Contrast field microscope capable of obtaining fluorescence image without dichroic mirror

Abstract

The present invention relates to a contrast field microscope capable of obtaining a bright field image and a dark field image without an additional light source and simultaneously obtaining a fluorescence image without a dichroic mirror, and comprises: a light source; a ring-forming optical system for converting light irradiated from the light source, into ring-shaped parallel light; an excitation filter for exciting the light converted by the ring-forming optical system; a through-hole mirror for reflecting the light transmitted through the excitation filter, to an object, and passing the light reflected from the object, through a through-hole; and a reflection type objective lens forming, on the object, a focal point of the light reflected from the through-hole mirror and irradiated toward the object; and a camera for obtaining an image of the object by receiving the light reflected from the object and passing through the through-hole of the through-hole mirror.

Description

Bright-field microscope capable of obtaining fluorescence images without a color-selective mirror

[0001] This invention claims the benefit of Korean Patent Application No. 10-2024-0037972 filed with the Korean Intellectual Property Office on March 19, 2024, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to a bright-field microscope, and more specifically, to a bright-field microscope capable of acquiring bright-field and dark-field images without an additional light source, and simultaneously acquiring fluorescence images without a color-selective mirror.

[0003]

[0004] A microscope is an optical instrument for observing microscopic objects difficult to observe with the naked eye. It is used in virtually all natural science fields, including biology, geology, physics, and chemistry. Generally, the term "microscope" refers to an optical microscope, which essentially consists of an objective lens and a condenser lens. The objective lens, which contacts the object to be observed, creates a magnified image of the nearby object, while the condenser lens focuses light onto the object, brightening it and enhancing the resolution of the objective lens. While optical microscopes may be equipped with eyepieces for visual observation, they may also be equipped with cameras capable of capturing images, enabling observations to be stored and documented beyond the human eye. While optical microscopes typically utilize visible light, electron microscopes utilize electron beams as a light source, allowing them to observe objects at the atomic level, which is much smaller and more minute than the objects in an optical microscope.

[0005] Meanwhile, the types of microscopes have become more diverse as microscopes are designed to optimally observe the target object depending on its characteristics. For example, for targets such as biosamples less than 20 μm in thickness, light in the visible wavelength range can easily penetrate the target object. Therefore, a transmission microscope is widely used, which observes the target object by observing the image obtained by placing a light source and a condenser lens below the target object and allowing the incident light to pass through the target object. On the other hand, for targets such as minerals containing metallic elements, the low transmittance of visible light makes it difficult to use a transmission microscope. On the other hand, metallic elements tend to strongly reflect or scatter light, so for these targets, a reflection microscope is widely used, which observes the target object by observing the image obtained by allowing light irradiated from a light source to reflect off the target object.

[0006] Microscopes can also be classified into phase contrast microscopes, interference microscopes, metallurgical microscopes, and fluorescence microscopes based on their optical structure, and into light-field microscopes and dark-field microscopes based on the type of illumination.

[0007] In particular, a fluorescence microscope is a microscope used to search for fluorescent substances contained in a sample, and an image can be obtained based on the phenomenon of luminescence when light of a specific wavelength is irradiated on the sample.

[0008] Also, bright-field microscopes refer to most microscopes that obtain a field of view by incident illumination obtained by a vertical illuminator. Dark-field microscopes are designed to observe objects that are difficult to observe with ordinary bright-field microscopes, such as microcracks or microinclusions, and use a special type of dark-field illumination different from that of bright-field microscopes. Dark-field illumination is generally formed in a ring shape, and rather than shining light directly on the object, it uses plain light (oblique light), that is, light that shines obliquely at an angle between the side and front of the object, which has the effect of contrasting the light in the outer boundary area of ​​the object. Accordingly, observation using dark-field illumination can obtain an image that can more clearly discern the shape of objects such as the above-mentioned microcracks or microinclusions.

[0009] As such, there are a wide variety of microscope types available, each capable of optimally observing the characteristics of a given object. However, the trend of convergence and advancement across various technological fields has intensified recently, leading to an increasing need for diverse methods of observing a given object. For example, while transmission-type bright-field microscopes utilizing H&E staining techniques were previously considered optimal for observing biological tissue, reflection-type or dark-field microscopes may be necessary to observe metal particles in biological tissue. Furthermore, obtaining fluorescence images typically requires the use of a separate fluorescence microscope.

[0010] The background technology described above is technical information that the inventor possessed for the purpose of deriving embodiments of the present invention or acquired during the derivation process, and cannot necessarily be said to be publicly known technology disclosed to the general public prior to the filing of the embodiments of the present invention.

