Microscope system for mobile phase particle analysis and method therefor

Abstract

The present disclosure relates to a microscope system and a method for mobile phase particle analysis. The microscope system is used to observe a transparent sample, and comprises a light source used to generate light to illuminate a sample surface, the sample surface being configured to carry the sample. An objective lens group comprises a pair of objective lenses having different numerical apertures. The objective lens group converges light from the light source onto the sample surface, and collects light from the sample surface, the light from the sample surface comprising surrounding waves not diffracted by the transparent sample and diffraction waves diffracted by the transparent sample. A diaphragm is disposed close to the objective lens group, the diaphragm being configured to adjust a ratio of diffracted waves and surrounding waves passing therethrough, the diffracted waves and the surrounding waves interfering with an imaging surface to generate a microscopic image of the transparent sample.

Description

A microscopic system for mobile phase particle analysis and method thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on and claims priority to the Chinese patent application with Chinese application number 202311474467.5 and application date November 7, 2023. The disclosed content of the Chinese patent application is hereby introduced as a whole into this application. Technical Field

[0003] The present disclosure relates to a microscopy system and method for mobile phase particle analysis. Background Art

[0004] For non-luminous, transparent objects, light is reflected or refracted at the interface due to the difference in refractive index between the object and the surrounding medium. This difference in reflected or refracted light and the background light allows the transparent object to be observed. However, in microscopic imaging, transparent objects are often difficult to observe due to the similar refractive index between the object and the surrounding medium.

[0005] In the field of life science research, if you want to gain a deeper understanding of the life processes within cells (for example, cells of yeast, algae, etc.), tissues, or organs, you need to first have a deep understanding of the cell's structure and organization. However, most of these samples are colorless and transparent, making it difficult to clearly observe their microstructures under brightfield illumination microscopy. 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.

[0006] Currently, brightfield microscopy uses macromolecular reagents such as trypan blue (TB), while fluorescence microscopy uses small molecule reagents such as acridine orange (AO), propidium iodide (PI), and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI). Both methods require pretreatment of the cell sample, which involves thoroughly mixing the reagents in a specific ratio, observing and comparing the cells under the microscope, and then analyzing the results. Currently, a variety of manual and fully automated cell analyzers based on chemical staining or biomarker methods are commercially available worldwide.

[0007] However, whether using stains, dyes, or fluorescent probes, all require sample pretreatment for microscopic observation, a complex process that can disrupt the sample's physiological state. Chemical staining or biolabeling are both biochemical methods that employ exogenous methods to enhance the imaging contrast of living cells, inducing certain biochemical reactions in the cells themselves. This makes it impossible to monitor cell status over long periods of time, nor can it truly reflect the biophysical properties of the cells themselves. Furthermore, the introduction of reagents to pretreat the sample during analysis limits detection speed and throughput, while also increasing the complexity of the microscopic system.

[0008] Summary of the Invention

[0009] Therefore, one object of the present disclosure is to provide a microscopy system that can directly improve the endogenous contrast of cells without chemical or biological pretreatment of the sample, thereby obtaining indicator results such as cell viability, diameter, and concentration; and can be combined with a liquid flow system for automatic and rapid sample loading to achieve fully automatic, high-throughput cell analysis functions.

[0010] According to some aspects of the present disclosure, a microscopic system for mobile phase particle analysis is provided, wherein the microscopic system is configured to detect a transparent sample, the microscopic system including a light source, the light source being configured to generate light to illuminate a sample surface, the sample surface being configured to carry a sample; an objective lens group, the objective lens group including a pair of objective lenses with different numerical apertures, the sample surface being located between the two objective lenses, the objective lens group being configured to converge light from the light source to the sample surface and collect light from the sample surface, wherein the light from the sample surface includes an ambient wave that is not diffracted by the transparent sample and a diffracted wave that is diffracted by the transparent sample; and an aperture, the aperture being arranged to be adjacent to the objective lens group, the aperture being configured to adjust a ratio of the diffracted wave and the ambient wave passing therethrough, wherein the diffracted wave and the ambient wave interfere at an imaging surface to generate a microscopic image of the transparent sample.

[0011] In some embodiments, the aperture includes an annular light-passing portion and a central light-shielding portion, wherein the central light-shielding portion corresponds to the surround wave that is not diffracted by the transparent sample, and the annular light-passing portion corresponds to the diffracted wave that is diffracted by the transparent sample, wherein the central light-shielding portion allows the surround wave to pass with attenuation, and the annular light-passing portion allows the diffracted wave to pass with basically no attenuation.

[0012] In some embodiments, the contrast C of the microscopic image of the transparent sample is related to the ratio of the light flux of the diffracted wave passing through the aperture to the light flux of the surrounding wave.

[0013] In some embodiments, the contrast of the microscopic image of the transparent sample is in is the luminous flux of the diffracted wave passing through the aperture, is the luminous flux of the surround wave passing through the aperture.

[0014] In some embodiments, the contrast C of the microscopic image of the sample is made to meet a predetermined condition by adjusting the ratio of the light flux of the diffracted wave passing through the aperture to the light flux of the surrounding wave.

[0015] In some embodiments, the contrast ratio 1≤C≤10 is adjusted by adjusting the ratio of the light flux of the diffracted wave passing through the aperture to the light flux of the surrounding wave.

