Observation method and apparatus using optical device

Abstract

An embodiment of the present disclosure provides an observation apparatus comprising: a first polarization unit for polarizing light emitted from a light source unit; a light splitting unit for splitting the light having passed through the first polarization unit into first reflection light and first transmission light; an objective lens for collecting light including information about an object on the basis of the first reflection light; an image sensor for detecting input light on the basis of the light collected by the objective lens; and a second polarization unit for blocking the first transmission light so that the first transmission light is not detected by the image sensor.

Description

Observation method and apparatus using an optical device

[0001] The present disclosure relates to an observation method and apparatus using an optical device. More specifically, the present disclosure relates to a method and apparatus for observing an object using a polarizing element that polarizes light.

[0002] Optical devices, such as microscopes, are used as important tools in various disciplines including biology, chemistry, and materials science because they allow for the magnification and observation of minute objects. In particular, optical systems equipped with in-line illumination allow light to be incident perpendicularly on the surface, reducing scattering and facilitating observation of objects without distortion caused by reflection. Furthermore, since the illumination and observation paths align, a separate mechanism for adjusting the illumination angle is not required, thereby simplifying the design of the optical device.

[0003] However, in-line illumination is primarily used for observing macroscopic objects such as metals and other objects, and in-line illumination systems are not utilized in microscopes for biological applications, such as the detailed structures of cells, tissues, proteins, and other biomaterials. This is because transmitting illumination is required to observe transparent objects like cells, necessitating appropriate control of light intensity; however, the light emitted by the light source from in-line illumination is too strong to see the light emitted by microscopic and transparent cells.

[0004] The present disclosure aims to solve the problem that in-line illumination cannot be used in an observation device for observing such cells, etc.

[0005]

[0006] [Prior Art Literature]

[0007] [Patent Literature]

[0008] Registered Patent Publication No. 10-0813915 (Date of publication: March 18, 2008)

[0009] One embodiment of the present disclosure is to provide an observation method and apparatus using an optical device.

[0010] One embodiment of the present disclosure may provide an observation device comprising: a first polarizing unit that polarizes light emitted from a light source unit; a light splitting unit that splits light passing through the first polarizing unit into a first reflected light and a first transmitted light; an objective lens that collects light containing information of an object based on the first reflected light; an image sensor that detects input light based on the light collected by the objective lens; and a second polarizing unit that blocks the first transmitted light so that the first transmitted light is not detected by the image sensor.

[0011] In one embodiment, the light detected by the image sensor may include at least one of the transmitted light in which the first reflected light is scattered above the object and the scattered light passes through the object, or the scattered light in which the first reflected light passes through the object and is scattered above the object; or the scattered light in which the first reflected light is scattered from the object.

[0012] In one embodiment, the light scattered from above the object by the first reflected light may include light scattered by at least one of the top plate of the container or the surface of the media.

[0013] In one embodiment, the light collected by the objective lens includes light reflected by at least one of the target, the container, or the surface of the media, and the reflected light may be blocked by the second polarizing unit if it is light that has not undergone a scattering process.

[0014] In one embodiment, the observation device may further include the light source unit provided inside the observation device.

[0015] In one embodiment, the polarization angle of the first polarizing part may be 90 degrees to the polarization angle of the second polarizing part.

[0016] In one embodiment, the second polarizing unit polarizes the light collected by the objective lens, and the image sensor can acquire the light polarized by the second polarizing unit.

[0017] In one embodiment, the object may be contained in a single-layer container or a multi-layer container.

[0018] In one embodiment, the multilayer container is CellSTACK™, HYPERStack™, Cell Factory™, Nunc™ EasyFill™ Cell Factory, Falcon ® Cell Culture Multi-Layer Flask, STACKMAX™, iCELLis™ Nano and iCELLis™ 500+, TripleFlask™ System, or HYPERFlask ® It may include.

[0019] In one embodiment, the object is contained in a container, and the container may be composed of a bottom plate and a top plate, or may be composed only of a bottom plate.

[0020] In one embodiment, the object is contained in a container, and the container may include an optically transparent material.

[0021] In one embodiment, the observation device may be a live cell imaging system.

[0022] One embodiment of the present disclosure may provide an observation method comprising: a step of emitting light by a light source unit; a step of polarizing the light emitted by the light source unit by a first polarizing unit; a step of splitting the light that has passed through the first polarizing unit into a first reflected light and a first transmitted light by a light splitting unit; a step of blocking the first transmitted light by a second polarizing unit so that the first transmitted light is not detected by an image sensor; a step of collecting light containing information of an object by an objective lens based on the first reflected light; and a step of detecting light input to the image sensor based on the light collected by the objective lens.

[0023] In one embodiment, the light detected by the image sensor may include at least one of the transmitted light in which the first reflected light is scattered above the object and the scattered light passes through the object, or the scattered light in which the first reflected light passes through the object and is scattered above the object; or the scattered light in which the first reflected light is scattered from the object.

[0024] In one embodiment, the light scattered from above the object by the first reflected light may include light scattered by at least one of the top plate of the container or the surface of the media.

[0025] In one embodiment, the light collected by the objective lens includes light reflected by at least one of the target, the container, or the surface of the media, and if the reflected light is light that has not undergone a scattering process, it may be blocked by the second polarizing part.

[0026] One embodiment of the present disclosure is an observation method comprising: a step of scattering at least a portion of light emitted from a light source unit onto an object or above the object; and a step of detecting at least one of scattered light scattered from the object, transmitted light that has passed through the object after being scattered above the object, or scattered light that has passed through the object and then scattered above the object.

[0027] One embodiment of the present disclosure includes a program stored on a recording medium to execute a method according to one embodiment of the present disclosure on a computer.

[0028] One embodiment of the present disclosure includes a computer-readable recording medium having a program for executing a method according to one embodiment of the present disclosure on a computer.

