Phase contrast microscopy system and dark field microscopy system for high resolution

Abstract

The invention relates to a phase contrast microscopic system and a dark field microscopic system for high resolution. The phase contrast microscopic system for detecting a transparent sample comprises a condenser for converging parallel light to a sample plane; the objective lens is used for collecting light from the sample plane, the light from the sample plane comprises surrounding waves not diffracted with the transparent sample and diffracted waves diffracted with the transparent sample, and the numerical aperture of the objective lens is larger than that of the collecting lens; the diaphragm is arranged on the rear focal plane of the objective lens and comprises a central part corresponding to the surrounding waves and an annular belt part corresponding to the diffracted waves, the surrounding waves pass through the central part of the diaphragm in an attenuated manner, and the ratio of the area of the central part to the area of the annular belt part is smaller than or equal to a preset threshold value; wherein the central portion applies a [lambda] / 4 phase delay to the wrap-around wave.

Description

Technical Field

[0001] The present invention relates to a phase contrast microscope system and a dark field microscope system for high resolution. Background Art

[0002] For non-luminous transparent objects, because there is a difference in refractive index between the non-luminous transparent object and the surrounding medium, light will be reflected and refracted at the interface between the two. It is through the difference between the reflected light or refracted light and the background light that the transparent object can be observed. However, in microscopic imaging, since the refractive index of the transparent object and the surrounding medium is relatively close, the transparent object is usually not easy to observe.

[0003] In the field of life science research, if you want to gain a deeper understanding of the life processes in cells (e.g., cells of yeast, algae, etc.), tissues, or organs, you need to first have a deep understanding of the structure and organization of the cells. However, most of these samples are colorless and transparent, and it is difficult to clearly observe their fine structures under a bright field illumination microscope. Therefore, stains and dyes are often used in the fields of biology and pharmacy to improve the visibility of biological tissues. Similarly, fluorescent probe labels have similar uses.

[0004] Whether it is staining, dyes or fluorescent probes, the samples need to be pretreated when performing microscopic observation. This process is complicated and will destroy the physiological state of the samples. Chemical staining or biomarkers are both biochemical methods. They always use exogenous methods to improve the imaging contrast of living cells and produce certain biochemical reactions on the cells themselves. They can neither monitor the cell state for a long time nor truly reflect the biophysical properties of the cells themselves. In addition, the introduction of reagents to pretreat the samples during the analysis process not only limits the detection speed and throughput, but also increases the complexity of the microscopic system.

[0005] Phase contrast microscopy is a microscopic imaging system that can enhance the contrast between the sample and the background. High-contrast images of transparent samples such as living cells, microorganisms, bacteria, yeast, algae, thin tissue sections, etc. can be obtained without staining the transparent samples. Phase contrast microscopy produces contrast in almost transparent samples by attenuating the surround wave (i.e., zero-order diffracted light, no diffraction) and introducing a relative phase shift between the surround wave and the diffracted light (i.e., higher-order diffracted light). Thus, the phase contrast microscopy system uses an optical mechanism to convert the slight change in phase into a corresponding change in light intensity, which can be interpreted as a difference in contrast in human vision. The main advantage of phase contrast microscopy is that living cells can be examined in their natural state without having to be killed, fixed, and stained in advance. Therefore, the dynamics of ongoing biological processes can be observed and recorded.

[0006] However, phase contrast microscopy is limited by the limited numerical aperture of the illumination condenser, which means that the maximum magnification is 20X, and the resolution limit is only 1.11 times the illumination wavelength. In addition, phase contrast systems are complicated to operate because they usually require adjusting the two diaphragms in front of the condenser and behind the objective to align them with each other before each operation. Summary of the invention

[0007] Therefore, an object of the present invention is to provide a phase contrast system in which the resolution is not limited by the numerical aperture of the illumination condenser, and which has a simple structure, reduced cost and simple operation.

[0008] According to some aspects of the present disclosure, a phase contrast microscopy system for high resolution is provided, which is configured to detect transparent samples, and includes, in sequence along an optical axis, a condenser, which converges parallel light to a sample plane; an objective lens, which collects light from the sample plane, wherein the light from the sample plane includes an ambient wave that is not diffracted by the transparent sample and a diffracted wave that is diffracted by the transparent sample, wherein a numerical aperture of the objective lens is larger than a numerical aperture of the condenser; an aperture, which is arranged at a rear focal plane of the objective lens, and includes a central portion corresponding to the ambient wave and an annular portion corresponding to the diffracted wave, wherein the ambient wave passes through the central portion of the aperture with attenuation, wherein a ratio of an area of ​​the central portion to an area of ​​the annular portion is less than or equal to a predetermined threshold, wherein the central portion applies a λ / 4 phase delay to the ambient wave, wherein the diffracted wave and the ambient wave passing through the aperture interfere on an imaging plane to generate a phase contrast microscopy image of the transparent sample.

[0009] In some embodiments, the condenser has a numerical aperture NA condensor , the objective lens has a numerical aperture NA objective and focal length f, where the area of ​​the central portion of the aperture corresponding to the surround wave is A S =π*NA condensor 2 *f 2 The area of ​​the annular zone portion of the aperture corresponding to the diffraction wave is A D =π*(NA objective +NA condensor )*(NA objective -NA condensor )*f 2 .

[0010] In some embodiments, the predetermined threshold is 1.5, 1.4, 1.3, 1.2 or 1.1.

[0011] In some embodiments, the central portion of the aperture passes less than 30% of the light flux of the surround waves.