[0016] In some embodiments, the objective lens group includes a first objective lens and a second objective lens along an optical axis from the sample plane to the imaging plane, the first objective lens being configured to converge light from a light source to the sample plane, and the second objective lens being configured to collect light from the sample plane.

[0017] In some embodiments, the first objective lens has a first numerical aperture and a first focal length, the second objective lens has a second numerical aperture and a second focal length different from the first numerical aperture, and when the first numerical aperture is smaller than the second numerical aperture, the aperture is arranged along the optical path after the second objective lens, wherein when the first numerical aperture is greater than the second numerical aperture, the aperture is arranged along the optical path before the first objective lens.

[0018] In some embodiments, the distance between the first objective lens and the sample surface is equal to a first focal length, and the distance between the second objective lens and the sample surface is equal to a second focal length.

[0019] In some embodiments, the objective lens with a larger numerical aperture among the first objective lens and the second objective lens in the objective lens group has a numerical aperture NA b and focal length f b , wherein the first objective lens and the second objective lens in the objective lens group having a smaller numerical aperture have a numerical aperture NA s and focal length f s The cross-sectional area of ​​the central light shielding portion of the aperture corresponding to the surround wave is S block =π·(NA s *f b ) 2 The cross-sectional area of ​​the annular light-passing portion of the aperture corresponding to the diffraction wave is S ring =π·(NA b *f b ) 2 -π·(NA s *f b ) 2 .

[0020] In some embodiments, the microscope system further includes a collimating lens group disposed between the light source and the objective lens group, wherein the collimating lens group is configured to collimate the light from the light source into a parallel beam to feed the parallel beam to the objective lens group.

[0021] In some embodiments, the microscope system further includes an imaging lens arranged between the objective lens group and the imaging surface, and an imaging component arranged at the imaging surface, wherein the objective lens group is configured to converge the light collected from the sample surface into parallel light, and the imaging lens is configured to converge the parallel light to the imaging component at the imaging surface.

[0022] In some embodiments, the imaging mirror includes a first positive lens and a first negative lens that are adjacently arranged.

[0023] In some embodiments, the central light shield of the aperture allows less than or equal to 30% of the surround waves to pass through the aperture.

[0024] In some embodiments, the transparent sample does not need to be chemically stained or biolabeled before being viewed.

[0025] In some embodiments, the microscope system further includes a control unit connected to the aperture, wherein the control unit is configured to adjust a ratio of the diffracted wave and the surrounding wave passing through the aperture.

[0026] In some embodiments, the sample surface includes a fluid channel for carrying the sample, and the fluid channel is connected in series with a fluid source, a pump, and a recovery vessel to achieve automatic sample loading.

[0027] According to other aspects of the present disclosure, a method for microscopic imaging for mobile phase particle analysis is also disclosed, which is used to detect transparent samples, and includes: a light source generates light to illuminate a sample surface, and the sample surface is configured to carry a sample; an objective lens group converges the light from the light source to the sample surface and collects the light from the sample surface, wherein the light from the sample surface includes an ambient wave transmitted from the transparent sample and a diffraction wave scattered by the transparent sample, the objective lens group includes a pair of objective lenses with different numerical apertures, the sample surface is located between the two objective lenses, and an aperture adjacent to the objective lens group adjusts the ratio of the diffraction wave and the ambient wave passing through the aperture, wherein the diffraction wave and the ambient wave interfere at an imaging surface to generate a microscopic image of the transparent sample.

[0028] According to other aspects of the present disclosure, a readable storage medium is also disclosed, comprising computer program instructions: when the computer program instructions are executed by at least one processor of an electronic device, the microscopic imaging method as described above is implemented.

[0029] According to other aspects of the present disclosure, an electronic device is also disclosed, 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 microscopic imaging method as described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The above and other objects and advantages of the present disclosure will be further described below in conjunction with specific embodiments and with reference to the accompanying drawings. In the accompanying drawings, identical or corresponding technical features or components will be represented by identical or corresponding reference numerals.

[0031] FIG1 is a schematic diagram of a microscopy system for mobile phase particle analysis according to some embodiments of the present disclosure;

[0032] FIG2 is a schematic diagram of a microscopy system for mobile phase particle analysis according to other embodiments of the present disclosure;

[0033] FIG3 is a schematic diagram of an aperture of a microscope system for mobile phase particle analysis according to some embodiments of the present disclosure;

[0034] FIG4 is a schematic diagram for calculating the light flux passing through an aperture according to some embodiments of the present disclosure;

[0035] 5A and 5B are schematic diagrams of application scenarios of a microscopic system for mobile phase particle analysis according to some embodiments of the present disclosure;

[0036] 6A and 6B are microscopic images at different contrasts C of a microscopic system for mobile phase particle analysis according to some embodiments of the present disclosure; FIG. 6C is a microscopic image of a conventional microscopic system;

[0037] FIG. 7 is a schematic diagram of an electronic device according to some embodiments of the present disclosure. DETAILED DESCRIPTION

[0038] 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 are not intended 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 intended only to enable a clear and consistent understanding of the present disclosure. In addition, descriptions of well-known structures, functions, and configurations may be omitted for clarity and brevity. Those skilled 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.