[0011]

[0012] The present invention provides a bright-field microscope capable of acquiring bright-field and dark-field images without an additional light source, and simultaneously acquiring fluorescence images without a color-selective mirror.

[0013]

[0014] The present invention relates to a brightfield microscope, which may include a light source, a ring-forming optical system that converts light irradiated from the light source into ring-shaped parallel light, an excitation filter that excites the light converted by the ring-forming optical system, an aperture mirror that reflects the light transmitted through the excitation filter toward an object and passes the light reflected from the object through an aperture, a reflective objective lens that forms a focus on the object of the light reflected from the aperture mirror and irradiated toward the object, and a camera that receives the light reflected from the object and passed through the aperture of the aperture mirror to obtain an image of the object.

[0015] According to one embodiment of the present invention, the filter may further include a movable module capable of moving the filter so as to be placed or removed on the optical path.

[0016] According to one embodiment of the present invention, a trigger board may be further included to turn on / off the light source and the camera by transmitting a pulse-shaped trigger signal to the light source and the camera.

[0017] According to one embodiment of the present invention, the device may further include a control unit that controls the moving module to remove the excitation filter from the optical path or place the excitation filter on the optical path during the off-period of the pulse in the trigger signal.

[0018] According to one embodiment of the present invention, the present invention may further include an image processing unit that processes a dark field image and a fluorescent image acquired through a camera into a single image.

[0019] According to one embodiment of the present invention, a collimator may be further included to make light irradiated from a light source into parallel light.

[0020] According to one embodiment of the present invention, the object may further include a white scattering plate disposed behind the object to reflect light.

[0021] According to one embodiment of the present invention, a white scattering plate may be provided at a certain distance from the rear of the object so as to be detachable.

[0022] According to one embodiment of the present invention, a bright-field microscope may be characterized in that, when a white scattering plate is placed behind an object, light reflected and scattered from the white scattering plate passes through the object and is received by a camera, thereby obtaining a bright-field image of the object, and when the white scattering plate is removed from behind the object, only light reflected and scattered from the object is received by the camera, thereby obtaining a dark-field image of the object.

[0023] According to one embodiment of the present invention, the light source may be characterized in that it is formed to emit white light.

[0024] According to one embodiment of the present invention, a ring-shaped optical system may further include a first collecting lens that collects light from a light source, a ring filter that converts the light collected by the first collecting lens into ring-shaped light, a collimating lens that collimates the light from the ring filter, and a second collecting lens that collects the light from the collimating lens and sends it to a through-hole mirror.

[0025] According to one embodiment of the present invention, a ring filter may be characterized by forming a ring shape by passing light through a mask in which a ring-shaped empty space is formed.

[0026] According to one embodiment of the present invention, the ring-forming optical system may further include a first aperture that controls the outermost size of light collected by the first focusing lens, and a second aperture that controls the brightness of the light controlled by the first aperture and sends it to the collimating lens.

[0027] According to one embodiment of the present invention, the ring-forming optical system may be characterized in that the focus of light coming from the first condenser lens is formed between the first aperture and the second aperture.

[0028] According to one embodiment of the present invention, a reflective objective lens may be characterized in that light reflected from a through-hole mirror and traveling to an object passes through an edge portion of the reflective objective lens, and light reflected from the object passes through a central portion of the reflective objective lens.

[0029] According to one embodiment of the present invention, a reflective objective lens may be characterized in that an edge portion and a central portion are each formed as separate, independent lenses.

[0030] According to one embodiment of the present invention, a brightfield microscope may further include a third aperture that removes noise from light passing through the aperture, and a light-receiving lens that receives the light from the third aperture and sends it to a camera.

[0031]

[0032] The present invention has the effect of enabling the acquisition of fluorescence images from a brightfield microscope. While fluorescence images typically obtained through fluorescence microscopes require components such as a color-selective mirror, the present invention enables the acquisition of fluorescence images even without such components.

[0033] In addition, the present invention has the advantage of being able to obtain both dark-field and bright-field images of a single object using a single optical path. In tissue analysis methods using H&E (Hematoxylin & Eosin), a staining and diagnostic method widely used in biological tissue analysis, there are many cases where both bright-field images of the same object are required. The present invention has the effect of being able to provide both bright-field images of the same object much more smoothly. In addition, since both bright-field images, which are advantageous for observing biological tissue, and dark-field images, which are advantageous for observing metal particles, can be obtained, there is the effect of being able to observe metal particles exposed to biological tissue.