[0029] One embodiment of the present disclosure includes a computer-readable recording medium that records a database used in one embodiment of the present disclosure.

[0030]

[0031] According to one embodiment of the present disclosure, an in-line illumination method can be used to observe microscopic objects such as cells.

[0032] In addition, according to one embodiment, a clear observation image can be obtained even when the bottom surface of a container containing an object of observation is opaque or uneven.

[0033] In addition, according to one embodiment, even when observing a sample contained in a cell culture medium or a container that does not have a ceiling plate, such as a lidless petri dish containing the object of observation, a clear observation image can be obtained without separately placing a reflective member above the container or the object of observation.

[0034]

[0035] FIG. 1 is a drawing showing an optical system using an in-line illumination method according to one embodiment of the present disclosure.

[0036] FIG. 2a is a diagram showing the process of light emitted from a light source unit according to one embodiment of the present disclosure reaching an object.

[0037] FIG. 2b is a diagram showing the process of light passing through an object of observation according to one embodiment of the present disclosure reaching an image acquisition unit.

[0038] FIG. 3 is a drawing showing an image obtained by observing a macroscopic object using an observation device according to one embodiment of the present disclosure.

[0039] FIGS. 4a and 4b are drawings showing images obtained by observing a microscopic object using an observation device according to one embodiment of the present disclosure.

[0040] FIG. 5a is a diagram illustrating a bright-field imaging method for the process in which light emitted from a light source unit reaches an object according to one embodiment of the present disclosure.

[0041] FIG. 5b is a diagram illustrating a bright-field imaging method for the process in which light scattered or reflected from an object reaches an image acquisition unit according to one embodiment of the present disclosure.

[0042] FIG. 6a is a diagram illustrating a dark-field imaging method for the process in which light emitted from a light source unit reaches an object according to one embodiment of the present disclosure.

[0043] FIG. 6b is a diagram illustrating a dark-field imaging method for the process in which light transmitted or reflected from an object reaches an image acquisition unit according to one embodiment of the present disclosure.

[0044] FIG. 7 is a drawing illustrating a bright-field imaging method for acquiring light scattered by an object and incident on an objective lens according to one embodiment of the present disclosure.

[0045]

[0046] To clarify the technical concept of the present disclosure, embodiments of the present disclosure will be described in detail with reference to the attached drawings. In describing the present disclosure, detailed descriptions of related known functions or components will be omitted if it is determined that such detailed descriptions would unnecessarily obscure the essence of the present disclosure. Components having substantially the same functional configuration among the drawings have been assigned the same reference numerals and symbols as much as possible, even if they are shown in different drawings. For convenience of explanation, devices and methods will be described together where necessary. Each operation of the present disclosure does not necessarily have to be performed in the order described and may be performed in parallel, selectively, or individually.

[0047] The terms used in the embodiments of this disclosure have been selected to be as widely used and general as possible, taking into account the functions of this disclosure; however, these terms may vary depending on the intent of those skilled in the art, case law, the emergence of new technologies, etc. Additionally, in specific cases, terms have been selected at the applicant's discretion, and in such cases, their meanings will be described in detail in the description of the relevant embodiments. Therefore, terms used in this specification should be defined not merely by their names, but based on their meanings and the overall content of this disclosure.

[0048] Throughout this disclosure, singular expressions may include plural expressions unless the context clearly indicates otherwise. Terms such as “comprising” or “having” are intended to specify the presence of features, numbers, steps, actions, components, parts, or combinations thereof, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof. That is, throughout this disclosure, when a part is described as “comprising” a certain component, it means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0049] Expressions such as "at least one" modify the entire list of components and do not modify the components of the list individually. For example, "at least one of A, B, and C" and "at least one of A, B, or C" refer to only A, only B, only C, both A and B, both B and C, both A and C, all of A, B, and C, or any combination thereof.

[0050] Additionally, terms such as “...part,” “...module,” etc., as described in this disclosure refer to a unit that processes at least one function or operation, and may be implemented in hardware or software, or a combination of hardware and software.

[0051] Throughout the entire disclosure, when a part is described as being “connected” to another part, this includes not only cases where they are “directly connected” but also cases where they are “electrically connected” with other elements interposed between them. Furthermore, when a part is described as “comprising” a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0052] As used throughout this disclosure, the expression “configured to” may be replaced, depending on the context, with, for example, “suitable for,” “having the capacity to,” “designed to,” “adapted to,” “made to,” or “capable of.” The term “configured to” may not necessarily mean only “specifically designed to” in hardware. Instead, in some situations, the expression “system configured to” may mean that the system is “capable of” together with other devices or components. For example, the phrase “a processor configured (or set) to perform A, B, and C” may mean a dedicated processor for performing said operations (e.g., an embedded processor), or a generic-purpose processor (e.g., a CPU or an application processor) capable of performing said operations by executing one or more software programs stored in memory.

[0053] The term “about” as used in this disclosure means within 10%, preferably within 5%, and more preferably within 1% of a given number or range.

[0054] As used herein, the term “sample” is used interchangeably with “sample” and may include anything known in the art that is observable by optical or light microscopes. For example, “sample” may include, but is not limited to, cells or tissues (e.g., cells or tissues of animals, plants, fungi, protists, bacteria, etc.—including both general and pathological cells / tissues), cell components (e.g., nucleus, cytoplasm, chloroplasts, mitochondria, etc.), microorganisms (e.g., bacteria, protists, some algae and fungi, etc.), organoids, etc.

[0055] As used throughout this disclosure, the term "in-line illumination" refers to an illumination method in which a light ray from a light source passes through an objective lens, passes through an object of observation, and then passes through the same objective lens again.

[0056] FIG. 1 is a drawing showing an observation device using an in-line illumination method according to one embodiment of the present disclosure.