[0039] Biological research often requires the observation of thin and transparent biological samples, such as living cells, microorganisms, and thin tissue sections. However, because the samples are thin and transparent, they are difficult to see directly under an optical microscope. Therefore, when observing cells, a specific dye is often used to stain the sample to improve the visibility of biological tissues. However, both chemical staining and biological labeling belong to biochemical methods, and they always use exogenous methods to improve the imaging contrast of living cells, causing 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 sample pretreatment that requires the introduction of reagent reactions during the analysis process limits the detection speed and throughput; it also increases the complexity of the microscopy system.

[0040] Biological research therefore requires a method for directly observing cells without pretreatment. The microscopy system for mobile phase particle analysis according to some embodiments of the present disclosure addresses this problem. With the microscopy system of the present disclosure, living cells can be examined in their natural state without the need for prior killing, fixation, and staining. This allows for the observation and recording of the dynamics of ongoing biological processes.

[0041] The disclosed microscope system utilizes a single-light-source illumination method. By incorporating a specially configured aperture plate into the microscope system, the incident light is split into two components: the first component is a substantially undeflected (non-diffracting; zero-order) surround wave (S wave), which passes through and surrounds the sample without interacting with it; the second component is a deflected (diffracting) diffracted wave (D wave), which passes through the sample and interacts with its internal structure, diffracting in various directions. After entering the objective lens, the surround wave and diffracted wave are projected onto different spatial locations on the objective's rear focal plane. The S and D waves are amplitude-modulated by the specially configured aperture plate at the rear focal plane. The S and D waves are then converged onto the image plane by an imaging lens to synthesize a particle wave (P wave). The synthesized particle wave (P wave) is generated entirely by the interference of the separated surround and diffracted wavefronts.

[0042] By controlling the ratio of the component intensities (amplitude squared) of the different surround and diffracted waves on the aperture, namely the amount of light transmitted by the S-wave and D-wave, different phase enhancement or attenuation effects are achieved, thereby enhancing the refractive index differences between different organelles within the cell and between the inside and outside of the cell. The microscopic system disclosed herein can directly enhance the endogenous contrast of cells without chemical or biological pretreatment of the sample, revealing subtle refractive index differences inside and outside the cell or between different cells, such as between dead and living cells, thereby obtaining results for indicators such as cell viability, diameter, and concentration. Furthermore, it can be combined with a liquid flow system for automatic and rapid sample loading, enabling fully automated, high-throughput cell analysis.

[0043] FIG1 is a schematic diagram of a microscopy system for mobile phase particle analysis according to some embodiments of the present disclosure. As shown in FIG1 , microscopy system 100 includes a light source 101, a collimating lens assembly 102, an objective lens assembly 103, a sample plane 104, an aperture 105, an imaging lens 106, and an imaging plane 107. The sample to be observed is placed at the sample plane 104. Exemplary samples include, but are not limited to, cells, microorganisms, or thin tissue sections. In some embodiments of the present disclosure, the sample is transparent and does not require chemical staining or biological labeling prior to observation.

[0044] In the following description, the direction from light source 101 to sample plane 104 and then to imaging plane 107 is considered the optical axis of microscope system 100, i.e., the Z direction. The direction perpendicular to the Z direction is considered the X direction. The direction perpendicular to both the Z and X directions is considered the Y direction. The sample stage (not shown) of sample plane 104 lies in the plane defined by the X and Y directions.

[0045] The light source 101 is configured to generate light to illuminate a sample surface 104. The sample is placed at a sample stage on the sample surface 104. The light source 101 can have any configuration, such as a halogen lamp or an LED lamp. The collimating lens group 102 is configured to collimate the light from the light source 101 into parallel beams. The collimating lens group 102 can have any configuration, such as a combination of a plano-convex lens and a pair of convex lenses arranged oppositely. The light source 101 is arranged at the focal point of the collimating lens group 102. The objective lens group 103 is arranged after the collimating lens group 102 along the optical axis Z.

[0046] The objective lens group 103 is configured to converge the collimated light from the light source 101 onto the sample surface and collect the light from the sample surface, wherein the light from the sample surface 104 includes an undeviated surround wave (S wave), which passes through the sample and surrounds the sample but does not interact with the sample; and also includes a deviated diffraction wave (D wave), which passes through the sample and interacts with the internal structure of the sample and is diffracted in various directions.

[0047] The objective lens assembly 103 is provided with a first objective lens 1031 and a second objective lens 1032, arranged in sequence along the optical axis. The distance between the first objective lens and the second objective lens is the sum of their respective focal lengths. The sample plane 104 is disposed between the first objective lens 1031 and the second objective lens 1032. The distance between the sample plane 104 and the first objective lens 1031 is the focal length of the first objective lens 1031, while the focal length between the sample plane 104 and the second objective lens 1032 is the focal length of the second objective lens 1032. The first objective lens 1031 is configured to converge light from the light source 101 onto the sample plane 104, and the second objective lens 1032 is configured to collect light from the sample plane 104. As described in the previous paragraph, the light collected from the sample plane 104 by the second objective lens 1032 is divided into two parts. The surround wave (S wave) indicated by the letter S and the corresponding auxiliary dotted line is a light beam formed by the light focused by the first objective lens 1031 onto the sample surface passing through the sample. The surround wave passes through the sample and surrounds the sample but does not interact with the sample. The second part indicated by the letter D and the corresponding auxiliary dotted line is the diffraction wave (D wave) surrounding the surround wave. The diffraction wave passes through the sample and interacts with the internal structure of the sample, diffracting in all directions. Since the diffraction wave interacts with the internal structure of the sample, the diffraction wave is slightly delayed in phase compared to the surround wave. In addition, because the sample surface 104 is located at the focal plane of the second objective lens 1032, both the diffraction wave and the surround wave will be collimated by the second objective lens 1032 into parallel light beams and fed to the subsequent aperture 105.