[0012] In some embodiments, no annular aperture is arranged in front of the condenser and thus the light beam entering the condenser is not an annular light cone.

[0013] In some embodiments, the diffracted waves from the sample plane are located at the periphery of the surrounding waves from the sample plane.

[0014] In some embodiments, the microscope system further includes a light source and a collimating lens group, and the collimating lens group collimates the light generated by the light source and feeds the collimated light to the condenser.

[0015] In some embodiments, the transparent sample does not need to be chemically stained or biolabeled prior to being viewed.

[0016] In some embodiments, the phase contrast microscopy system further comprises a control unit connected to the aperture, wherein the control unit is configured to adjust the attenuation of the surround wave passing through the aperture.

[0017] According to other aspects of the present disclosure, a method for high-resolution phase contrast microscopy imaging is provided, which is used to detect transparent samples, and includes the condenser converging parallel light to a sample plane; the objective lens collecting light from the sample plane, wherein the light from the sample plane includes an ambient wave that is not diffracted by the transparent sample and a diffracted wave that is diffracted by the transparent sample, wherein the numerical aperture of the objective lens is larger than the numerical aperture of the condenser; the ambient wave passes through a central portion of an aperture with attenuation, wherein the aperture is arranged at a rear focal plane of the objective lens, the aperture includes a central portion corresponding to the ambient wave and an annular portion corresponding to the diffracted wave, wherein the ratio of the area of ​​the central portion to the area of ​​the annular portion is less than a predetermined value, wherein the central portion applies a λ / 4 phase delay to the ambient wave, wherein the diffracted wave and the ambient wave passing through the aperture interfere on an imaging plane to generate a phase contrast microscopy image of the transparent sample.

[0018] According to other aspects of the present disclosure, a readable storage medium is provided, comprising computer program instructions: when the computer program instructions are executed by at least one processor of an electronic device, the phase contrast microscopy imaging method as described above is implemented.

[0019] According to other aspects of the present disclosure, an electronic device is provided, including a memory and a processor; the memory is configured to store computer program instructions; the processor is configured to execute the computer program instructions to implement the phase contrast microscopy imaging method as described above.

[0020] According to other aspects of the present disclosure, a dark field microscope system for high resolution is provided, the dark field microscope system is configured to detect transparent samples, and the dark field microscope system includes a condenser along the optical axis, the condenser converges parallel light to the sample plane; an objective lens, the objective lens collects light from the sample plane, wherein the light from the sample plane includes an ambient wave that is not diffracted by the transparent sample and a diffracted wave that is diffracted by the transparent sample, wherein the numerical aperture of the objective lens is greater than the numerical aperture of the condenser; and an aperture, the aperture is arranged at the back focal plane of the objective lens, the aperture includes a central portion corresponding to the ambient wave and an annular portion corresponding to the diffracted wave, wherein the central portion substantially completely blocks the passage of the ambient wave. The diffracted wave passing through the aperture generates a dark field microscope image of the transparent sample under a dark field background.

[0021] In some embodiments, the aperture does not impose a phase delay on the diffracted waves or the surrounding waves.

[0022] In some embodiments, the central portion is a light-impermeable layer. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The above and other purposes and advantages of the present disclosure are further described below in conjunction with specific embodiments and with reference to the accompanying drawings. In the accompanying drawings, the same or corresponding technical features or components will be represented by the same or corresponding reference numerals.

[0024] Figure 1 is a schematic diagram of a phase contrast microscope system in the prior art;

[0025] Figure 2 Schematic diagram of a microscopic system for mobile phase particle analysis according to some other embodiments of the present invention;

[0026] Figure 3 A schematic diagram of a high-resolution dark-field microscopy system according to some embodiments of the present invention;

[0027] Figure 4 A schematic diagram of an application scenario of a microscopic system for mobile phase particle analysis according to some embodiments of the present invention; and

[0028] Figure 5 A schematic diagram of the structure of an electronic device provided according to some embodiments of the present disclosure. DETAILED DESCRIPTION

[0029] The following detailed description is made with reference to the accompanying drawings, and the following detailed description is provided to assist in a comprehensive understanding of the various exemplary embodiments of the present disclosure. The following description includes various details to assist in understanding, but these details are considered to be examples only and not to limit the present disclosure, which is defined by the appended claims and their equivalents. The words and phrases used in the following description are only used to enable a clear and consistent understanding of the present disclosure. In addition, for the sake of clarity and brevity, descriptions of well-known structures, functions, and configurations may be omitted. Those of ordinary skill in the art will recognize that various changes and modifications may be made to the examples described herein without departing from the scope of the present disclosure.

[0030] When a beam of parallel light passes through the sample being observed, due to the different absorption of light at each point in the sample, images of each object point with different grayscale (i.e., brightness) can be seen in the field of view of a traditional microscope. This is due to the different amplitudes of light. However, if the object to be observed in the sample is nearly transparent, no obvious grayscale difference can be seen in the field of view.

[0031] At this time, it is necessary to use a phase contrast microscope system to observe transparent samples. When the light beam passes through a transparent sample, the light waves are diffracted and refracted by each object point in the sample, causing the light waves to deviate due to delay, and there is a certain difference in the optical path, that is, a phase difference. The human eye cannot distinguish this phase difference, but it can try to convert this phase difference into an amplitude difference of light intensity. A phase contrast microscope is a microscope device that uses the principle of light interference to convert the phase difference into an amplitude difference (i.e., the difference between light and dark).

[0032] However, existing phase contrast microscopy systems have many disadvantages.