[0034] Meanwhile, in the past, when acquiring dark-field images using dark-field illumination, there was a problem that large-area analysis was difficult due to limitations in the light irradiation angle. However, according to the present invention, since the dark-field microscope is implemented as a reflective type, it has the effect of enabling analysis of much larger areas than before. In addition, because it is formed as a reflective type, it also has the effect of fundamentally solving the problem that in the past, in the case of a transmission type, light did not properly penetrate excessively thick biological tissue, making observation difficult.

[0035] Furthermore, according to the present invention, the three-dimensional structure of biological tissue can be observed by utilizing the scattering intensity generated from the density difference in a dark-field image, and thus, there is also the effect of enabling label-free analysis without staining. While the staining method widely used in biological tissue analysis has the advantage of allowing easy visual recognition of the internal structure of the tissue, there is a problem that the tissue may be damaged or lost during the staining process. In this case, performing label-free analysis using the present invention fundamentally solves this problem.

[0036] The effects that can be obtained from the invention are not limited to the effects mentioned above, and other effects not mentioned can be clearly understood by a person having ordinary skill in the art to which the present invention belongs from the description below.

[0037]

[0038] FIG. 1 illustrates a configuration diagram for obtaining a fluorescence image in a bright-field microscope according to one embodiment of the present invention.

[0039] Figure 2 illustrates a block diagram of a bright-field microscope according to one embodiment of the present invention.

[0040] FIG. 3 illustrates a pulse signal of a trigger signal in a bright-field microscope according to an embodiment of the present invention.

[0041] Figures 4 and 5 illustrate dark-field images and fluorescence images observed with a bright-field microscope according to one embodiment of the present invention.

[0042] FIG. 6 illustrates a configuration capable of acquiring a bright-field image in a bright-field microscope according to one embodiment of the present invention.

[0043] Figure 7 illustrates a configuration capable of acquiring a dark field image in a bright field microscope according to one embodiment of the present invention.

[0044] FIG. 8 illustrates a bright / dark field image of a biological tissue containing metal particles observed with a bright-field microscope according to an embodiment of the present invention.

[0045] FIGS. 9 and 10 illustrate large-area dark-field images observed with a bright-field microscope according to one embodiment of the present invention.

[0046] FIG. 11 illustrates a bright / dark field image of unstained biological tissue observed with a bright field microscope according to one embodiment of the present invention.

[0047] ※ Explanation of symbols

[0048] 100: Brightfield microscope 110: Light source

[0049] 120: Collimator 130: Ring forming optical system

[0050] 131: First condenser lens 132: First aperture

[0051] 133: Second aperture 134: Ring filter

[0052] 135: Collimating lens 136: Second condensing lens

[0053] 140: Through-hole mirror 145: Through-hole

[0054] 150: Reflective objective lens 160: White plate

[0055] 170: Third aperture 180: Photoreceptor lens

[0056] 190: Camera 200: Filter here

[0057] 300: Trigger board 500: Object

[0058]

[0059] The present invention will become clearer with reference to the embodiments described below in detail together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms. These embodiments are provided only to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention. The present invention is defined solely by the scope of the claims. Meanwhile, the terminology used in this specification is for the purpose of describing the embodiments and is not intended to limit the present invention.

[0060] Throughout this specification, singular forms also include plural forms unless specifically stated otherwise in the text.

[0061] Throughout this specification, the terms "comprises" and / or "comprising" do not exclude the presence or addition of one or more other components, steps, operations and / or elements, and do not exclude other components unless specifically stated to the contrary, but rather include other components.

[0062] Terms such as “first” or “second” used throughout this specification may be used simply to distinguish the corresponding component from other corresponding components and do not limit the corresponding components in any other respect (e.g., importance or order).

[0063] Additionally, terms such as “unit” described throughout this specification mean a unit that processes at least one function or operation, which may be implemented as hardware or software, or a combination of hardware and software.

[0064] Additionally, when it is said throughout this specification that a part is "connected" to another part, this includes not only cases where it is "directly connected" but also cases where it is connected "with another structure in between."

[0065]

[0066] Hereinafter, the present invention will be described in more detail.

[0067] The present invention relates to a method for obtaining fluorescence images without a dichromatic mirror in a brightfield microscope. In particular, the present invention is characterized by being able to obtain darkfield images and fluorescence images separately and / or simultaneously. Below, a configuration capable of obtaining darkfield and fluorescence images is described, followed by a configuration capable of obtaining brightfield images.

[0068] FIG. 1 illustrates a configuration diagram of a bright-field microscope (100) capable of obtaining a fluorescence image according to an embodiment of the present invention. The bright-field microscope (100) according to an embodiment of the present invention may include a light source (110), a ring-forming optical system (130), an excitation filter (200), a through-hole mirror (140), a reflective objective lens (150), and a camera (190). In addition, the microscope may additionally include a third aperture (170) and a light-receiving lens (180).