[0048] The aperture 105 is positioned immediately adjacent to the objective lens assembly 103 along the optical axis Z and is configured to adjust the ratio of the surrounding wave to the diffracted wave passing therethrough. When the numerical aperture of the first objective lens 1031 is smaller than that of the second objective lens 1032, as in the configuration shown in FIG1 , the aperture 105 is positioned after the second objective lens 1032 along the optical axis. Conversely, because the light source, sample plane, and imaging plane each form a conjugate plane, and based on the principle of reversibility of the optical path, as shown in FIG2 , when the numerical aperture of the first objective lens 2031 is larger than that of the second objective lens 2032, the aperture 205 is positioned before the first objective lens 2031 along the optical axis. FIG2 schematically illustrates the situation in which the numerical aperture of the first objective lens 2031 is larger than that of the second objective lens 2032. That is, the aperture 105 is always positioned adjacent to the objective lens with the larger numerical aperture in the objective lens assembly 103, because the back focal plane of the objective lens with the larger numerical aperture contains relatively complete information about both the surround and diffracted waves. In the embodiment of FIG1 , the aperture 105 is positioned after the second objective lens 1032 along the optical axis. Typically, the light intensity of the central surround wave emitted from the second objective lens 1032 is higher than the light intensity of the surrounding diffracted waves emitted from the second objective lens 1032. As will be explained more clearly below in conjunction with FIG3 , the aperture 105 is configured to block a large portion of the central surround wave, allowing only a small portion of the central surround wave to pass through the aperture 105, while allowing nearly all of the surrounding diffracted waves to pass through the aperture 105. In conjunction with FIG1 , the aperture 105 blocks a large portion of the central surround wave, represented by S, while allowing nearly all of the surrounding diffracted waves, represented by D, to pass through. The apertures 105 and 205 shown in Figures 1 and 2 are schematic only. The specific geometric structures of apertures 105 and 205 are illustrated in Figure 3. Therefore, aperture 105, located at the rear focal plane of the second objective lens 1032, receives relatively complete information about both the ambient and diffracted waves. Its unique geometric structure modulates the ratio of the ambient and diffracted waves passing through it from the objective lens assembly, thereby adjusting the ratio between the high-frequency signal (i.e., the diffracted wave) representing the detailed features of the observed sample and the low-frequency signal (i.e., the ambient wave) representing the outline of the observed sample. By mixing the high-frequency and low-frequency signals in a specific ratio, high-definition rendering of transparent samples is achieved, enabling the observation of living cells in their natural state without the need for prior fixation and staining. Adjusting the ratio of the diffracted and ambient waves passing through can also adjust the final imaging effect, for example, choosing to emphasize internal details or external contours.

[0049] FIG2 is a schematic diagram of a microscopy system for mobile phase particle analysis according to other embodiments of the present disclosure. As shown in FIG2 , when the numerical aperture of the first objective lens 2031 is greater than the numerical aperture of the second objective lens 2032, the aperture 205 is positioned along the optical axis before the first objective lens 2031. In general, the aperture is always positioned immediately adjacent to the objective lens with the larger numerical aperture in the objective lens group. The aperture 205 is configured to block a large portion of the central surround wave, represented by S, allowing only a small portion of the surround wave to pass through the aperture 205, while allowing all of the surrounding light (represented by the letter D) intended to be diffracted by the sample to pass through and enter the second objective lens 2032 as a diffracted wave. Similar to the aperture 105 in FIG1 , the aperture 205 in FIG2 is configured to modulate the ratio of the diffracted wave to the surround wave through its unique geometric structure.

[0050] Figure 3 is a schematic diagram of an aperture 305 of a microscopy system for mobile phase particle analysis according to some embodiments of the present disclosure. For ease of illustration and description, aperture 305 is depicted together with the objective lens assembly 303, which includes a first objective lens 3031 and a second objective lens 3032, and the sample plane 304. To clearly illustrate the relationship between the different parts of aperture 305 and the numerical apertures and focal lengths of the two objective lenses, the aperture in Figure 3 is viewed in the Z direction, while the first and second objective lenses are viewed in the X direction. In the embodiment of Figure 3, aperture 305 is positioned immediately adjacent to objective lens assembly 303. Specifically, in Figure 3, along the optical path, first objective lens 3031 has a smaller numerical aperture, while second objective lens 3032 has a larger numerical aperture. Based on the above description, aperture 305 is positioned immediately adjacent to the objective lens 3032 in the objective lens assembly with the larger numerical aperture. Clearly, the technical solutions of the present disclosure also encompass situations where the first objective lens 3031 has a larger numerical aperture and the second objective lens 3032 has a smaller numerical aperture. According to the principle of optical path reversibility, the geometric arrangement of the diaphragm in FIG2 is also applicable to the description of the diaphragm structure and the explanation of the principle in FIG3 .

