problem still cannot be effectively solved, namely two important characteristic parameters of the spatial resolution and the spectral resolution of the confocal brillouin spectral detection system cannot be considered all the time, because the two parameters are mutually contradictory under the condition of limited numerical aperture. The scattered light in the collection angle range of the objective lens can be collected, the Brillouin scattering frequency shift is directly related to the scattering angle, the spectrum result detected by the Brillouin spectrum detection system is the integral result of all the scattering angles in the numerical aperture range of the objective lens, therefore, under the condition of limited numerical aperture, the spectral broadening effect is inevitably caused by the mutual superposition of the spectrum distributions corresponding to different scattering angles, the broadening effect is more obvious along with the increase of the numerical aperture of the objective lens, the spectral resolution and the measurement precision are seriously reduced, the spectral broadening effect can be effectively reduced by using the objective lens with low numerical aperture, but the spatial resolution and the spectral resolution of the confocal Brillouin spectrum detection system are also reduced, therefore, how to effectively consider the spatial resolution and the spectral resolution of the Brillouin spectrum detection system, and further ensure the spectral measurement accuracy of the Brillouin spectrum detection system, is always a hot problem for research in the field of spectral microscopic imaging.
At present, several spectral analysis methods and experimental devices are available to effectively reduce the spectrum broadening effect in Brillouin scattering, but the former generally requires the establishment of a theoretical model according to parameters such as numerical apertures of a sample and an objective lens, and not only is the calculation process complicated, but also accurate calculation cannot be performed on an unknown sample; the optical path of the device is complex, the device with different numerical apertures needs to be redesigned and calculated in a large amount, and importantly, the device can not realize high-spatial resolution Brillouin spectral microscopic imaging generally, so that the further application of a confocal Brillouin spectral detection system in the field of microscopic spectral imaging is severely limited.
The innovation points of the invention are as follows: the invention utilizes the annular pupil to simultaneously modulate the illumination point diffusion function and the collection point diffusion function, the wavefront of the illumination beam is modulated by the annular diaphragm before the illumination beam is focused by the measurement objective, so that the beam only keeps a high numerical aperture part, the transverse half-height width of the illumination point diffusion function is reduced, the transverse size of a focusing light spot is compressed, meanwhile, the annular pupil further modulates the wavefront of the collection beam, so that the half-height width of the collection point diffusion function is also compressed, and the transverse resolution of the system is improved because the three-dimensional point diffusion function of the confocal microscope system is the product of the illumination point diffusion function and the collection point diffusion function. In addition, the system can effectively reduce the Brillouin spectrum broadening effect by reducing the illumination angle and the collection angle range, improve the spectral resolution of the system, and further effectively give consideration to the spatial resolution and the spectral resolution of the traditional confocal Brillouin system.
The technical solutions provided by the embodiments of the present invention are described in detail below with reference to the accompanying drawings.
Fig. 1 is an embodiment of an annular pupil confocal brillouin microscope system, which can simultaneously improve the spatial resolution and spectral resolution of the confocal brillouin microscope system, and as an embodiment of the present invention, an annular pupil confocal brillouin microscope system, which is used for a sample 6 to be measured, includes: the device comprises an illumination module 1, a beam splitting plain film 2, an annular diaphragm 3, a measurement objective 4 and a spectrum detection module 5.
The illumination module is used for generating linearly polarized laser with selectable polarization direction as incident light; the incident light is reflected by the light splitting flat sheet to enter the annular diaphragm, and is focused on a sample to be measured through the measuring objective lens after being shaped by the annular diaphragm; brillouin scattered light generated by a sample to be detected after excitation is collected by the measuring objective lens, enters the annular diaphragm, is modulated by the annular diaphragm and then is transmitted to the spectrum detection module through the light splitting flat sheet; the spectrum detection module is used for collecting and detecting the Brillouin scattering light spectrum of the sample to be detected.
In the embodiment of the present invention, the annular diaphragm is implemented by at least one of the following modes: a digital micro-mirror array, a spatial light modulator, or a light barrier.