[0069] The light source (110) emits light to illuminate the object (500) so that the object (500) can be easily observed. In particular, since the bright-and-dark field microscope (100) of the present invention is primarily intended for analyzing biological tissue, the light source (110) is configured to emit white light, which is primarily used for analyzing biological tissue. An example of the light source (110) may be an LED.

[0070] The ring-forming optical system (130) is configured to form light irradiated from a light source (110) into a ring shape, and in one embodiment, may pass the light through a mask in which a ring-shaped empty space is formed. More specifically, the ring-forming optical system (130) may be configured in a form in which a first condensing lens (131), a first aperture (132), a second aperture (133), a ring filter (134), a collimating lens (135), and a second condensing lens (136) are sequentially arranged. The first condensing lens (131) serves to condense light from the light source (110). Noise can be removed from the condensed light as it passes through the first aperture (132) and the second aperture (133). The first aperture (132) mainly plays a role of controlling the outermost size of the light collected by the first condenser lens (131), and the second aperture (133) mainly plays a role of controlling the brightness of the light controlled by the first aperture (132). The ring filter (134) plays a role of making the light from the second aperture (133) into ring-shaped light, and as described above, the light is made into a ring shape by passing it through a mask in which a ring-shaped empty space is formed. The collimating lens (135) plays a role of collimating the light from the ring filter (134), and the second condensing lens (136) plays a role of collecting the light from the collimating lens (135) and sending it to the aperture mirror (140).

[0071] In addition, the present invention further includes first and second apertures (132, 133) to further remove noise and directly control the size and brightness of light while refining light with a lens system arranged before and after the ring filter (134). As described above, the first aperture (132) mainly functions to control the size of light, and the second aperture (133) functions to control the brightness of light. Additionally, in order for the first and second apertures (132, 133) to function properly, as illustrated in FIG. 1, it is preferable that the focus of the light coming from the first condenser lens (131) be formed between the first aperture (132) and the second aperture (133).

[0072] Since the bright-and-dark field microscope (100) of the present invention is intended for use in analyzing biological tissue, the light source (110) is formed to emit white light. Conventional dark-field microscopes mostly use a laser light source with a long coherence length. In the case of a laser light source, not only is it possible to know the wavelength of the laser in advance and set it to a desired range to some extent, but it is also relatively easy to create light with little noise and uniformity by using the laser wavelength known in advance when forming light into a ring shape. However, since white light has a much shorter coherence length than laser light and is a mixture of light of all wavelengths, it is not possible to create light that is uniform and stable enough for observation simply by passing it through a ring-shaped mask. Accordingly, in the present invention, in order to create uniform and stable ring-shaped light even when using white light, a ring-forming optical system (130) composed of several optical components is configured as illustrated in FIG. 1.

[0073] In addition, if a laser light with a long coherence length is used as a light source (110), there is no major problem in creating ring-shaped light with only one ring filter (134). However, since white light has a relatively much shorter coherence length than laser light, if only one ring filter (134) is used, a significant amount of noise is generated in the light passing through the ring filter (134). Accordingly, as the light travels, the outer / inner edges of the ring gradually spread apart, which has a significant negative effect on the analysis results, such as blurring the focus when acquiring an image of the target (500). Therefore, in the present invention, in order to suppress the light from spreading out as much as possible, a first condenser lens (131), a collimating lens (135), and a second condenser lens (136) are arranged before and after the ring filter (134), so as to purify the light as much as possible.

[0074] Here, the filter (200) is configured to excite the parallel light converted in the ring-forming optical system (130), and can excite the transmitted light. The filter (200) can selectively transmit only light of a specific wavelength among the various wavelengths of light irradiated. When the light of this specific wavelength is irradiated on an object (500), the fluorescent material included in the object (500) absorbs the light, transitions to a high energy state, and then returns to a low energy state, thereby emitting light of a predetermined wavelength. By obtaining an image of the light of the emitted wavelength through a camera (190), the presence, location, and size of a specific component can be confirmed. The filter (200) can be included as a separate component, or can be mounted on one surface of the through-hole mirror (140).

[0075] In particular, the present invention relates to a device capable of obtaining a fluorescence image from a field-of-view microscope. In order to obtain a field-of-view image, the excitation filter (200) needs to be removed. Therefore, a movement module may be further included so that the excitation filter (200) is placed on the optical path only during the process of obtaining a fluorescence image. The movement module is configured to be able to place or remove the excitation filter (200) from the optical path. A method for controlling the excitation filter (200) from the movement module will be described below.