[0051] As shown in Figure 3, the aperture 305 includes a central light shielding portion 3051 and an annular light-transmitting portion 3052. In some embodiments of the present disclosure, the transmittance of the central light shielding portion 3051 is adjustable to adjust the ratio of diffracted waves to ambient waves. In some embodiments of the present disclosure, the central light shielding portion 3051 includes a translucent film that allows a small portion of the ambient light incident on the central light shielding portion to pass through, for example, less than or equal to 30% of the ambient light. In some embodiments of the present disclosure, the central light shielding portion 3051 is formed of an electrochromic material, and its transmittance can be adjusted by applying an external electric field. In some embodiments of the present disclosure, the central light shielding portion 3051 includes multiple through-holes dispersed therein or a single through-hole located in the center, so as to allow only a small portion of the ambient light to pass through. In some embodiments of the present disclosure, the through-holes include, but are not limited to, square, circular, rectangular, triangular, trapezoidal, etc.

[0052] Parallel light is received from the second objective lens 3032 located in front of the aperture through the aperture 305. This beam of parallel light includes a central surround wave S and surrounding diffracted waves D. The aperture 305 is configured to block a large portion of the central surround wave S, allowing only a small portion of the surround wave S to pass through, while allowing substantially all of the surrounding diffracted waves D to pass through. The area of ​​the central light-shielding portion 3051 corresponds to the circular projection area of ​​the surround wave S on the second objective lens 3032, and the area of ​​the annular light-passing portion 3052 corresponds to the annular projection area of ​​the diffracted wave D on the second objective lens 3032. Thus, the area of ​​the central light-shielding portion 3051 corresponds to the light transmitted from the sample without deviation, while the area of ​​the annular light-passing portion 3052 corresponds to the light diffracted from the sample with deviation. In some embodiments of the present disclosure, the central light-shielding portion 3051 of the aperture may also be connected to the outer portion of the aperture 305 via one or more connecting portions (not shown). The one or more connecting portions span the annular light-passing portion 3052.

[0053] According to the principle of trigonometric function, the area of ​​the central light shielding portion 3051 of the aperture 305 is: S s ≤π·(NA s *f b ) 2 Formula 1

[0054] where NA s is the numerical aperture of the objective lens with a small numerical aperture (i.e., the objective lens 3031 in FIG3 ), f b is the focal length of the objective lens with a large numerical aperture (ie, the objective lens 3032 in FIG3 ).

[0055] The area of ​​the annular light-passing portion 3052 of the aperture 305 is S D =π·(NA b *fb ) 2 -π·(NA s *f b ) 2 Formula 2

[0056] where NA b is the numerical aperture of the large numerical aperture objective lens (i.e., objective lens 3032 in FIG3 ), NA s is the numerical aperture of the objective lens with a small numerical aperture (i.e., the objective lens 3031 in FIG3 ), f b is the focal length of the objective lens with a large numerical aperture (ie, the objective lens 3032 in FIG3 ).

[0057] Note that, since the diaphragm is located adjacent to the objective lens with the larger numerical aperture, both the area of ​​the central light-shielding portion 3051 of the diaphragm and the area of ​​the annular light-passing portion 3052 of the diaphragm are equal to the focal length f of the objective lens 3032 with the larger numerical aperture. b It is related to the focal length f of the objective lens with a small numerical aperture. s Not relevant.

[0058] In some embodiments of the present disclosure, when the numerical aperture of the first objective lens along the optical path is greater than the numerical aperture of the second objective lens, the aperture 305 will be arranged before the first objective lens along the optical path, and Formulas 1 and 2 are still applicable to the calculation of the areas of the central light-shielding portion and the annular light-passing portion of the aperture 305.

[0059] Referring again to FIG. 1 , after passing through aperture 105 , the diffracted wave and the surround wave are converged onto imaging surface 107 by imaging lens 106 . The diffracted wave is diffracted by the sample (e.g., a cell) and is subjected to a certain phase delay by the substances within the sample. In contrast, the surround wave, although also passing through the interior of the sample, has little interaction with the substances within the sample, and therefore has little phase delay. Thus, the diffracted wave and the surround wave constitute coherent light with a phase difference from each other, causing the diffracted wave and the surround wave to interfere at imaging surface 107 to generate a microscopic image of the sample with a higher light-dark contrast. This allows the endogenous contrast of the cell to be directly improved without chemical or biological pretreatment of the sample, thereby obtaining results of indicators such as cell viability, diameter, and concentration.