Specifically, in the process of constructing the annular pupil confocal brillouin microscopic system, the annular diaphragm can be realized in multiple modes such as a digital micro-mirror array, a spatial light modulator and a light barrier, and can be added and adjusted in the last link of the system construction, so that the advantages of convenience in disassembly and assembly and convenience in use are brought, the influence of the concentricity error of the annular diaphragm and the optical axis on the transverse resolution of the system is small, but the influence on the broadening effect of a spectral curve measured by the system is large, and the adjustment error of the annular diaphragm is smaller than 1% of the excircle radius of the annular diaphragm under a general condition.
The embodiment of the invention provides a confocal Brillouin microscope system based on an annular pupil, wherein the annular pupil is added to the part of the overlapped light path of an illumination light path and a collection light path, so that annular light beam illumination and annular light beam collection can be realized on the basis of the light path of the traditional confocal Brillouin microscope system, and the confocal Brillouin microscope system has obvious advantages in cost and instrument complexity.
Fig. 2 is an embodiment of an annular pupil confocal brillouin microscope system including a brillouin spectrometer, which is an implementation manner of the annular pupil confocal brillouin microscope system with high spatial resolution and high spectral resolution of the present invention, and as an embodiment of the present invention, an annular pupil confocal brillouin microscope system, which is used for a sample 6 to be measured, includes: the device comprises an illumination module 1, a beam splitting plain film 2, an annular diaphragm 3, a measurement objective 4, a spectrum detection module 5 and a three-dimensional translation table 7.
The lighting module includes: the device comprises a continuous laser 11, a beam expanding system 12, a half wave plate 13 and a polarizer 14; the spectrum detection module comprises: analyzer 51, collecting lens 52, confocal pinhole 53, brillouin spectrometer 54.
The laser emitted by the continuous laser passes through a beam expanding system to expand beams, a half wave plate and a polarizer to obtain linearly polarized laser with selectable polarization direction as incident light; the incident light is reflected by the light splitting flat sheet to enter the annular diaphragm, and is focused on a sample to be measured through the measuring objective lens after being shaped by the annular diaphragm; brillouin scattered light generated by a sample to be detected after excitation is collected by the measuring objective lens and is further modulated by the annular diaphragm, and the Brillouin scattered light is converged in a confocal pinhole after passing through the beam splitter, the analyzer and the collecting lens, and is finally collected and detected by the Brillouin spectrometer.
The three-dimensional translation platform is used for placing a sample to be tested and can drive the sample to be tested to move in a three-dimensional mode.
In the embodiment of the present invention, the normalized pinhole radius of the confocal pinhole is 2.5 ± 0.2, that is, the normalized pinhole radius of the confocal pinhole is greater than or equal to 2.3 and less than or equal to 2.7, for example, the normalized pinhole radius of the confocal pinhole is 2.5. The normalized pinhole radius of a typical confocal brillouin microscope system is 3, since the focused spot at the pinhole is compressed after modulation by the annular stop.
In the embodiment of the present invention, the annular pupil coefficient is less than 0.9, and as shown in fig. 2, the annular pupil coefficient ∈ is a/b, where a is the inner circle radius of the annular diaphragm, and b is the outer circle radius of the annular diaphragm. It should be noted that the annular pupil coefficient must be greater than 0, i.e., 0 < ε < 0.9.
The embodiment of the invention provides a high-spatial-resolution and high-spectral-resolution annular pupil confocal Brillouin microscope system, which can realize high-spatial-resolution and high-spectral-resolution Brillouin spectrum detection and provides a new way for micro-area spectrum detection and geometric measurement. According to the embodiment of the invention, on the basis of the traditional confocal Brillouin microscopic system, the annular diaphragm is added to modulate the illuminating light beam and the collecting light beam, so that high-precision mechanical performance imaging is realized.
Fig. 3 is a schematic diagram of an illumination beam and a collection beam of a conventional confocal brillouin microscope system, and fig. 3(b) is a schematic diagram of an illumination beam and a collection beam of an annular pupil confocal brillouin microscope system, wherein the annular pupil can effectively reduce the collection angle range of scattering elements.
FIG. 3(a) is a diagram of an illumination beam and a collection beam of a conventional confocal Brillouin microscope system according to an embodiment of the beams, where α is the range of the incident angle and the collection angle, and FIG. 3(b) is a diagram of an illumination beam and a collection beam of an annular pupil confocal Brillouin microscope system according to an embodiment of the beams, where β is1+β2For the range of incident angles and collection angles, alpha > beta for the same measurement objective1+β2Namely, under the action of the annular pupil, the collection angle range of the scattering elements is compressed, so that the spectrum broadening effect can be greatly reduced, the spectrum resolution and the spectrum fitting precision are improved, and the confocal Brillouin spectrum imaging with high spatial resolution and high spectral resolution is further realized.