[0033] One disadvantage of existing phase contrast microscopy systems is that their resolution is limited. The working distance WD of the phase contrast microscopy system at the illumination end decreases as the numerical aperture of the condenser at the illumination end increases. The larger the numerical aperture of the condenser, the smaller the working distance of the illumination end, that is, the closer the optical device is to the biological culture dish. When the numerical aperture NA of the condenser at the illumination end is > 0.65, its working distance WD < 0.17 mm, which may not be able to meet the application scenarios of biological culture dishes. Therefore, the numerical aperture NA of the condenser at the illumination end of the phase contrast microscopy system is usually condensor The maximum value does not exceed 0.6, for example, it is usually between 0.55 and 0.6. In addition, since the condenser needs to project the annular light cone to the sample at a large angle to enhance the diffraction effect, the numerical aperture of the condenser usually needs to be greater than or equal to the numerical aperture of the objective. According to the derivation of the Rayleigh criterion of formula 1, the resolution of the microscope system is determined by the sum of the numerical aperture of the condenser and the numerical aperture of the objective.

[0034]

[0035] Based on the above constraints, the resolution of the phase contrast microscope system in the prior art is actually limited by the limited numerical aperture of the condenser at the illumination end. Since the maximum numerical aperture of the condenser can be 0.6, and the numerical aperture of the objective lens must be less than or equal to the numerical aperture of the condenser, the resolution limit can only be around 1.11λ.

[0036] Another disadvantage of the existing phase contrast microscopy system is that the operation of the phase contrast system is complicated. Figure 1 As shown in the figure, the phase contrast system includes a pair of diaphragms, namely the illumination diaphragm before the condenser and the objective diaphragm after the objective lens, and sometimes there may be two objective diaphragms. Before observing the phase contrast system, the bright ring of the illumination diaphragm and the annular ring of the objective diaphragm must be adjusted each time to make them coincide, so that the phase contrast microscope system can be effective. Otherwise, the optical path of the surround wave and the diffracted light will be disordered, the light that should be absorbed cannot be effectively absorbed, and the light that should be phase delayed cannot be effectively phase delayed, which will directly lead to the failure of the phase contrast microscope system.

[0037] In order to solve the above technical problems, the phase contrast microscopy system of the present invention sets the numerical aperture of the objective lens to be larger than the numerical aperture of the condenser, and modulates the intensity of the surround waves and diffracted light to be interfered with each other by setting a corresponding aperture only at the rear focal plane position of the objective lens with a large numerical aperture (rather than setting an illumination aperture in front of the condenser) to achieve high-resolution phase contrast microscopy imaging, and slightly improves the similar principle to achieve high-resolution dark-field microscopy imaging.

[0038] Based on this, the present invention does not need to generate an annular light cone that illuminates the sample at a large angle to enhance the diffraction effect. Therefore, there is no need to set an illumination diaphragm before the condenser at the illumination end, which simplifies the overall structure of the microscope system. Therefore, there is no need for the operator to adjust the illumination diaphragm and the objective diaphragm to strictly coincide before each observation.

[0039] In addition, since the present invention does not need to generate a large-angle annular light cone, the numerical aperture of the condenser can be smaller than the numerical aperture of the objective lens. Therefore, the system resolution is no longer limited by the numerical aperture of the condenser at the illumination end, while the numerical aperture of the condenser at the illumination end cannot be made very large due to the limitation of the working distance.

[0040] Figure 1 FIG. 1 is a schematic diagram of a phase contrast microscope system 100 of the prior art. Figure 1 , a light source (not shown) emits illumination light, which is guided by a collimating lens (not shown) and focused on an annular illumination aperture 101 located in front of a condenser 102 to form an annular light cone. Figure 1A cross-sectional view of an annular illumination aperture 101 in the XY plane is separately shown next to the microscope system 100, and the annular illumination aperture 101 includes a shading portion represented by a shade and a light-transmitting portion represented by a blank. The shading portion blocks the passage of the light beam, and the light-transmitting portion allows the light beam to pass through. Thus, the parallel light beam from the collimating lens passes through the annular illumination aperture 101 to form an annular light cone. The annular light cone ensures that the sample at the sample plane 103 can be illuminated at a large angle, thereby forming an ideal diffraction effect. The annular light cone is focused by the condenser 102 and illuminated on the sample plane 103 including the sample. The annular light cone is divided into two components when passing through the sample. One component is a plane wavefront that passes through the sample without deviation (no diffraction; zero order), usually referred to as a surround wave (S wave), which passes through the sample and surrounds the sample but does not interact with the sample. The other component is the spherical wavefront that passes through the sample with a deviation (higher-order diffraction), called the diffracted wave (D wave), which passes through the sample and diffracts with the sample in multiple directions. Depending on the change in the optical path between the sample and its surrounding medium, the diffracted wave will be delayed in phase compared to the surround wave. For cells cultured in tissues, there is usually a phase difference of about 1 / 4 wavelength with the background. The diffracted wave (D wave) and surround wave (S wave) are respectively Figure 1 . The distribution (number and position) of the diffracted waves (D waves) depends on the number, size and refractive index difference of the objects in the sample. For most samples, only a small part of the incident light wave is diffracted by the sample, and most of the incident light does not interact with the sample to form surround waves. Afterwards, the diffracted waves (D waves) and surround waves (S waves) are projected to different positions at the rear focal plane of the objective lens 104. In order to maximize the intensity difference between the sample and the background in the image plane, it is necessary to advance the phase of the surround waves by 1 / 4 wavelength, and preferably also reduce the light intensity of the surround waves. Therefore, a λ / 4 phase plate 105 is set at the rear focal plane of the objective lens 104. The shaded area of ​​the phase plate 105 corresponds to the area where the surround waves are projected. The shaded area advances the surround waves by 1 / 4 wavelength, for example by having a thinner thickness than other areas. It should be noted that the light-passing portion of the annular illumination aperture 101 at the front focal plane of the condenser needs to be conjugate with the shadow area of ​​the λ / 4 phase plate 105 at the rear focal plane of the objective lens in the optical path, so that the annular light cone generated by the annular illumination aperture 101 corresponds exactly to the shadow area of ​​the λ / 4 phase plate 105 after passing through the condenser 102, the sample plane 103 and the objective lens 104. At the imaging plane 106, after two phase transformations, the phase difference between the diffracted wave (D wave) and the surround wave (S wave) reaches 1 / 2λ, causing destructive or constructive interference, so that the phase change can be converted into a change in light intensity. Since the human eye interprets the difference in light intensity as contrast, the sample can be observed by the human eye at the imaging plane.