[0060] Furthermore, since the surround wave usually has a relatively strong brightness, if the surround wave and the diffracted wave are directly interfered, the stronger surround wave signal will drown out the weaker diffracted wave signal, making it impossible to reflect the relatively weak changes in the amplitude and direction of the diffracted wave. Therefore, the present disclosure reduces the light flux of the surround wave by using an aperture with a central light-shielding portion, and uses a combination of a specially configured objective lens group and an aperture to increase the ratio of the light flux of the diffracted wave to the light flux of the surround wave, thereby increasing the sensitivity of the microscope system to relatively weak refractive index changes in transparent samples. By blocking most of the surround waves and allowing almost all of the diffracted waves to pass through, the diffracted wave Φ can be adjusted. D and surround wave Φ S The proportional relationship between the light flux and the contrast C needs to satisfy the following formula 3, so that different organelles in the cell and the relatively weak refractive index differences inside and outside the cell can be observed, making the edges of transparent samples clearer and improving the observation effect:

[0061] Among them Ф D is the luminous flux of the diffracted wave passing through the aperture; Ф S As shown in Formula 3, preferably, the contrast C is between 1 and 10.

[0062] FIG4 is a schematic diagram for calculating a luminous flux Φ involving an aperture 400 according to some embodiments of the present disclosure.

[0063] Referring to Formula 4, the unit luminous flux dφ is equal to the unit solid angle dω multiplied by the luminous intensity per unit area, L·ds·cosU. Integrating Formula 4 over the solid angle of the aperture yields Formula 5, where L is the illumination intensity of the light source and sinU is the numerical aperture of the objective lens with the larger numerical aperture adjacent to the aperture. dφ = L·ds·cosU·dω = L·ds·cosU·sinUdU·dθ Formula 4 φ = L·ds·∫∫cosU·sinU dU·dθ = π·L·ds·sin2U Formula 5

[0064] Substituting the area of ​​the central light-shielding portion of the aperture into Formula 5 and multiplying it by the luminous flux attenuation coefficient will give Φ S , that is, the luminous flux of the surrounding wave passing through the center of the aperture; substituting the area of ​​the annular light-passing portion of the aperture into formula 5 will give Ф D , that is, the luminous flux of the surrounding diffracted waves passing through the aperture. s and the luminous flux of the diffracted wave Φ D Substituting into Formula 3, we obtain the contrast expression C of the final image. Preferably, the contrast C is not greater than 10 and not less than 1. Preferably, the contrast C is 1.

[0065] Returning to FIG. 1 , the microscope system 100 further includes an imaging lens 106 disposed between the objective lens group 103 and the imaging plane 107. The second objective lens 1032 in the objective lens group 103 is configured to converge the light collected from the sample surface 104 into parallel light, which includes a central surround wave and diffracted waves surrounding the surround wave. The imaging lens 106 is configured to converge the parallel light onto an imaging component at the imaging plane 107. In other words, both the surround wave and the diffracted wave, which are coherent light, are converged onto the imaging component at the focal plane of the imaging lens. In some embodiments of the present disclosure, the imaging lens 106 includes a first positive lens and a second negative lens disposed adjacent to each other. A curved surface of the first positive lens is aligned with a curved surface of the second negative lens. The microscope system 100 further includes an imaging plane 107 disposed at the exit light path of the imaging lens 106, the imaging plane 107 including an imaging component, wherein the diffracted wave and the surround wave are interfered and imaged at the imaging component. The imaging component 107 may include a charge coupled device (CCD) or a complementary metal oxide semiconductor device (CMOS). In some embodiments, the optical detection device may further include a memory, a processor, etc. communicatively connected to the imaging component to automatically store and process the image information formed by the imaging component, thereby further facilitating detection.

[0066] Figures 5A and 5B are schematic diagrams illustrating application scenarios of a microscopy system for mobile phase particle analysis according to some embodiments of the present disclosure. In the embodiments of Figures 5A and 5B, a sample is placed in a channel located on a sample surface 504. In some embodiments of the present disclosure, the channel is a fluidic channel. The channel, combined with a fluidics system comprising a fluid source 509, a pump 511, and a recovery vessel 510, enables automated and rapid sample loading, enabling fully automated, high-throughput cell analysis. This allows the microscopy system to observe real-time microscopic images of transparent samples flowing through the fluidic channel. In the embodiment of Figure 5A, the numerical aperture of the first objective lens 5031 is larger than that of the second objective lens 5032. Therefore, the diaphragm 505 is positioned along the optical path before the first objective lens 5031, which has a larger numerical aperture. In the embodiment of Figure 5B, the numerical aperture of the first objective lens 5031 is smaller than that of the second objective lens 5032. Therefore, the diaphragm 505 is positioned along the optical path after the second objective lens 5032, which has a larger numerical aperture. In the embodiments of Figures 5A and 5B , the structure of the aperture 505 is designed to allow substantially all of the diffracted waves to pass through the aperture, while allowing only a small portion of the ambient waves to pass through the aperture. The diffracted and ambient waves are collected by the second objective lens 5032 and converged by the imaging lens 506 onto an imaging assembly at the imaging plane 507 to form a microscopic image of the sample. A processor 508 is connected to the optical components before and after the sample plane 504 to obtain imaging information at the imaging plane 507 and / or control the aperture 505 and / or the light source 501. Optionally, the ratio of the diffracted and ambient waves passing through the aperture 505 can be automatically or semi-automatically adjusted based on the imaging information, for example by adjusting the transmittance of the electrochromic material in the central light shielding portion of the aperture 505, or by adjusting the size, number, and / or shape of the through-holes in the central light shielding portion (not shown) of the aperture 505. Optionally, the position of the aperture 505 in three-dimensional space is automatically or semi-automatically adjusted according to the imaging information to substantially align the annular light-passing portion and the central light-shielding portion with the diffracted wave and the surrounding wave, respectively.