[0057] Referring to FIG. 1, an observation device (100) using in-line lighting may include a light source (110), a light splitting unit (120) (e.g., a beam splitter), an image sensor (130), an objective lens (150), a tube lens (160), a condensing lens, etc. The observation device (100) using in-line lighting may include an optical device (100) that integrates lighting into an optical assembly including an image sensor (130), an objective lens (150), a tube lens (160), etc., using the light source (110) and the light splitting unit (120). That is, the observation device (100) using in-line lighting may not require separate external lighting by emitting the light necessary for imaging internally. However, a method using external lighting may also be considered.

[0058] In one embodiment, when a beam splitter is used as a light splitter (120) by positioning it at a specific angle, some of the light emitted from the light source (110) may be transmitted and the rest may be reflected. For example, a 50:50 beam splitter may be positioned at a 45-degree angle. In this case, half of the light emitted from the light source (110) may be transmitted and the other half may be reflected. However, this is merely an example, and the transmission:reflection ratio of the light splitter (120) does not necessarily have to be 50:50, but may be selected as an appropriate value between, for example, 0:100 and 100:0. Likewise, the positioning angle of the light splitter (120) does not necessarily have to be 45 degrees, but may be selected as an appropriate angle to transmit some of the light and reflect the rest.

[0059] In one embodiment, in an observation device (100) using in-line illumination, the light splitter (120) may be located between the objective lens and the tube lens. Additionally, the light splitter (120) may be located between the objective lens and the image sensor. Thus, the observation device (100) using in-line illumination can be made smaller than observation devices using other types of illumination.

[0060] However, despite the advantages of such in-line lighting, it may be difficult to use an observation device (100) that uses in-line lighting when observing microscopic objects such as cells. This is because the light from the light source (110) emitted from the in-line lighting is too strong compared to the light passing through a microscopic and transparent object such as a cell. Throughout the present disclosure, light passing through an object may include light that reaches the object, light that passes through the object, light scattered from the object, light reflected from the object, etc.

[0061] That is, when observing a transparent object such as a cell, the shape of the object can be observed using light that has passed through the object. However, due to the characteristics of the observation device (100) using in-line lighting in which a light splitting unit (120) is placed in the imaging train path, there is a problem in that the image sensor (130) cannot properly detect the light that has passed through the transparent and microscopic object, because there is strong light emitted from the light source unit (110) that passes through the light splitting unit (120) and enters the image sensor (130) directly, in addition to the light that has passed through the transparent object.

[0062] Below, an observation device (200) that solves these problems is described.

[0063] FIG. 2a is a diagram showing the process of light emitted from a light source unit according to one embodiment of the present disclosure reaching an object.

[0064] Referring to FIG. 2a, the observation device (200) may include a light source unit (210), a light splitting unit (220), an image sensor (230), a first polarizing unit (270), a second polarizing unit (280), an objective lens, a tube lens, a condensing lens, etc. However, not all components shown in FIG. 2a are essential components of the observation device (200). The observation device (200) may be implemented with more components than those shown in FIG. 2a, or with fewer components than those shown in FIG. 2a.

[0065] In one embodiment, the observation device (200) according to one embodiment of the present invention may be equipped with an inline lighting method. The light splitting unit (220) may be located between the objective lens and the tube lens. Additionally, the light splitting unit (220) may be located between the objective lens and the image sensor (230). Additionally, the condensing lens may be located between the light source unit (210) and the first polarizing unit (270). Additionally, the tube lens may be located between the second polarizing unit (280) and the image sensor (230).

[0066] In one embodiment, the observation device (200) is a device capable of observing the “object of observation” or “object of observation” as defined herein, and may include, but is not limited to, a microscope, a live cell imaging device, etc. In another embodiment, the observation device may also be used for the inspection of parts or components used in industrial fields other than bio-uses, such as semiconductor or display equipment and related industries, in addition to the “object of observation” as defined herein.

[0067] In one embodiment, the light source unit (210) may emit light. The light source unit (210) may be located inside the observation device (200) or outside, as shown in FIG. 2a. In one embodiment, when the light source unit (210) is located outside the observation device (200), the observation device (200) and the light source unit (210) may be located on the same plane relative to the object being observed. For example, when the observation device (200) is located below the object being observed, the light source unit (210) may also be located below the object being observed.

[0068] Throughout the entire disclosure, the term “object of observation” or “object of observation” may include the term “sample” or “sample” as defined herein, which is a target object to be observed through an observation device, such as cells, tissues, microorganisms, microparticles, plasma, etc. Here, microparticles include red blood cells, platelets, white blood cells, circulating tumor cells, stem cells, inactivated stored erythrocytes, T-cells derived from autologous T-cell expansion, organic microparticles, inorganic microparticles, organometallic microparticles, metallic microparticles, aerosol particles, bacteria, yeast, fungi, algae, viruses, microinvertebrates or their eggs, pollen, cell or tissue fragments, cell masses, cell debris (e.g., cell debris associated with DNA or RNA purification), bioreactor-produced cells or granules, proteins, protein aggregates, prions, vesicles, liposomes, precipitates (e.g., precipitates from blood or blood fractions, industrial process precipitates, wastewater precipitates, etc.), granules or cells from fermented foods (e.g., granules or cells from fermented beverages), macromolecules, macromolecular aggregates, DNA, organelles, spores, bubbles, droplets, and exosomes, etc. It can be included.

[0069] In this document, "object of observation" and "object of observation" are used interchangeably and are concepts included within "object," and are sometimes simply referred to as "object." That is, while "object" refers to the "object of observation" or "object of observation" as defined herein, it should be noted that, depending on the context, it may also refer to objects to which light is reflected, transmitted, or scattered, such as vessels, vessel lids, dust, etc.