[0041] In the above description, the direction from the light source to the sample plane 103 and then to the imaging plane 106 is considered to be the optical axis of the microscope system 100, that is, the Z direction. The direction perpendicular to the Z direction is considered to be the X direction. The direction perpendicular to both the Z direction and the X direction is considered to be the Y direction. The sample stage (not shown) of the sample plane 103 is in the plane formed by the X direction and the Y direction.

[0042] Depend on Figure 1 It can be seen that, compared with a general bright field microscope, the phase contrast microscope system of the prior art needs to place an annular illumination aperture 101 at the front focal plane of the condenser at the illumination end, and a λ / 4 phase plate 105 needs to be placed at a position conjugate with the annular aperture 101 behind the objective lens. The shadow area on the λ / 4 phase plate 105 has a light intensity attenuation function; the area outside the shadow area on the phase plate 105 has a phase difference of 1 / 4 wavelength with the light pass function of the shadow area. This requires that before using the phase contrast microscope system for observation each time, the operator needs to adjust the annular illumination aperture 101 and the phase plate 105 in the Z direction so that they are at the appropriate front / back focal plane, and also needs to adjust the annular illumination aperture 101 and the phase plate 105 in the XY direction so that the light-transmitting portion of the annular illumination aperture 101 and the shadow area on the λ / 4 phase plate 105 are conjugate on the optical path. Therefore, the phase contrast microscope system of the prior art poses a challenge to the operator.

[0043] In addition, the imaging resolution of the phase contrast microscope system is closely related to the numerical aperture NA of the condenser. condensor and the numerical aperture NA of the objective objective The sum of the numerical apertures of the two is related, see formula 1. The larger the sum of the numerical apertures of the two, the higher the resolution of the microscope. However, the numerical aperture of the objective lens of the traditional phase contrast microscopy system needs to be less than or equal to the numerical aperture of the condenser, and the numerical aperture of the condenser of the traditional phase contrast microscopy system cannot be very large due to the limitation of the working distance, usually only between 0.55 and 0.6. Therefore, according to the derivation of the Rayleigh criterion of formula 1, the resolution of the traditional phase contrast system is mainly limited by the numerical aperture of the condenser, resulting in a resolution limit of only about 1.11λ. Therefore, the resolution of the phase contrast microscopy system is limited.

[0044] In order to overcome the above technical problems of complex operation and limited resolution, the present invention sets the numerical aperture of the objective lens to be larger than the numerical aperture of the condenser, and only sets a corresponding aperture at the rear focal plane of the objective lens with a large numerical aperture, so that the ratio of the area through which the surround wave passes to the area through which the diffracted wave passes in the aperture is less than a predetermined value, so that the surround wave and the diffracted wave interfere in the imaging plane to achieve high-resolution phase contrast microscopy imaging. The system of the present invention is not subject to the numerical aperture limit of the illumination condenser, and has a simple structure and economical cost.

[0045] Figure 2 Schematic diagram of a high-resolution phase contrast microscope system according to some embodiments of the present invention. Figure 2 and Figure 1 Shown side by side to show the difference. Figure 2 As shown, the phase microscope system 200 includes a condenser 201, a sample plane 202, an objective lens 203, an aperture 204, and an imaging plane 205. The sample to be observed is placed at the sample plane 202. Exemplary samples include, but are not limited to, cells, microorganisms, or thin tissue sections. In one embodiment of the present invention, the sample is transparent and does not need to be chemically stained or biolabeled before being observed.

[0046] In the following description, the direction from the condenser 201 to the sample plane 202 and then to the imaging plane 205 is considered to be the optical axis of the microscope system 200, that is, the Z direction. The direction perpendicular to the Z direction is considered to be the X direction. The direction perpendicular to both the Z direction and the X direction is considered to be the Y direction. The sample stage (not shown) of the sample plane 202 is in the plane formed by the X direction and the Y direction.

[0047] The condenser 201 receives parallel light from a light source (not shown). In some embodiments of the present invention, the light source may have any configuration, such as a halogen lamp or an LED lamp. In some embodiments of the present invention, a collimating lens group (not shown) is configured to collimate the light from the light source into a parallel light beam. The collimating lens group may have any configuration, such as a combination of a plano-convex lens and a pair of convex lenses disposed oppositely. In some embodiments of the present invention, the light source is disposed at the focus of the collimating lens group. The condenser 201 is disposed after the collimating lens group along the optical axis Z. Compared with the phase contrast microscopy system of the prior art, the phase contrast microscopy system of some embodiments of the present invention Figure 2 The high-resolution phase contrast microscope system shown does not require an illumination diaphragm in front of the condenser. What enters the condenser 201 is a collimated light beam, not an annular light cone.