[0067] Figures 6A and 6B are microscopic images of a microscopic system for mobile phase particle analysis according to some embodiments of the present disclosure at different contrasts C. In contrast, Figure 6C is a microscopic image of a conventional microscopic system. Figures 6A, 6B, and 6C are imaged for the same object field of view. According to Formula 3, when the surrounding wave is attenuated to a certain extent, for example, when the contrast C=1 is reached, a microscopic image as shown in Figure 6A is obtained, in which the transparent sample presents a significantly brighter outline relative to the background; when the surrounding wave is further attenuated, for example, when the contrast C=10 is reached, a microscopic image as shown in Figure 6B is obtained, in which the transparent sample presents a significantly darker outline relative to the background.

[0068] FIG7 is a schematic diagram of the structure of an electronic device provided according to some embodiments of the present disclosure. Referring to FIG7 , the electronic device 700 provided in this embodiment includes: a memory 701 and a processor 702. The memory 701 may be an independent physical unit and may be connected to the processor 702 via a bus 703. The processor 702 may include, but is not limited to, a central processing unit (CPU), a hardware microprocessor, a hardware processor, a multi-core processor, a single-core processor, a microcontroller, an application-specific integrated circuit (ASIC), a digital signal processor (DSP), or other similar processing devices, capable of executing any type of instructions, algorithms, or software for the operations and functions of microscopic imaging according to the embodiments described in this disclosure. The processor 702 may 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 702 may include, for example, an integrated circuit (IC), part 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. Processor 702 may be a CPU, a network processor (NP), or a combination of a CPU and an NP. Processor 702 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.

[0069] The memory 701 can be integrated with the processor 702 and implemented through hardware, etc. The memory 701 is used to store program instructions, and the processor 702 calls the program instructions to perform the operations of any of the above method embodiments. The memory 701 may include a volatile memory (volatile memory), such as a 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 (HDD) or a solid-state drive (SSD); the memory may also include a combination of the above types of memory. The memory 701 can be used to store any type of instructions, software or algorithm, including instructions for controlling the general functions and operations of the electronic device.

[0070] Optionally, when part or all of the methods of the above embodiments are implemented by software, the electronic device 700 may also include only the processor 702. The memory 701 for storing programs is located outside the electronic device 700, and the processor 702 is connected to the memory via circuits / wires to read and execute the programs stored in the memory.

[0071] The present disclosure may be implemented as any combination of an apparatus, a system, an integrated circuit, and a computer program or program product on a non-transitory computer-readable medium.

[0072] 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 and are therefore not described again. 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.

[0073] 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 implementation 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.

[0074] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not limiting. Therefore, other examples of the exemplary embodiments may have different values.

[0075] The terms "front," "back," "top," "bottom," "above," "below," and the like, if any, in the specification and claims, are used for descriptive purposes and are not necessarily intended to describe invariant relative positions. It is understood that the terms 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.

[0076] 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 is not necessarily to be construed as preferred or advantageous over other implementations. Furthermore, the present disclosure is not to be bound by any expressed or implied theory presented in the preceding technical field, background, summary, or detailed description.

[0077] As used herein, the word "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 word "substantially" also allows for deviations from a perfect or ideal condition due to parasitic effects, noise, and other practical considerations that may be present in actual implementations.

[0078] The description may indicate elements or nodes or features that are "connected" or "coupled" together. As used herein, unless expressly stated otherwise, "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 expressly stated otherwise, "coupled" means that one element / node / feature can be mechanically, electrically, logically, or otherwise connected to another element / node / feature, directly or indirectly, to allow interaction, even though the two features may not be directly connected. In other words, "coupled" is intended to encompass both direct and indirect connections of elements or other features, including connections utilizing one or more intermediate elements.

[0079] 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.

[0080] Those skilled in the art will appreciate that the boundaries between the above-mentioned operations are merely illustrative. Multiple operations can be combined into a single operation, a single operation can be distributed among additional operations, and operations can be performed at least partially overlapping in time. Moreover, alternative embodiments can include multiple instances of specific operations, and the order of operations can be changed in various other embodiments. However, other modifications, variations, and replacements are also possible. Therefore, this specification and the accompanying drawings should be considered illustrative, not restrictive.

[0081] Although some specific embodiments of the present disclosure have been described in detail by way of examples, 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 microscopic system for mobile phase particle analysis, the microscopic system being configured to detect a transparent sample, the microscopic system comprising: a light source configured to generate light to illuminate a sample surface configured to carry a sample; an objective lens group, the objective lens group comprising two objective lenses with different numerical apertures, the sample plane being located between the two objective lenses, the objective lens group being configured to converge light from a light source to the sample plane and to collect light from the sample plane, wherein the light from the sample plane comprises a surround wave that is not diffracted by the transparent sample and a diffracted wave that is diffracted by the transparent sample, and an aperture, the aperture being disposed adjacent to the objective lens group, the aperture being configured to adjust a ratio of the diffracted wave and the surrounding wave passing therethrough, The diffracted wave and the surrounding wave passing through the aperture interfere with each other on an imaging surface to generate a microscopic image of the transparent sample.