[0070] In one embodiment, light emitted from a light source unit (210) may be polarized by a first polarizing unit (270) that polarizes the light in a first direction. The first polarizing unit (270) may include a polarizing plate, a polarizing filter, an analyzer, etc., that polarizes the light emitted from the light source unit (210) in a first direction. For example, the light emitted from the light source unit (210) may include light vibrating in all directions. When such light encounters the first polarizing unit (270), only light vibrating in the first direction passes through the first polarizing unit (270), and all others are blocked, so that the light passing through the first polarizing unit (270) may include only light vibrating in the first direction.

[0071] In one embodiment, light that has passed through the first polarizing unit (270) (light of ① in FIG. 2a) may be separated or divided into a first reflected light (light of ② in FIG. 2a) and a first transmitted light (light of ③ in FIG. 2a) by a light splitting unit (220). At this time, the first reflected light (light of ② in FIG. 2a) and the first transmitted light (light of ③ in FIG. 2a) are divided from the light that has passed through the first polarizing unit (270) (light of ① in FIG. 2a), and may have the same period, frequency, and wavelength as the light that has passed through the first polarizing unit (270).

[0072] In one embodiment, the first transmitted light (the light of ③ in FIG. 2a) may travel toward the image sensor (230). However, the first transmitted light (the light of ③ in FIG. 2a) may include light that must not be incident on the image sensor (230) in order to clearly observe the object being observed. Accordingly, a second polarizing unit (280) may be placed in the path of the first transmitted light (the light of ③ in FIG. 2a) that has passed through the light splitting unit (220) toward the image sensor (230). Accordingly, the first transmitted light (the light of ③ in FIG. 2a) may be blocked by the second polarizing unit (280) so that it is not detected by the image sensor (230). To this end, the second polarizing unit (280) may include a polarizing plate, a polarizing filter, an analyzer, etc., that polarizes light in a second direction to block the first transmitted light (the light of ③ in FIG. 2a). However, this is merely an example, and it goes without saying that the second polarizing unit (280) can be replaced with another device for blocking the first transmitted light in addition to the device for performing polarization.

[0073] For example, if the first polarizing section (270) is a transverse polarizing plate, the light emitted from the light source section (210) passes through the first polarizing section (270) and only the light vibrating in the transverse direction (light of ① in FIG. 2a) passes through, and this light can become the first transmitted light (light of ③ in FIG. 2a) vibrating in the transverse direction as it passes through the light splitting section (220). To block this first transmitted light (light of ③ in FIG. 2a), a longitudinal polarizing plate may be used in the second polarizing section (280). As another example, if a longitudinal polarizing plate is used in the first polarizing section (270), a transverse polarizing plate may be used in the second polarizing section (280). However, this is merely an example, and various devices may be used in the second polarizing section (280) to block the first transmitted light (light of ③ in FIG. 2a).

[0074] In one embodiment, the first reflected light (the light of ② in FIG. 2a) may pass through an objective lens and reach the object of observation. This first reflected light (the light of ② in FIG. 2a) can serve as the illumination required when observing the object of observation. For example, the first reflected light passing through the objective lens is scattered from above the object of observation, and among the light scattered from above the object of observation, the light that passes through the object of observation can be collected back into the objective lens as light containing information about the object of observation. Here, the light scattered from above the object of observation may include light directly scattered from above the object (e.g., the surface of the media), light that is scattered from above the object (e.g., the surface of the media) and then reflected back from further above (e.g., the top plate of the container), etc. This will be described in more detail later with reference to FIG. 5a, 5b, and 7. As another example, the first reflected light passing through the objective lens is scattered from the object being observed, and at least some of the scattered light can be collected back into the objective lens. This will be described in more detail later with reference to FIGS. 6a and 6b.

[0075] At this time, since the first reflected light (light ② in FIG. 2a) is light that has passed through the first polarizing unit (270), it may contain only light in the direction polarized by the first polarizing unit (270). When the first reflected light, which has only the directionality of the first direction, reaches an object, it may be reflected or scattered. The reflected light still has the directionality of the first direction, but the scattered light may lose its directionality and have a random directionality. Unlike conventional technology that views light scattering as light loss, one embodiment of the present disclosure aims to provide an observation method that uses scattered light as illumination. Subsequently, the process of the light having a random directionality entering the objective lens again will be described later with reference to FIG. 2b.

[0076] FIG. 2b is a diagram showing the process of light passing through an object of observation according to one embodiment of the present disclosure reaching an image acquisition unit.

[0077] Referring to FIG. 2b, the first reflected light can pass through the objective lens again and the light scattered from the object of observation or from the object of observation.

[0078] In one embodiment, light scattered from above an object of observation may contain information about the object of observation as at least a portion of it is transmitted or reflected by the object of observation after being scattered from above the object of observation.

[0079] Throughout the entire disclosure, "above the object of observation" refers to a direction above the object of observation, that is, the opposite direction of the observation device relative to the object of observation. For example, if the observation device intends to observe an object of observation contained in a media (or culture medium) from below, the above the object of observation may include the surface of the media (or culture medium), the lid of the container, dust, air, etc., which are located above the object of observation.

[0080] Throughout this disclosure, the term "light containing information of an object" means light that, through interaction (e.g., absorption, reflection, refraction, scattering, fluorescence expression, etc.) with an object of observation (e.g., cell, tissue, or other biological sample, etc.), comes to contain information such as the shape, location, structure, composition, concentration, or changes over time of the object of observation. Such light is light generated or modified as a result of physical, chemical, or biological interactions with the object of observation, and may contain information that enables the inference or determination of the physical properties or biological state of the object of observation.

[0081] In another embodiment, light scattered from the object of observation may also contain information about the object of observation.