[0048] The condenser 201 is configured to converge a collimated light beam from a light source to the sample plane, and the objective lens 203 is configured to collect light from the sample plane 202, wherein the light from the sample plane 202 includes a plane wavefront that passes through the sample without deviation (no diffraction; zero order), which is usually called a surround wave (S wave); and a spherical wavefront that passes through the sample with deviation (higher-order diffraction), which is called a diffracted wave (D wave). The surround wave passes through the sample and surrounds the sample but does not interact with the sample, while the diffracted wave passes through the sample and diffracts with the sample in multiple directions. Due to the difference in refractive index between the transparent sample and the surrounding medium, the diffracted wave generated by diffraction with the transparent sample is delayed in phase compared to the surround wave that does not diffract with the transparent sample. For cells cultured in tissues, the two usually produce a phase difference of about 1 / 4 wavelength. For most samples, only a small part of the incident light wave is diffracted by the sample, and most of the incident light does not interact with the sample to form a surround wave. The diffracted wave (D wave) and the surround wave (S wave) are respectively at Figure 2 In the phase contrast microscopy system of the prior art, the surround wave is located outside the diffraction wave; in contrast, the diffraction wave of the high-resolution phase contrast microscopy system according to some embodiments of the present invention is located outside the surround wave. The diffraction wave and the non-diffracted surround wave occupy different positions on the rear focal plane of the objective lens, that is, the surround wave and the diffraction wave are basically separated in the XY space of the rear focal plane of the objective lens, so any wave component (surround wave or diffraction wave) can be selectively manipulated without affecting the other.

[0049] As mentioned above, the condenser 201 and the objective lens 203 are successively arranged along the optical axis Z. The sample plane 202 is arranged between the condenser 201 and the objective lens 203. The distance between the sample plane 202 and the condenser 201 is the focal length of the condenser 201. The condenser 201 is configured to converge the light from the light source to the sample plane 202, and the objective lens 203 is configured to collect the light from the sample plane 202. The objective lens 203 converges the light beam including the central surround wave and the surrounding diffraction wave to the imaging plane 205. An aperture 204 is arranged between the objective lens 203 and the imaging plane 205. As mentioned above, the surround wave and the diffraction wave are basically separated in the XY space of the rear focal plane of the objective lens. Here, the surround wave and the diffraction wave are fed to the central part and the annular part of the aperture 204, respectively. Based on the principle of trigonometric function approximation, the area A of the central part of the aperture 204 corresponding to the surround wave is s It can be approximated as:

[0050] A S =π*NA condensor 2 *f 2 Formula 2

[0051] where NA condensoris the numerical aperture of the condenser 201, and f is the focal length of the objective lens 203.

[0052] Similarly, the area A of the annular zone portion of the aperture 204 corresponding to the diffraction wave is D It can be approximated as:

[0053] A D =π*(NA objective +NA condensor *(NA objective +NA condensor )*f 2 Formula 3

[0054] where NA objective is the numerical aperture of the objective lens 203.

[0055] Note that, since the aperture 204 is disposed adjacent to the objective lens 203 having a larger numerical aperture, the area A of the central portion of the aperture 204 is s Or the area A of the annular zone of the aperture 204 D They are all related to the focal length f of the objective lens 203 with a large numerical aperture, but have nothing to do with the focal length of the condenser lens with a small numerical aperture.

[0056] The central part of the aperture 204 blocks part of the surround waves and applies a phase delay of λ / 4 to the surround waves. In some embodiments of the present invention, the transmittance of the central part is adjustable to adjust the luminous flux of the surround waves passing through. In some embodiments of the present invention, the central part has a translucent film, and the translucent film allows a small part of the surround waves incident on the central part to pass through, for example, less than or equal to 30% of the surround waves. In some embodiments of the present invention, the central part is formed of an electrochromic material, and the transmittance of the central part can be adjusted by an external electric field. In some embodiments of the present invention, a controller is coupled to the aperture 204 to control the transmittance of the central part of the aperture. In some embodiments of the present invention, the central part includes a plurality of through holes dispersed therein or a single through hole located in the center to allow only a small part of the surround waves to pass through. In some embodiments of the present invention, the through holes include but are not limited to square, circular, rectangular, triangular, trapezoidal, etc.

[0057] The area of ​​the center part A s The area A of the annular zone D satisfy:

[0058] A s ≤1.3A D Formula 4

[0059] The microscopy system 200 also includes an imaging plane 205, where the imaging plane 205 includes an imaging component, wherein the surround wave and the diffracted light are interferometrically imaged at the imaging component. The imaging component may include a charge coupled device (CCD) or a complementary metal oxide semiconductor device (CMOS). In some embodiments, the phase contrast microscopy system may also include a memory, a processor, etc. that are communicatively connected to the imaging component to automatically store and process the image information formed by the imaging component, thereby further facilitating detection.