[0076] Meanwhile, a collimator (120) may be further included between the light source (110) and the ring-forming optical system (130). The collimator (120) is a configuration that converts the light irradiated from the light source (110) into parallel light.

[0077] The aperture mirror (140) reflects light transmitted through the filter (200) here to an object (500) and illuminates the object (500) to obtain an image. An aperture (145) is formed in the center of the aperture mirror (140), and light reflected from the object (500) passes directly through the aperture (145) and exits.

[0078] The reflective objective lens (150) serves to focus the light reflected from the aperture mirror (140) and irradiated toward the object (500) on the object (500). In other words, the reflective objective lens (150) is a component that directly acquires the image of the object (500). At this time, the reflective objective lens (150) has a slightly different configuration from a general objective lens. In the case of a general objective lens, the path for sending light to the object (500) and the path for the light reflected from the object (500) to pass through the objective lens again are formed in the same manner. However, in the case of the reflective objective lens (150), the light itself sent to the object (500) is formed in a ring shape, which is different in shape from the light directly irradiated to the object (500).

[0079] In order to be able to respond well to this type of light, the reflective objective lens (150) is formed so that the light reflected from the aperture mirror (140) and traveling to the object (500) passes through the edge portion of the reflective objective lens (150), and the light reflected from the object (500) passes through the center portion of the reflective objective lens (150). That is, the reflective objective lens (150) is formed so that the edge portion and the center portion are each formed as separate, independent lenses. By forming the reflective objective lens (150) in this way, the ring-shaped light is first reflected on the mirror portion of the aperture mirror (140), passes through the edge portion of the reflective objective lens (150), and is smoothly irradiated to the object (500). In addition, the light reflected from the target (500) passes through the central portion of the reflective objective lens (150), and thus can smoothly pass through the aperture (145) formed in the center of the aperture mirror (140).

[0080] The camera (190) receives light reflected from the object (500) and passes through the aperture (145) to acquire an image. At this time, in order to improve the quality of the image entering the camera (190), a third aperture (170) that removes noise from light passing through the aperture (145) and a light receiving lens (180) that receives light from the third aperture (170) and sends it to the camera (190) may be further provided between the aperture mirror (140) and the camera (190).

[0081]

[0082] Below, the principles of obtaining a contrast-field image and a fluorescence image according to the configuration are described.

[0083] In order to obtain a dark-field image, light irradiated from a light source (110) is converted into ring-shaped parallel light while passing through a ring-forming optical system (130), and this light is reflected through a through-hole mirror (140) and reaches a target (500) through a reflective objective lens (150), and the light reflected and scattered from the target (500) passes through the through-hole (145) of the through-hole mirror (140), a third aperture (170), and a light-receiving lens (180) and is received by a camera (190) to obtain an image. To explain more clearly, light that passes through the edge of the reflective objective lens (150) is irradiated to the target (500), and light that is reflected and scattered from the target (500) and passes through the center of the reflective objective lens (150) is received by the camera (190). Here, the light passing through the edge of the reflective objective lens (150) plays the same role as the light illuminated by the ring-shaped dark field illumination on the target object (500) in the dark field microscope.

[0084] Here, in order to obtain dark field illumination, the filter (200) can be removed from the optical path so that white light is irradiated to the object (500).

[0085]

[0086] Meanwhile, in order to obtain a fluorescence image, it is necessary to place the configuration of the excitation filter (200) on the optical path in the configuration for obtaining a dark-field image. More specifically, it is formed so that the light converted into parallel light in the ring-forming optical system (130) can be transmitted through the excitation filter (200) and converted into excitation light. Thereafter, the excitation light forms the same optical path as the configuration for obtaining a dark-field image and is received by the camera (190), thereby obtaining a fluorescence image. In this case, there is an advantage in that an image can be obtained even without installing a color-selective mirror in a microscope capable of obtaining a fluorescence image.

[0087] In addition, the present invention may further include a trigger board (300) that forms a trigger signal. Fig. 2 illustrates a block diagram of a bright-field microscope according to an embodiment of the present invention, and Fig. 3 illustrates a pulse signal of a trigger signal in a bright-field microscope according to an embodiment of the present invention.

[0088] Referring to FIG. 2, a trigger signal can generate a pulse signal at a specific timing to turn on / off different configurations according to the pulse signal. In the present invention, a pulse-shaped trigger signal generated from a trigger board (300) can be transmitted to a light source (110) and a camera (190) to turn them on / off.

[0089] Referring to FIG. 3, the trigger board (300) sets one cycle and generates a trigger signal at a predetermined interval in the cycle. For example, one cycle is set to 15 minutes, and a pulse width of 150 msec (hereinafter, the first pulse) and 1 sec (hereinafter, the second pulse) is generated. The interval between the first pulse and the second pulse can be set to 0.5 sec. This trigger signal is transmitted to the camera (190) and the light source (110), and the first pulse and the second pulse of the trigger signal are input to the camera (190) and the light source (110).