FIG. 4 is a schematic diagram of an embodiment of the transverse point spread function distribution and the spectral distribution at different annular pupil parameters.
Fig. 4 shows the transverse point spread function distribution and the spectral distribution under different annular pupil parameters calculated by simulation, taking methanol as an example, it can be seen that as the annular pupil coefficient epsilon increases continuously, the transverse point spread function distribution of the system is compressed, i.e. the transverse resolution is improved, and meanwhile, under the condition of the same objective numerical aperture, the increase of the annular pupil coefficient epsilon can also effectively inhibit the spectral broadening effect.
However, due to the limitation of spectral excitation and spectral collection efficiency, the annular pupil coefficient epsilon cannot be too large, so that in the system construction process, the selection of parameters such as the diameter of laser emitted by a laser, the magnification factor of a beam expanding system, the diameter of the back pupil of an objective lens, the size of an annular diaphragm and the like should be matched with each other to ensure the Brillouin spectral excitation efficiency and fully utilize the effective numerical aperture of the objective lens.
The theoretical value range of the annular pupil coefficient is 0 < epsilon < 1, in the embodiment of the present invention, 0 < epsilon < 0.9, it should be noted that the value of the annular pupil coefficient may be 0 < epsilon < 0.9 in the embodiment of the present invention, or may be other ranges, which are not particularly limited herein.
It is to be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
The above description is only an example of the present invention, and is not intended to limit the present invention. Various modifications and alterations to this invention will become apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.
Claims (8)
1. An annular pupil confocal brillouin microscopy system, comprising: the device comprises an illumination module, a light splitting plain film, an annular diaphragm, a measuring objective lens and a spectrum detection module;
the illumination module is used for generating linearly polarized laser with selectable polarization direction as incident light;
the incident light is reflected by the light splitting flat sheet to enter the annular diaphragm, and is focused on a sample to be measured through the measuring objective lens after being shaped by the annular diaphragm;
brillouin scattered light generated by a sample to be detected after excitation is collected by the measuring objective lens, enters the annular diaphragm, is modulated by the annular diaphragm and then is transmitted to the spectrum detection module through the light splitting flat sheet;
the spectrum detection module is used for collecting and detecting the Brillouin scattering light spectrum of the sample to be detected.
2. The annular pupil confocal brillouin microscopy system of claim 1, wherein the illumination module comprises: the device comprises a continuous laser, a beam expanding system, a half wave plate and a polarizer;
the laser emitted by the continuous laser passes through the beam expanding system to expand the beam, the half wave plate and the polarizer to obtain the linearly polarized laser with the optional polarization direction.
3. The annular pupil confocal brillouin microscopy system of claim 1, wherein the spectral detection module comprises: the device comprises an analyzer, a collecting lens, a confocal pinhole and a Brillouin spectrometer;
brillouin scattered light generated by the sample to be tested after excitation is collected by the measuring objective lens, enters the annular diaphragm, is modulated by the annular diaphragm, then passes through the beam splitter, the analyzer and the collecting lens, then is converged in the confocal pinhole, and then is collected and detected by the Brillouin spectrometer.
4. The annular pupil confocal brillouin microscopy system of claim 1, further comprising: and the three-dimensional translation table is used for placing a sample to be detected and performing three-dimensional movement.
5. The annular pupil confocal brillouin microscopy system according to claim 1, wherein the annular diaphragm is implemented by at least one of: a digital micro-mirror array, a spatial light modulator, or a light barrier.
6. The annular pupil confocal brillouin microscope system according to claim 1, wherein an adjustment error of the annular diaphragm is 1% or less of an outer radius of the annular diaphragm.
7. The annular pupil confocal brillouin microscopy system of claim 1, wherein the pupil coefficient of the annular stop should be less than 0.9.
8. The annular pupil confocal brillouin microscopy system of claim 5, wherein the confocal pinhole has a normalized pinhole radius of 2.5 ± 0.2.
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