[0082] In this way, light containing information about the object of observation may face a second polarization unit (280) that polarizes it in a second direction. At this time, among the light containing information about the object of observation, only light vibrating only in the second direction passes through the second polarization unit (280), and all others are blocked, so that the light passing through the second polarization unit (280) may contain only light vibrating in the second direction. For example, the second polarization unit (280) can polarize in the second direction not only the first transmitted light but also the first reflected light that enters the objective lens after being reflected or scattered by the object of observation, container, media surface, etc. Since the reflected light retains the directionality polarized by the first polarization unit (270), it cannot pass through the second polarization unit (280), and only light that has lost its directionality through scattering can pass through the second polarization unit (280). Accordingly, the image sensor (230) can acquire scattered light polarized by the second polarizing unit (280). In one embodiment, the light detected by the image sensor (230) may include the first reflected light (light of ② in FIG. 2a) scattered from above the target, transmitted light that has passed through the target, scattered light that has scattered from the target, etc.

[0083] Accordingly, since the image sensor (230) can detect only scattered light at least once, it is hardly affected by the light source (210) and can image only a small amount of light containing information of the object of observation, thereby enabling imaging of a small level of weak light.

[0084] In one embodiment, the path from the light splitting section of the light detected by the image sensor (230) to the second polarization section may coincide with the path from the light splitting section of the first transmitted light to the second polarization section.

[0085] FIG. 3 is a drawing showing an image obtained by observing a macroscopic object using an observation device according to one embodiment of the present disclosure.

[0086] Referring to FIG. 3, the result of observing a macroscopic object using an observation device (200) is illustrated. In one embodiment, when a first reflected light meets a macroscopic object and is reflected or scattered and enters the objective lens again, an image sensor detects the light that has passed through the light splitting unit (220) and the second polarization unit (280) among the light reflected or scattered by the macroscopic object, so that the background can be imaged as a dark color and the object as a bright color. Therefore, when observing a macroscopic object, an image with a principle similar to a dark-field microscope can be obtained as shown in FIG. 3.

[0087] FIGS. 4a and 4b are drawings showing images obtained by observing a microscopic object using an observation device according to one embodiment of the present disclosure.

[0088] FIGS. 4a and 4b illustrate the results of observation when observing a flat portion, such as the central portion of a vessel, in the case of observing a media containing a microscopic object, such as a cell, according to one embodiment of the present disclosure. In this case, since light reflection or scattering occurs at the boundary surface of the media, the boundary surface of the media acts like a mirror and performs the role of illumination. Therefore, as shown in FIGS. 4a and 4b, the background appears as a bright color and the microscopic object, such as a cell, can be observed as a dark color.

[0089] FIG. 5a is a diagram illustrating a bright-field imaging method for the process in which light emitted from a light source unit reaches an object according to one embodiment of the present disclosure.

[0090] In FIG. 5a, an observation method according to an embodiment of brightfield imaging is illustrated, such as when the object to be observed is contained in a container or immersed in a medium. Light emitted from a light source unit (210) can pass through a condensing lens (260). The condensing lens (260) can perform the function of collecting and irradiating the light emitted from the light source unit (210). The light passing through the condensing lens (260) can be polarized by a first polarizing unit (270). For example, the first polarizing unit (270) can allow only the light vibrating in a first direction among the light passing through the condensing lens (260) to pass through, and block other lights. Therefore, as illustrated in FIG. 5a, among the light vibrating in all directions before passing through the first polarizing unit (270), only the light vibrating in the first direction (light ① in FIG. 5a) can reach the light splitting unit (220).

[0091] In one embodiment, light reaching the light splitting unit (220) (light of ① in FIG. 5a) may be split into a first reflected light (light of ② in FIG. 5a) and a second transmitted light (light of ③ in FIG. 5a). Both the first reflected light (light of ② in FIG. 5a) and the second transmitted light (light of ③ in FIG. 5a) may be light that vibrates in a first direction after passing through the first polarization unit (270).

[0092] In one embodiment, the second polarizing unit (280) can block the second transmitted light (light of ③ in FIG. 5a) vibrating in the first direction. At this time, the second transmitted light passes through the first polarizing unit and is transmitted in the same direction as ① in FIG. 5a, and then may head toward the second polarizing unit through a reflection process from a wall of a structure, etc. To this end, the second polarizing unit (280) may be a device that polarizes light in the second direction rather than the first direction. For example, the second direction may form an angle of 90 degrees with the first direction. However, this is merely an example, and the second direction only needs to be different from the first direction and does not always have to form a 90-degree angle.

[0093] In one embodiment, the first reflected light (light ② in FIG. 5a) can pass through the objective lens (250) and reach the target. Here, the target may include not only objects of observation such as cells or samples, but also a concept including a vessel, a vessel lid, dust, etc. In one embodiment, the first reflected light (light ② in FIG. 5a) can be reflected or scattered from above the object of observation and proceed back toward the objective lens. For example, light traveling toward the objective lens may include, as shown in the example of FIG. 5a, (a) light that is first scattered when the first reflected light (light ② in FIG. 5a) is reflected from the top of the media, (b) light that is secondarily scattered when the first reflected light reaches the top plate of the container (e.g., the lid of the container) and is reflected, (c) light that is reflected or scattered when some of the light that is first scattered from the top of the media (e.g., the surface of the media) reaches the top plate of the container (e.g., the lid of the container), and (d) light that is scattered when the first reflected light encounters dust, etc. These lights (a) to (d) may pass through the object of observation while traveling toward the objective lens and may contain information regarding the object of observation.

[0094] In one embodiment, the container may be composed only of a bottom plate and a top plate, or may be composed only of a bottom plate.

[0095] According to one embodiment of the present disclosure, if a top plate of the container (e.g., a lid of the container) is present, the objective lens can collect more light reflected or scattered from the top plate of the container, so that a brighter image can be obtained. Light containing information regarding the object of observation can be collected again by the objective lens (250). This will be explained further with reference to FIG. 5b.