[0090] In particular, in the present invention, the light irradiated to the object (500) can be switched by removing or placing the excitation filter (200) on the optical path between the first pulse and the second pulse. More specifically, when the excitation filter (200) is removed from the optical path during the first pulse, the light irradiated to the object (500) from the light source (110) corresponds to white light. Therefore, the image obtained from the camera (190) corresponds to a dark field image. When the excitation filter (200) is placed on the optical path between the first pulse and the second pulse, the light irradiated to the object (500) from the light source (110) corresponds to excitation light. Therefore, the image obtained from the camera (190) corresponds to a fluorescence image.

[0091] Finally, by including a control unit that controls the movement module to remove the excitation filter (200) from the optical path or place the excitation filter (200) on the optical path during the off-period of the pulse in the trigger signal, i.e., between the first pulse and the second pulse, it is possible to simultaneously acquire a dark-field image and a fluorescence image.

[0092] In addition, it has the advantage of being able to check multiple images as one by further including an image processing unit that processes dark field images and fluorescent images acquired through a camera (190) into a single image.

[0093] Hereinafter, the effect of the bright-field microscope (100) of the present invention will be described in more detail through dark-field images and fluorescence images actually obtained through the bright-field microscope (100) of the present invention.

[0094] Figures 4 and 5 illustrate dark-field images and fluorescence images observed with a bright-field microscope according to one embodiment of the present invention.

[0095] Referring to Fig. 4, the photograph relates to the transfer of mitochondria between cells through tunneling nanotubes, and corresponds to images of MSCs (Mitotracker Red) cultured with A-RPE for 48 hours, taken at 10-second intervals for 1 hour. The image on the left corresponds to a fluorescence image obtained by forming an excitation filter (200) on the optical path, and the image on the right corresponds to a dark-field image obtained by removing the excitation filter (200) on the optical path. The present invention has the effect of simultaneously obtaining the two images without a color-selective mirror.

[0096] Referring to FIG. 5, the advantages of images obtainable from the brightfield microscope (100) of the present invention can be confirmed. More specifically, from the upper left to the lower center, a darkfield image (reflective), a MitoTracker Green (GFP) image in ARPE-19, a MitoTracker Red (TxRed) image in MSC, a two-channel fluorescence image in which GFP and TxRed images are merged, and a three-channel image in which GFP, TxRed, and darkfield images are merged are shown. Through the brightfield microscope (100) of the present invention, not only a darkfield image but also a fluorescence image can be obtained, and a multi-channel image in which multiple images are combined can be confirmed.

[0097]

[0098] In addition, the bright-field microscope (100) of the present invention can obtain a bright-field image.

[0099] In order to obtain a bright field image, as explicitly illustrated in FIG. 6, a white scattering plate (160) that is placed behind the object (500) and reflects light may be further included. By means of the white scattering plate (160), the bright field microscope (100) of the present invention can obtain a bright field image of the object (500) as needed.

[0100] As illustrated in FIG. 6, when the white scattering plate (160) is placed behind the object (500), a bright field image can be acquired. When the white scattering plate (160) is placed behind the object (500), light transmitted through the object (500) is reflected and scattered by the white scattering plate (160), as indicated by a small arrow in FIG. 6. When the reflected and scattered light passes through the object (500) again and is received by the camera (190), an image formed by the light reflected and scattered by the white scattering plate (160) is acquired by the camera (190). Here, the light reflected and scattered by the white scattering plate (160) plays the same role as light directly irradiated to the object (500) in a transmission-type bright field microscope. Therefore, when a white scattering plate (160) is placed behind the target object (500) as shown in Fig. 6, the image acquired from the camera (190) becomes a bright field image.

[0101] To elaborate, from the above description, it can be inferred that in the state of FIG. 6, the dark field image obtained by the reflective objective lens (150) is also formed on the camera (190) by the same principle as in the state of FIG. 7. However, in the state of FIG. 6, the bright field image obtained by the light reflected and scattered by the white scattering plate (160) is also obtained, and strictly speaking, the camera (190) forms an image in which the bright field image and the dark field image are overlapped. However, since the bright field image is much brighter than the dark field image (in other words, because it is a much stronger signal), even if the bright field image and the dark field image are overlapped, no significant change occurs in the bright field image, and therefore, it is safe to regard it as a bright field image as is.