[0060] exist Figure 2 In the high-resolution phase contrast microscopy system shown in FIG. 1 , the numerical aperture NA of the objective lens 203 of the present invention is different from that of the prior art phase contrast microscopy system. objective Designed to be larger than the numerical aperture NA of the illumination condenser 201 condensor Therefore, the resolution of the microscope system is not limited by the numerical aperture of the illumination condenser 201. If a "dry lens" is used as the objective lens, the maximum numerical aperture of the objective lens can be 0.9; if an "oil lens" is used as the objective lens, the maximum numerical aperture of the objective lens can be 1.3. According to the Rayleigh criterion of formula 1 When the numerical aperture of the illumination condenser is between 0.55 and 0.6, if a "dry lens" is used as the objective lens, the limit of resolution can be increased to 0.81λ; if an "oil lens" is used as the objective lens, the limit of resolution can be increased to 0.65λ. Compared with the resolution of 1.11λ of the phase contrast microscope system in the prior art, the resolution is increased by 27% to 42%.

[0061] In addition, since the present invention does not need to set any annular illumination aperture in front of the condenser 201, it is not necessary to align the annular illumination aperture in front of the condenser 201 and the objective aperture 204 behind the objective lens 203 before each operation.

[0062] In addition, since the present invention does not set an annular illumination aperture in front of the condenser 201, the illumination light from the light source can enter the condenser in the form of a collimated light beam with almost no obstruction, thereby increasing the illumination efficiency, which is very beneficial for high-resolution imaging.

[0063] Figure 3 Schematic diagram of a high-resolution dark-field microscopy system according to some embodiments of the present invention. Figure 2 The high-resolution phase-contrast microscope shown can be slightly modified for use in darkfield microscopy. Darkfield microscopy involves blocking most of the light that passes through and around the specimen, allowing only oblique light to interact with the specimen to form a bright image of the specimen superimposed on a dark background. Darkfield microscopy uses oblique illumination to enhance the contrast of specimens that would not be well imaged under normal lighting conditions. Figure 3In an embodiment of a dark field microscope system, the undeflected surround waves that constitute the bright background light and pass through the sample are completely blocked by the aperture, and only the deflected diffracted light that is diffracted by the sample is allowed to enter the imaging plane, thereby presenting a bright sample on a black background.

[0064] Specifically, Figure 2 The equivalent microscopic system is similar to that of Figure 3 The dark field microscope system includes a condenser 301, a sample plane 302, an objective lens 303, a diaphragm 304, and an imaging plane 305. The sample to be observed is placed at the sample plane 302. Figure 2 Similarly, the direction from the condenser 301 to the sample plane 302 and then to the imaging plane 305 is considered to be the optical axis of the microscope system 300, that is, the Z direction. The direction perpendicular to the Z direction is considered to be the X direction. The direction perpendicular to both the Z direction and the X direction is considered to be the Y direction. The sample stage (not shown) of the sample plane 302 is in the plane formed by the X direction and the Y direction.

[0065] and Figure 2 The phase contrast microscope system is still similar in that the condenser 301 receives parallel light from a light source (not shown). The condenser 301 is configured to converge a collimated light beam from the light source to the sample plane 302, and the objective lens 303 is configured to collect light from the sample plane 302. The light from the sample plane 302 includes a plane wavefront that passes through the sample without deviation (no diffraction; zero order), which is usually called a surround wave (S wave); and a spherical wavefront that passes through the sample with deviation (higher-order diffraction), which is called a diffracted wave (D wave). The surround wave passes through the sample and surrounds the sample but does not interact with the sample, while the diffracted wave passes through the sample and diffracts with the sample in multiple directions. The diffracted wave (D wave) and the surround wave (S wave) are respectively Figure 3 In most samples, only a small part of the incident light wave is diffracted by the sample, and most of the incident light does not interact with the sample and forms a surround wave. The undiffracted surround wave and the diffracted wave occupy different positions on the rear focal plane of the objective lens, that is, the surround wave and the diffracted wave are basically separated in the XY space of the rear focal plane of the objective lens, so any wave component (surround wave or diffracted wave) can be selectively manipulated without affecting the other.

[0066] An aperture 304 is provided between the objective lens 303 and the imaging plane 305, and the surround wave and the diffracted wave are transmitted to the central part and the annular part of the aperture 304, respectively. Figure 2 The difference between the phase contrast microscope system and the Figure 3 The central part of the aperture 304 of the dark field microscope system blocks all the surrounding waves, for example, by covering the area A in the central part corresponding to the surrounding waves. sThe light-proof coating is used at the annular zone, and all diffracted waves are allowed to pass along the annular zone. As a result, the surround waves (S waves) that constitute the bright background light are completely blocked, and only the diffracted waves (D waves) with offset enter the imaging plane. In addition, since the dark field microscope system does not rely on the interference imaging of diffracted waves and surround waves (even the surround waves are directly blocked by the aperture), there is no need for Figure 2 Thus, the aperture 304 does not apply any phase delay to the diffracted wave and the surrounding wave.

[0067] For reasons similar to the high resolution of phase contrast microscopy, Figure 3 Dark-field microscopy systems also have high resolution because they are not limited by the numerical aperture of the condenser.

[0068] Figure 4 Schematic diagram of an application scenario of a microscopic system for mobile phase particle analysis according to some embodiments of the present invention. Figure 4 In an embodiment of the present invention, the processor is connected to the optical devices before and after the sample plane 402, respectively, to obtain imaging information at the image 405 and / or control the aperture 404 and / or control the light source (not shown). Optionally, the transmittance of the central part of the aperture 404 is automatically or semi-automatically adjusted according to the imaging information, for example, by adjusting the transmittance of the electrochromic material in the central part of the aperture 404. Optionally, the position of the aperture 404 in three-dimensional space is automatically or semi-automatically adjusted according to the imaging information to substantially align the central part and the annular part with the surround wave and the diffracted wave from the objective lens 403, respectively.