[0096] FIG. 5b is a diagram illustrating a bright-field imaging method for the process in which light scattered or reflected from an object reaches an image acquisition unit according to one embodiment of the present disclosure.

[0097] Referring to FIG. 5b, the objective lens (250) can collect a portion of the light scattered from above the object of observation, such as from the object of observation, the lid of a container, or the surface of a media. This light can contain information about the object of observation by passing through the object of observation before or after scattering. Additionally, the light collected by the objective lens (250) can proceed toward the image sensor (230). That is, the image sensor (230) can detect the light input to the image sensor (230) based on the light collected by the objective lens (250).

[0098] However, since a second polarizing unit (280) exists in front of the image sensor (230), the image sensor (230) can detect only light that has lost its directionality due to scattering. For example, the image sensor (230) can detect transmitted light that has passed through the target after the first reflected light is scattered from above the target. In contrast, the objective lens (250) also collects light reflected by the target, container, media surface, etc., but since the polarization properties of such reflected light do not change unless it undergoes a scattering process, it is blocked by the second polarizing unit (280), so the image sensor (230) cannot detect light that has not undergone such a scattering process.

[0099] In one embodiment, the image sensor is a sensor that converts light entering through a tube lens (290) into an electrical digital signal and may include a CMOS (Complementary Metal Oxide Semiconductor), etc. For example, light entering through an objective lens (250) may pass through a light splitting unit (220), a second polarizing unit (280), and a tube lens (290) in sequence and then reach the image sensor (230). That is, the light splitting unit (220) may be located between the objective lens (250) and the tube lens (290). Accordingly, the image sensor (230) can acquire a bright-field image in which information about the object of observation is displayed in a dark color against a bright background.

[0100] FIG. 6a is a diagram illustrating a dark-field imaging method for the process in which light emitted from a light source unit reaches an object according to one embodiment of the present disclosure.

[0101] Referring to FIG. 6a, unlike FIG. 5a, an observation method according to one embodiment is illustrated in dark-field imaging, such as when the object to be observed is not in the media but is on a container without media.

[0102] In one embodiment, light emitted from a light source (210) passes through a condensing lens (260), and the light passing through the condensing lens (260) can be polarized by a first polarizing unit (270). For example, the first polarizing unit (270) can allow only light vibrating in a first direction to pass through among the light vibrating in all directions that passes through the condensing lens (260), and block other light. Therefore, as shown in FIG. 6a, among the light vibrating in all directions before passing through the first polarizing unit (270), only the light vibrating in a first direction (light ① in FIG. 6a) can reach the light splitting unit (220).

[0103] In one embodiment, light reaching the light splitting unit (220) (light ① in FIG. 6a) may be split into a first reflected light (light ② in FIG. 6a) and a second transmitted light (light ③ in FIG. 6a). Both the first reflected light (light ② in FIG. 6a) and the second transmitted light (light ③ in FIG. 6a) may be light that vibrates in a first direction after passing through the first polarization unit (270).

[0104] In one embodiment, the second polarizing unit (280) can block the second transmitted light (light of ③ in FIG. 6a) vibrating in the first direction. At this time, the second transmitted light passes through the first polarizing unit and is transmitted in the same direction as ① in FIG. 5a, and then may head toward the second polarizing unit through a reflection process from a wall of a structure, etc. To this end, the second polarizing unit (280) may be a device that polarizes light in the second direction rather than the first direction.

[0105] In one embodiment, the first reflected light (the light of ② in FIG. 6a) may pass through the objective lens (250) and reach the object of observation. Here, the object of observation may refer to an object to be observed placed on a vessel, or an object to be observed placed directly on the objective plate without a vessel (not shown). In one embodiment, the first reflected light (the light of ② in FIG. 6a) may reach the object of observation and be transmitted, reflected, or scattered, and some of the scattered light may proceed toward the objective lens. Therefore, the light proceeding toward the objective lens is light scattered by the object of observation, as in the example of FIG. 6a, and may contain information regarding the object of observation. Thus, unlike FIG. 5a, darkfield imaging may be performed in FIG. 6a. The process from the point where the light scattered from the object of observation proceeds toward the objective lens will be explained further with reference to FIG. 6b.

[0106] FIG. 6b is a diagram illustrating a dark-field imaging method for the process in which light transmitted or reflected from an object reaches an image acquisition unit according to one embodiment of the present disclosure.

[0107] Referring to FIG. 6b, the objective lens (250) can collect a portion of the scattered light as it is transmitted or reflected from the object of observation. This scattered light may contain information about the object of observation, as described above with reference to FIG. 6a. Additionally, the light collected by the objective lens (250) can proceed toward the image sensor (230). That is, the image sensor (230) can detect the light input to the image sensor (230) based on the light collected by the objective lens (250).

[0108] However, since a second polarization unit (280) exists in front of the image sensor (230), the image sensor (230) can detect only light that has lost its directionality due to scattering. For example, the image sensor (230) can detect scattered light that is scattered from the target by the first reflected light.

[0109] In one embodiment, light entering through the objective lens (250) may pass through the light splitting unit (220), the second polarizing unit (280), and the tube lens (290) in sequence and then reach the image sensor (230). Accordingly, the image sensor (230) can acquire a dark-field image in which information about the object of observation is displayed in a bright color.

[0110] FIG. 7 is a drawing illustrating a bright-field imaging method for acquiring light scattered by an object and incident on an objective lens according to one embodiment of the present disclosure.

[0111] In FIG. 7, light scattered by a target and incident on an objective lens in the bright-field imaging method of FIG. 5a and 5b according to one embodiment is depicted more specifically.