2. The microscope system according to claim 1, wherein the aperture comprises an annular light-passing portion and a central light-shielding portion, the central light-shielding portion corresponds to a surround wave that is not diffracted by the transparent sample, and the annular light-passing portion corresponds to a diffracted wave that is diffracted by the transparent sample, wherein the central light-shielding portion allows the surround wave to pass with attenuation, and the annular light-passing portion allows the diffracted wave to pass with essentially no attenuation.

3. The microscope system according to claim 1, wherein the contrast C of the microscope image of the transparent sample at the imaging plane is related to the ratio of the light flux of the diffracted wave passing through the aperture to the light flux of the surrounding wave.

4. The microscopic system according to claim 3, wherein the contrast of the microscopic image of the transparent sample at the imaging surface is in is the luminous flux of the diffracted wave passing through the aperture, is the luminous flux of the surround wave passing through the aperture. 5 . The microscope system according to claim 4 , wherein the contrast C of the microscope image of the sample satisfies a predetermined condition by adjusting the ratio of the light flux of the diffracted wave passing through the aperture to the light flux of the surrounding wave.

6. The microscope system according to claim 5, wherein the contrast ratio 1≤C≤10 is adjusted by adjusting the ratio of the light flux of the diffracted wave passing through the aperture to the light flux of the surrounding wave.

7. The microscope system according to claim 1, wherein the objective lens group includes a first objective lens and a second objective lens along an optical axis from the sample plane to the imaging plane, the first objective lens being configured to converge light from a light source to the sample plane, and the second objective lens being configured to collect light from the sample plane.

8. A microscope system according to claim 7, wherein the first objective lens has a first numerical aperture and a first focal length, the second objective lens has a second numerical aperture and a second focal length different from the first numerical aperture, when the first numerical aperture is smaller than the second numerical aperture, the aperture is arranged along the optical path after the second objective lens, wherein when the first numerical aperture is larger than the second numerical aperture, the aperture is arranged along the optical path before the first objective lens.

9. The microscope system according to claim 7, wherein the distance between the first objective lens and the sample surface is equal to a first focal length, and the distance between the second objective lens and the sample surface is equal to a second focal length.

10. The microscope system according to claim 7, wherein the objective lens with a larger numerical aperture among the first objective lens and the second objective lens in the objective lens group has a numerical aperture NA b and focal length f b , wherein the objective lens with a smaller numerical aperture among the first objective lens and the second objective lens in the objective lens group has a numerical aperture NA s and focal length f s , where the cross-sectional area of ​​the central light shielding portion of the aperture corresponding to the surround wave is S s ≤π·(NA s *f b ) 2 The cross-sectional area of ​​the annular light-passing portion of the aperture corresponding to the diffraction wave is S D =π·(NA b *f b ) 2 -π·(NA s *f b ) 2 .

11. The microscope system according to claim 1, wherein the microscope system further comprises a collimating lens group disposed between the light source and the objective lens group, the collimating lens group being configured to collimate the light from the light source into a parallel light beam to be fed to the objective lens group.

12. The microscope system according to claim 1, wherein the microscope system further comprises an imaging lens arranged between the objective lens group and the imaging plane, and an imaging component arranged at the imaging plane, wherein the objective lens group is configured to converge the light collected from the sample plane into parallel light, and the imaging lens is configured to converge the parallel light to the imaging component at the imaging plane.

13. The microscope system according to claim 12, wherein the imaging lens comprises a first positive lens and a first negative lens which are adjacently arranged.

14. The microscope system of claim 1, wherein a central light shielding portion of the aperture allows less than or equal to 30% of the surround waves to pass through the aperture.

15. The microscopy system of claim 1, wherein the transparent sample does not need to be chemically stained or biologically labeled before being observed. 16 . The microscope system according to claim 1 , further comprising a control unit connected to the aperture, the control unit being configured to adjust a ratio of the diffracted wave and the surrounding wave passing through the aperture.

17. The microscopy system according to claim 1, wherein the sample surface comprises a fluid channel for carrying the sample, and the fluid channel is connected in series with a fluid source, a pump, and a recovery vessel to achieve automatic sample loading.

18. A method for microscopic imaging of mobile phase particle analysis, the method being applied to detect transparent samples, the method comprising: a light source generating light to illuminate a sample face, the sample face being configured to carry a sample; an objective lens group that converges light from a light source onto the sample surface and collects light from the sample surface, wherein the light from the sample surface includes a surrounding wave that is not diffracted by the transparent sample and a diffracted wave that is diffracted by the transparent sample, the objective lens group includes a pair of objective lenses with different numerical apertures, and the sample surface is located between the two objective lenses; and The aperture next to the objective lens group adjusts the ratio of the diffracted wave and the surrounding wave passing through the aperture. The diffracted wave and the surrounding wave interfere at an imaging plane to generate a microscopic image of the transparent sample.

19. 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 microscopic imaging method of claim 18 is implemented.

20. 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 microscopic imaging method of claim 18.

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