[0069] Figure 5 Schematic diagram of the structure of an electronic device provided according to some embodiments of the present disclosure. Figure 5As shown, the electronic device 500 provided in this embodiment includes: a memory 501 and a processor 502. Among them, the memory 501 can be an independent physical unit, and the processor 502 can be connected through a bus 503. The processor 502 may include but is not limited to a CPU, a hardware microprocessor, a hardware processor, a multi-core processor, a single-core processor, a microcontroller, an application-specific integrated circuit (ASIC), a DSP or other similar processing devices, and can execute any type of instructions, algorithms or software for the operation and function of microscopic imaging according to the embodiments described in this disclosure. The processor 502 can be various implementations of a digital circuit system, an analog circuit system or a mixed signal (a combination of analog and digital) circuit system that performs functions in a computing system. The processor 502 may include, for example, an integrated circuit (IC), a portion or circuit of a separate processor core, an entire processor core, a separate processor, a programmable hardware device such as a field programmable gate array (FPGA), and / or a system including multiple processors. The processor 402 may be a central processing unit (CPU), a network processor (NP) or a combination of a CPU and a NP. The processor 402 may further include a hardware chip. The hardware chip may be an application-specific integrated circuit (ASIC), a programmable logic device (PLD) or a combination thereof. The PLD may be a complex programmable logic device (CPLD), a field-programmable gate array (FPGA), a generic array logic (GAL) or any combination thereof.

[0070] The memory 501 can be integrated with the processor 502 and implemented by hardware, etc. The memory 401 is used to store program instructions, and the processor 502 calls the program instructions to perform the operations of any of the above method embodiments. The memory 501 may include a volatile memory (volatile memory), such as a random-access memory (random-access memory, RAM); the memory may also include a non-volatile memory (non-volatile memory), such as a flash memory (flash memory), a hard disk drive (hard disk drive, HDD) or a solid-state drive (solid-state drive, SSD); the memory may also include a combination of the above types of memory. The memory 501 can be used to store any type of instructions, software or algorithms, including instructions for controlling the general functions and operations of electronic devices.

[0071] Optionally, when part or all of the methods of the above embodiments are implemented by software, the above electronic device 500 may also only include a processor 502. The memory 401 for storing programs is located outside the electronic device 500, and the processor 502 is connected to the memory through circuits / wires to read and execute the programs stored in the memory.

[0072] The present disclosure may be implemented as any combination of an apparatus, a system, an integrated circuit, and a computer program or a program product on a non-transitory computer-readable medium.

[0073] It should be understood that the computer executable instructions in the computer readable storage medium or program product according to the embodiments of the present disclosure can be configured to perform operations corresponding to the above-mentioned device and method embodiments. When referring to the above-mentioned device and method embodiments, the embodiments of the computer readable storage medium or program product are clear to those skilled in the art, so they are not repeatedly described. Computer readable storage media and program products for carrying or including the above-mentioned computer executable instructions also fall within the scope of the present disclosure. Such storage media may include, but are not limited to, floppy disks, optical disks, magneto-optical disks, memory cards, memory sticks, and the like.

[0074] In addition, it should be understood that the above series of processes and devices can also be implemented by software and / or firmware. In the case of being implemented by software and / or firmware, the storage medium of the relevant device stores the corresponding program constituting the corresponding software, and when the program is executed, various functions can be performed.

[0075] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limiting. Therefore, other examples of the exemplary embodiments may have different values.

[0076] The words "front", "rear", "top", "bottom", "above", "below", etc., if present, in the specification and claims are used for descriptive purposes and are not necessarily used to describe invariant relative positions. It should be understood that the words so used are interchangeable under appropriate circumstances, such that the embodiments of the disclosure described herein, for example, are capable of operation in other orientations than those illustrated or otherwise described herein.

[0077] As used herein, the word "exemplary" means "serving as an example, instance, or illustration" rather than as a "model" to be precisely copied. Any implementation described as exemplary herein is not necessarily to be construed as being superior to other implementations.

[0078] Furthermore, the present disclosure is not limited by any expressed or implied theory given in the above technical field, background technology, summary of the invention or detailed description.

[0079] As used herein, the term "substantially" is intended to include any minor variations due to design or manufacturing imperfections, device or component tolerances, environmental influences, and / or other factors. The term "substantially" also allows for deviations from a perfect or ideal condition due to parasitic effects, noise, and other practical considerations that may exist in actual implementations.

[0080] The description may indicate an element or node or feature that is "connected" or "coupled" together. As used herein, unless otherwise expressly stated, "connected" means that one element / node / feature is directly connected (or directly communicates) with another element / node / feature electrically, mechanically, logically or otherwise. Similarly, unless otherwise expressly stated, "coupled" means that one element / node / feature can be mechanically, electrically, logically or otherwise connected to another element / node / feature in a direct or indirect manner to allow interaction, even if the two features may not be directly connected. In other words, "coupled" is intended to include direct and indirect connections of elements or other features, including connections using one or more intermediate elements.

[0081] It should also be understood that when the term “include / comprises” is used in this document, it indicates the presence of the specified features, integers, steps, operations, units and / or components, but does not exclude the presence or addition of one or more other features, integers, steps, operations, units and / or components and / or their combinations.

[0082] Those skilled in the art will appreciate that the boundaries between the above operations are merely illustrative. Multiple operations can be combined into a single operation, a single operation can be distributed in additional operations, and operations can be performed at least partially overlapping in time. Moreover, alternative embodiments may include multiple instances of specific operations, and the order of operations may be changed in other various embodiments. However, other modifications, variations, and replacements are equally possible. Therefore, this specification and accompanying drawings should be considered illustrative, not restrictive.