[0102] In this way, the bright-field / dark-field microscope (100) of the present invention can easily obtain bright-field / dark-field images of the same object simply by the presence or absence of a white scattering plate (160). To summarize briefly, when the white scattering plate (160) is placed, the light reflected from the white scattering plate (160) acts like illumination of a transmission-type bright-field microscope, so that a bright-field image can be obtained, and when the white scattering plate (160) is removed, the light passing through the edge of the reflection-type objective lens (150) acts like illumination for dark-field, so that a dark-field image can be obtained.

[0103] In the case of conventional bright-field / dark-field microscopes, they are generally formed as a transmission type, and accordingly, a method of acquiring bright-field / dark-field images of an object (500) by replacing bright-field / dark-field illumination was used. However, as explained above, in this process, there is the inconvenience of replacing the illumination and the risk of the object (500) moving during the replacement process. However, the bright-field microscope (100) of the present invention is basically formed as a reflection type, and it is possible to acquire bright-field / dark-field images with just a simple operation of placing or removing a white scattering plate (160) behind the object (500). In addition, since the white scattering plate (160) is sufficiently spaced from the object (500), the risk of the object (500) moving or being damaged when placing / removing the white scattering plate (160) is also significantly reduced. Accordingly, according to the present invention, it is possible to realize the provision of both bright-field and dark-field images of the same object much more smoothly.

[0104] Hereinafter, the effects of the bright-field microscope of the present invention will be described in more detail through bright-field images and dark-field images actually obtained through the bright-field microscope of the present invention.

[0105] Figure 8 is a bright / dark field image of biological tissue containing metal particles. More specifically, Figure 8 is a photograph of rat lung tissue containing TiO2 particles.

[0106] As shown in the bright-field image on the left, the lung tissue of the rat containing the TiO2 particles (500) is stained to allow easy visual identification of the structure of the biological tissue. However, while the structure of the lung tissue itself is clearly identified as a purple line due to the staining, the TiO2 particles, which appear as dark gray dots, appear somewhat unclear. Intuitively, it can be inferred that it will not be easy to clearly determine the distribution of the TiO2 particles not only through visual observation but also through image processing.

[0107] The dark-field image on the right is a dark-field image of the same object (the shapes that appear as purple lines on a white background in the bright-field image and the shapes that appear as red lines on a dark-blue background in the dark-field image are the same). As explained above, dark-field images are advantageous for identifying metal particles because their reflected or scattered light signals are very strong. As explained above, the TiO2 particles appear as very bright dots (very bright white in the image) in the dark-field image on the right, unlike the dark gray ones in the bright-field image on the left. In other words, it has been confirmed that the distribution of TiO2 particles can be identified very clearly and accurately using dark-field images.

[0108] In this way, according to the present invention, it is possible to very easily obtain bright-field images and dark-field images of the same object. Since both bright-field images, which are advantageous for observing biological tissues, and dark-field images, which are advantageous for observing metal particles, can be obtained, the bright-field microscope according to the present invention can be very effectively utilized for tasks such as determining the distribution status of metal particles when metal particles are mixed in biological tissues, as in the example of Fig. 8.

[0109] Figures 9 and 10 are comparative illustrations of a large-area dark field image and a partial enlarged image of the image.

[0110] As previously explained, conventional dark-field microscopes are formed as transmission-type. That is, dark-field illumination that irradiates light in a ring shape is placed behind the object (500), and a dark-field image of the object (500) is acquired by the light irradiated by this dark-field illumination. However, when using dark-field illumination, the problem of difficulty in large-area analysis due to the limitation of the light irradiation angle has been consistently raised in the past.

[0111] On the other hand, the dark-field microscope of the present invention is configured as a reflection type. Accordingly, the limitations of conventional transmission types can be overcome, and it is possible to obtain images of a much larger area than conventional methods, as shown in the left drawings of FIGS. 9 and 10. The right drawings of FIGS. 9 and 10 are partial enlarged images of the left drawings, respectively. It can be intuitively confirmed that even when observing a large area as on the left and then enlarging it as on the right, a considerably high resolution can be sufficiently secured.

[0112] Figure 11 is a bright / dark field image of unstained biological tissue. As shown in the example in Figure 8, staining is a very common practice when observing biological tissue. This widely used staining method for biological tissue analysis has the advantage of allowing easy visual identification of the internal structure of the tissue. However, this staining process carries the risk of tissue damage or loss, leading to a growing demand for label-free analysis that does not require staining.

[0113] The bright-field image on the left is of unstained rat brain tissue. As can be seen, only a hazy, cloudy appearance is discernible, making it nearly impossible to visually discern the internal structure of the tissue.