[0112] In one embodiment, light polarized in a first direction is reflected by a light splitter, and the light reflected by the light splitter passes through an objective lens (250) and can reach the entire field of view (opening) of the observation device (200). At this time, the field of view (opening) may refer to an area where the observation target (700) can be observed.

[0113] In one embodiment, light passing through the objective lens (250) may be scattered from the surface (710) of the media. A portion of the light scattered from the surface (710) of the media (hereinafter, 'light A') may travel upward, that is, toward the underside of the vessel lid, and this light A may be reflected from the vessel lid and travel back toward the objective lens (250). The objective lens (250) can collect this light A when the light A reflected from the vessel lid travels back toward the objective lens (250). Additionally, another portion of the light scattered from the surface (710) of the media (hereinafter, 'light B') may travel downward, that is, toward the objective lens (250). The objective lens (250) can collect the light B that reaches the objective lens (250) after being reflected or scattered from the surface (710) of the media.

[0114] In one embodiment, light passing through the objective lens (250) may be scattered as it is reflected from the bottom surface (720) of the container lid. A portion of the light scattered as it is reflected from the bottom surface (720) of the container lid (hereinafter 'light C') travels back toward the objective lens (250), and the objective lens (250) can collect this light C.

[0115] In one embodiment, light passing through the objective lens (250) may be scattered as it is reflected from the upper surface (730) of the container lid. A portion of the light scattered as it is reflected from the upper surface (730) of the container lid (hereinafter referred to as 'light D') then travels back toward the objective lens (250), and the objective lens (250) can collect this light D.

[0116] In one embodiment, light passing through the objective lens (250) may be reflected or scattered from the surface of the media, the bottom surface of the container lid, or the top surface of the container lid, and may reach the top surface (740) of the container, and may be reflected and scattered from the top surface (740) of the container. A portion of the light (hereinafter 'light E') that is reflected or scattered from the top surface (740) of the container may then travel back toward the objective lens (250), and the objective lens (250) may collect this light E.

[0117] In one embodiment, light passing through the objective lens (250) may be reflected from the surface of the media, the bottom surface of the container lid, or the top surface of the container lid and reach the bottom surface (750) of the container, and such light may be reflected or scattered from the bottom surface (750) of the container. A portion of the light (hereinafter 'light F') that is reflected or scattered from the bottom surface (750) of the container after passing through the object of observation (700) may then proceed toward the objective lens (250), and the objective lens (250) may collect such light F.

[0118] In one embodiment, light passing through the objective lens (250) may encounter foreign substances (760), such as dust, and be scattered. A portion of the light scattered upon encountering foreign substances (760) (hereinafter 'light G') may enter the objective lens (250) again.

[0119] In one embodiment, the objective lens (250) can collect light A to light G containing information about the observation target (700) by passing through the observation target (700). However, this is merely an example, and it is obvious that light other than light A to light G can be collected, or only some of these can be collected. Light collected by the objective lens, such as light A to light G, can contain information about the observation target (700) while passing through the observation target (700). The light generated by passing through the observation target can enable brightfield imaging, which makes the observation target appear dark as the light passes through it.

[0120] An observation method according to one embodiment of the present disclosure may include: a step of scattering at least a portion of light emitted from a light source unit onto an object or above the object; and a step of detecting at least one of scattered light scattered from the object or transmitted light that has passed through the object after being scattered above the object.

[0121] According to one embodiment of the present disclosure, scattered light is collected, so a clear observation image can be obtained even when the bottom surface of a container containing an object of observation is opaque or uneven.

[0122] According to one embodiment of the present disclosure, a container holding an object of observation may include an optically transparent material. The container includes any type of container capable of holding and observing an object of observation, and is not particularly limited in its structure or shape.

[0123] According to one embodiment, the container may provide a surface for culturing, growing, and functioning adherent cells, suspension cells, stem cells, tumor cells, immune cells, neuronal cells, or other mammalian-derived cells. In addition to cells, it may be used without limitation for culturing, growing, and functioning any subject included in the "object of observation" as defined herein.

[0124] In one embodiment, the container may include various types of culture vessels. Depending on the structure and culture method, the container may be classified into a monolayer vessel, a multi-layer vessel, a 3D culture vessel, or a bioreactor-based vessel, each of which may include, but is not limited to, the following examples.

[0125] (1) A monolayer vessel is a structure for culturing cells in a single plane, such as a T-flask, Petri dish, slide glass, multi-well plate, chamber slide, microfluidic chip, cover glass, etc., but is not limited thereto.

[0126] (2) A multi-layer vessel has multiple cell culture surfaces in a vertical or inclined direction, and each layer functions as an individual culture space, and has a structure in which optical access from the outside is limited. In such a structure, it is difficult to observe cells in a specific layer with a conventional optical system, and the optical system of the present invention is designed to solve this problem.

[0127] These multilayer containers may include, but are not limited to, Corning’s CellSTACK™, HYPERStack™, HYPERFlask®, TripleFlask™, Falcon® Cell Culture Multi-Layer Flask, Thermo Fisher Scientific’s Cell Factory™, Nunc™ EasyFill™, Sartorius’ STACKMAX™, Cytiva’s iCELLis™ Nano and iCELLis™ 500+.

[0128] (3) A 3D culture vessel is a structure that provides an environment in which cells can grow in three dimensions, and may include spheroids, organoids, hydrogel-based culture systems, structures using scaffolds, or suspension cultures. These may also be difficult to observe optically as the cells are distributed at various depths.

[0129] (4) Bioreactor-based vessels are devices for continuous culture environment control and mass culture, and may include spinner flasks, wave-type bioreactors, or small-scale bioreactor chambers.

[0130] An optical system according to one embodiment of the present invention can overcome structural and optical constraints occurring in such multilayer containers and three-dimensional cell culture systems, and by enabling effective observation of cells present in each layer or depth, it can improve the accuracy and reproducibility of cell culture and analysis.