[0083] Although some specific embodiments of the present disclosure have been described in detail by way of example, it should be understood by those skilled in the art that the above examples are for illustration only and are not intended to limit the scope of the present disclosure. The various embodiments disclosed herein may be combined in any manner without departing from the spirit and scope of the present disclosure. It should also be understood by those skilled in the art that various modifications may be made to the embodiments without departing from the scope and spirit of the present disclosure. The scope of the present disclosure is defined by the appended claims.

Claims

1. A phase contrast microscopy system for high resolution, the phase contrast microscopy system being configured to detect a transparent sample, the phase contrast microscopy system comprising, in sequence along an optical axis: a condenser lens that converges parallel light onto a sample plane; an objective lens that collects light from the sample plane, wherein the light from the sample plane includes a surround wave that is not diffracted by the transparent sample and a diffracted wave that is diffracted by the transparent sample, wherein a numerical aperture of the objective lens is greater than a numerical aperture of the condenser lens; an aperture, the aperture being arranged at the rear focal plane of the objective lens, the aperture comprising a central portion corresponding to the surround wave and an annular portion corresponding to the diffracted wave, wherein the surround wave passes through the central portion of the aperture with attenuation, wherein the ratio of the area of ​​the central portion to the area of ​​the annular portion is less than or equal to a predetermined threshold, wherein the central portion applies a λ / 4 phase delay to the surround wave, The diffracted wave and the surrounding wave passing through the aperture interfere on an imaging surface to generate a phase contrast microscopic image of the transparent sample.

2. The phase contrast microscopy system according to claim 1, wherein the condenser has a numerical aperture NA condensor , the objective lens has a numerical aperture NA objective and focal length f, where the area of ​​the central portion of the aperture corresponding to the surround wave is A s =π*NA condensor 2 *f 2 The area of ​​the annular zone portion of the aperture corresponding to the diffraction wave is A D =π*(NA objective +NA condensor )*(NA objective -NA condensor )*f 2 .

3. The phase contrast microscopy system according to claim 1, wherein the predetermined threshold is 1.5, 1.4, 1.3, 1.2 or 1.

1.

4. The phase contrast microscopy system of claim 1, wherein the central portion of the aperture allows less than 30% of the light flux of the surround wave to pass therethrough.

5. The phase contrast microscope system according to claim 1, wherein no annular aperture is arranged in front of the condenser and thus the light beam entering the condenser is not an annular light cone.

6. The phase contrast microscopy system according to claim 1, wherein the diffracted waves from the sample plane are located at the periphery of the surround waves from the sample plane.

7. The phase contrast microscope system according to claim 1, wherein the microscope system further comprises a light source and a collimating lens group, and the collimating lens group collimates the light generated by the light source and then feeds the collimated light to the condenser.

8. The phase contrast microscopy system of claim 1, wherein the transparent sample does not need to be chemically stained or biologically labeled before being observed. 9 . The phase contrast microscopy system according to claim 1 , further comprising a control unit connected to the aperture, the control unit being configured to adjust the attenuation of the surround wave passing through the aperture.

10. A method for high-resolution phase contrast microscopy imaging, the method being used to detect a transparent sample, the method comprising: The condenser focuses parallel light onto the sample plane; An objective lens collects light from the sample plane, wherein the light from the sample plane includes a surround wave that is not diffracted by the transparent sample and a diffracted wave that is diffracted by the transparent sample, wherein a numerical aperture of the objective lens is greater than a numerical aperture of the condenser lens; The surround wave passes through the central part of the aperture with attenuation, wherein the aperture is arranged at the rear focal plane of the objective lens, the aperture comprises a central part corresponding to the surround wave and an annular part corresponding to the diffracted wave, wherein the ratio of the area of ​​the central part to the area of ​​the annular part is less than or equal to a predetermined value, wherein the central part applies a λ / 4 phase delay to the surround wave, The diffracted wave and the surrounding wave passing through the aperture interfere on an imaging surface to generate a phase contrast microscopic image of the transparent sample.

11. A readable storage medium comprising computer program instructions: When the computer program instructions are executed by at least one processor of an electronic device, the phase contrast microscopy imaging method according to claim 10 is implemented.

12. An electronic device comprising a memory and a processor; The memory is configured to store computer program instructions; The processor is configured to execute the computer program instructions to implement the phase contrast microscopy imaging method according to claim 10.

13. A dark field microscopy system for high resolution, the dark field microscopy system being configured to detect a transparent sample, the dark field microscopy system comprising, in sequence along an optical axis: a condenser lens that converges parallel light onto a sample plane; an objective lens that collects light from the sample plane, wherein the light from the sample plane includes a surround wave that is not diffracted by the transparent sample and a diffracted wave that is diffracted by the transparent sample, wherein a numerical aperture of the objective lens is greater than a numerical aperture of the condenser lens; An aperture is arranged at the rear focal plane of the objective lens, and the aperture includes a central part corresponding to the surrounding waves and an annular part corresponding to the diffracted waves, wherein the central part substantially completely blocks the passage of the surrounding waves. The diffraction waves passing through the aperture generate a dark field microscopic image of the transparent sample under a dark field background.

14. The dark field microscopy system according to claim 13, wherein the aperture does not apply phase delay to the diffracted wave or the surrounding wave.

15. The dark field microscopy system according to claim 13, wherein the central portion is a light-impermeable layer.

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