[0114] The dark-field image on the right is a dark-field image of the same object. As explained several times before, the dark-field image shows strong reflected light and scattered light signals. At this time, the difference in density according to the internal structure of the tissue, which is the object (500), causes a difference in scattering intensity, and this difference appears as a distinct contrast in the dark-field image. Unlike the bright-field image on the left, where only a hazy and blurry stain was recognized, it is clearly confirmed that the location of large wrinkles and the shape of small unit structures are quite distinct in the dark-field image on the right.

[0115] Thus, according to the present invention, high-quality bright / dark field images of the same object can be obtained very easily and smoothly, and using these, the internal structure of biological tissue can be observed and analyzed quite well even in a label-free manner, as shown in the example of Figure 9. Therefore, the present invention can serve as a fundamental solution to the problem of damage and loss of objects due to dyeing.

[0116] Although the present invention has been described above with reference to limited embodiments, the present invention is not limited thereto, and it is obvious that various modifications and variations are possible within the scope of the technical idea of ​​the present invention and the equivalent scope of the patent claims to be described below by a person having ordinary skill in the art to which the present invention pertains.

Claims

1. Light source; A ring-forming optical system that converts light irradiated from the above light source into parallel light in a ring shape; An excitation filter that excites the light converted in the above ring-forming optical system; A through-hole mirror that reflects light passing through the above-described filter onto an object and passes light reflected from the object through an through-hole; A reflective objective lens that forms a focus on the object of light reflected from the above-mentioned through-mirror and irradiated toward the object; and A bright-field microscope including a camera that receives light reflected from the object and passes through the aperture of the aperture mirror to obtain an image of the object.

2. In paragraph 1, A brightfield microscope further comprising a movable module capable of moving the above-described filter so as to be placed or removed from the optical path.

3. In paragraph 2 A brightfield microscope further comprising a trigger board that transmits a pulse-shaped trigger signal to the light source and the camera to turn the light source and the camera on and off.

4. In paragraph 3, A brightfield microscope further comprising a control unit that controls the moving module to remove the excitation filter from the optical path or to place the excitation filter on the optical path during the off-period of the pulse in the trigger signal.

5. In paragraph 4, A bright-field microscope further comprising an image processing unit that processes dark-field images and fluorescence images acquired through the above camera into a single image.

6. In paragraph 1, A bright-field microscope further comprising a collimator that converts light irradiated from the above light source into parallel light.

7. In paragraph 1, A bright-field microscope further comprising a white scattering plate positioned at the rear of the object to reflect light.

8. In paragraph 7, A bright-and-dark field microscope, wherein the white scattering plate is detachably provided at a certain interval behind the object.

9. In paragraph 8, The above bright-field microscope is, When the white scattering plate is placed behind the object, light reflected and scattered from the white scattering plate passes through the object and is received by the camera, thereby obtaining a bright field image of the object. A bright-field microscope characterized in that when the white scattering plate is removed from the rear of the object, only light reflected and scattered from the object is received by the camera, thereby obtaining a dark-field image of the object.

10. In paragraph 1, The above light source is, A bright-field microscope characterized by being formed to emit white light.

11. In paragraph 1, The above ring forming optical system, A bright-and-dark field microscope further comprising a first condenser lens for condensing light from the light source, a ring filter for forming the light condensed by the first condenser lens into a ring-shaped light, a collimating lens for collimating the light condensed from the ring filter, and a second condenser lens for condensing the light condensed from the collimating lens and sending it to the aperture mirror.

12. In paragraph 11, The above ring filter, A bright-field microscope characterized in that it forms a ring shape by passing light through a mask in which a ring-shaped empty space is formed.

13. In paragraph 11, The above ring forming optical system, A bright-and-dark field microscope, characterized in that it further includes a first aperture that controls the outermost size of light collected by the first focusing lens, and a second aperture that controls the brightness of the light controlled by the first aperture and sends it to the collimating lens.

14. In paragraph 13, The above ring forming optical system, A bright-and-dark field microscope characterized in that the focus of light coming from the first condenser lens is formed between the first aperture and the second aperture.

15. In paragraph 1, The above reflective objective lens is, A bright-and-dark field microscope characterized in that light reflected from the above-mentioned through-hole mirror and traveling to the target passes through the edge portion of the above-mentioned reflective objective lens, and light reflected from the above-mentioned target passes through the center portion of the above-mentioned reflective objective lens.

16. In paragraph 15, The above reflective objective lens is, A bright-field microscope characterized in that the edge and central portions are each formed by separate, independent lenses.

17. In paragraph 1, The above bright-field microscope is, A bright-and-dark field microscope characterized by further comprising a third aperture for removing noise from light passing through the above aperture, and a light-receiving lens for receiving light from the third aperture and sending it to the camera.

Images