[0131] A container according to one embodiment of the present invention may have a structure including a ceiling plate or a lid, or may have a structure without a ceiling plate or a lid. For example, a structure without a ceiling plate may include an open-top container such as a standard Petri dish, slide glass, or multi-well plate. In such a structure, the top of the container is open, so no reflective member is required above the container or above the sample, even in environments where it is difficult to install a separate reflective member.

[0132] An observation device according to one embodiment of the present invention may be a device that images an object of observation in real time or at regular intervals to visually observe it or analyze image data. The observation device may include a configuration capable of monitoring morphological changes, migration, differentiation, or functional responses of an object of observation, such as cells, tissues, or biological samples, over time.

[0133] According to one embodiment, the observation device may be a device for a live cell imaging system, which may be a system designed to observe the state of a cell over a long period of time while maintaining the survival environment of the live cell.

[0134] In one embodiment, when the observation device according to one embodiment of the present invention is a live cell imaging system, it generally operates in an environment where CO₂, temperature, and humidity are controlled, and can typically be installed and operated inside a commercially available incubator. In another embodiment, it may include a separately mountable incubation module so that the observation device itself may perform imaging while providing an independent culture environment for live cells.

[0135] In addition, the observation device may include an optical system such as an optical microscope, a fluorescence microscope, a phase contrast or confocal microscope, and may be composed of various components such as an image sensor (CMOS, CCD, etc.), an illumination unit, a lens system, a focusing device, and image processing software.

[0136] The above system can support various observation modes, such as static imaging, time-lapse shooting, Z-stack shooting, and high-speed video shooting.

[0137] One embodiment of the present disclosure may also be implemented in the form of a recording medium comprising computer-executable instructions, such as program modules executed by a computer. A computer-readable medium may be any available medium accessible by a computer and includes both volatile and non-volatile media, and both removable and non-removable media. Additionally, a computer-readable medium may include both computer storage media and communication media. A computer storage medium includes both volatile and non-volatile, removable and non-removable media implemented by any method or technique for storing information, such as computer-readable instructions, data structures, program modules, or other data. A communication medium typically includes computer-readable instructions, data structures, or program modules and includes any information transmission medium.

[0138] The foregoing description of the present disclosure is for illustrative purposes only, and those skilled in the art will understand that modifications can be easily made to other specific forms without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single unit may be implemented in a distributed manner, and components described as distributed may likewise be implemented in a combined form.

[0139] The scope of the present disclosure is defined by the claims set forth below rather than by the detailed description above, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of the present disclosure.

Claims

1. As an observation device, A first polarizing unit that polarizes light emitted from a light source unit; A light splitting unit that splits the light passing through the first polarizing unit into a first reflected light and a first transmitted light; An objective lens that collects light containing information of an object based on the first reflected light above; An image sensor that detects input light based on light collected by the above objective lens; and A second polarizing unit that blocks the first transmitted light so that the first transmitted light is not detected by the image sensor. An observation device including 2. In paragraph 1, the light detected by the image sensor is, The first reflected light is scattered from above the object and the scattered light is transmitted light that passes through the object; or The scattered light of the first reflected light scattered from the object including, Observation device.

3. In Paragraph 2, The light scattered from above the object by the first reflected light is, An observation device comprising light scattered by at least one of the top plate of a container or the surface of a media.

4. In paragraph 3, the light collected by the objective lens is, It includes light reflected by at least one of the above object, the above container, or the above media surface, and An observation device in which the above reflected light is blocked by the second polarizing part when it is light that has not undergone a scattering process.

5. In paragraph 1, the observation device is, An observation device further comprising the light source unit provided inside the observation device.

6. In paragraph 1, the polarization angle of the first polarizing part is, An observation device characterized by forming a 90-degree angle with the polarization angle of the second polarization part.

7. In paragraph 1, the second polarizing unit is, Polarizing the light collected by the above objective lens, The above image sensor is, An observation device that acquires light polarized by the second polarizing unit.

8. In paragraph 1, the object is contained in a single-layer container or a multi-layer container, Observation device.

9. In claim 8, the multilayer container is CellSTACK™, HYPERStack™, Cell Factory™, Nunc™ EasyFill™ Cell Factory, Falcon ® Cell Culture Multi-Layer Flask, STACKMAX™, iCELLis™ Nano and iCELLis™ 500+, TripleFlask™ System, or HYPERFlask ® person, Observation device.

10. In paragraph 1, the object is contained in a container, and The above container is composed of a bottom plate and a top plate, or is composed only of a bottom plate. Observation device.

11. In paragraph 1, the object is contained in a container, and The above container comprises an optically transparent material, Observation device.

12. In paragraph 1, the observation device is, An observation device that is a live cell imaging system.

13. Regarding the method of observation, A step of emitting light by a light source; A step of polarizing the light emitted by the light source unit by the first polarizing unit; A step of dividing the light that has passed through the first polarizing unit into a first reflected light and a first transmitted light by means of a light splitting unit; A step of blocking the first transmitted light by the second polarizing unit so that the first transmitted light is not detected by the image sensor; A step of collecting light containing information of an object by means of an objective lens based on the first reflected light; An observation method comprising the step of detecting light input to the image sensor based on light collected by the objective lens.

14. In paragraph 13, the light detected by the image sensor is, The first reflected light is scattered from above the object and the scattered light is transmitted light that passes through the object; or The scattered light of the first reflected light scattered from the object An observation method that includes 15. As a method of observation, A step in which at least a portion of the light emitted from a light source is scattered on an object or above the object; and An observation method comprising the step of detecting scattered light scattered from the object, or transmitted light scattered from above the object and then transmitted through the object.

Images