KR20240079551A - Automatic excitation light control device for optical microscope and automatic excitation light control method - Google Patents

Abstract

The present invention relates to an automatic excitation light control device and method for automatic excitation light control for an optical microscope that can maintain the irradiation position of excitation light that excites fluorescent molecules even if the distance to the objective lens changes due to movement of the sample area. The present invention includes: a first light source installed in the light source unit of an optical microscope and generating excitation light to excite phosphors of a sample; a second light source that generates a guide beam to form a focus on the sample surface; a beam combining unit that matches irradiation directions of the excitation light and the guide beam; and a third beam control unit that adjusts the irradiation range and irradiation position of the excitation light according to changes in the focus position of the guide beam formed on the sample surface. It provides an automatic excitation light control device for an optical microscope.

Description

Automatic excitation light control device for optical microscope and automatic excitation light control method}

The present invention relates to an automatic excitation light control device and an automatic excitation light control method for an optical microscope. More specifically, the irradiation position of the excitation light that excites fluorescent molecules is determined even if the distance from the objective lens changes due to movement of the sample area. It relates to an automatic excitation light control device and method for automatic excitation light control for an optical microscope that can be maintained.

An optical microscope is an optical device that creates enlarged images of very small objects or structures that cannot be seen or are not visible to the naked eye. Optical microscopes are divided into fluorescence microscopes, metallurgical microscopes, polarizing microscopes, interference microscopes, phase contrast microscopes, dark field microscopes, and bright field microscopes according to their principles. A dual fluorescence microscope is an optical microscope that uses fluorescence for imaging. When a specific wavelength absorbed by a fluorescent substance present in a sample is illuminated, the light absorbed by the fluorescent substance detects long-wavelength light that comes out in the form of fluorescence. This allows imaging.

Fluorescence microscopes use wavelength-specific filters to detect emitted light that is much weaker than the emitted light. A fluorescence microscope consists of a light source using xenon, mercury, LED, or laser, an excitation filter, a dichroic mirror, and an emission filter. The light from the light source passes only the wavelengths that can be absorbed by the fluorescent material through the excitation light filter, and illuminates the sample through the dichroic mirror. The fluorescent substance in the sample absorbs light of this specific wavelength and emits long-wavelength light in the form of fluorescence. The light emitted in this way is not reflected by the dichroic mirror but is detected by a detector. In the case of a multicolor fluorescence microscope, fluorescent substances of various colors present in a sample can be imaged by mounting excitation light filters and dichroic mirrors corresponding to each color (see Figure 2).

Such fluorescence microscopes are used to image intracellular organelles and proteins, and include confocal microscopes and total internal reflection fluorescence microscopes.

This fluorescence microscope measures the type and amount of the biomarker by attaching a fluorescent substance to the biomarker and then exciting and observing the fluorescent substance. However, in the case of existing microscopes, excitation light is supplied from the direction of the objective lens, and as a result, auto-fluorescence due to the excitation light is generated in the objective lens, which has the disadvantage of lowering the signal-to-noise ratio and making accurate observation difficult. In addition, the excitation light supplied in this way has the disadvantage of intensifying photobleaching of the sample (see Figure 3).

To improve this, a microscope that supplies excitation light from a direction opposite to the objective lens based on the sample is used. This type of microscope has the advantage of minimizing the excitation light incident on the objective lens by supplying the excitation light from the top of the sample and at the same time supplying the excitation light at a constant angle (see FIG. 4). In addition, the excitation light incident on the objective lens can be reduced to close to 0 by additionally placing a prism on top of the sample to completely reflect the excitation light.

However, when excitation light is supplied from the other side of the objective lens, the excitation light irradiation position may change as the sample surface moves, making accurate observation difficult (see Figure 5).

Therefore, there is a need for a new method of excitation light supply device to improve the shortcomings of these microscopes.

(0001) Republic of Korea Patent No. 10-0942195 (0002) Republic of Korea Patent No. 10-2290325

In order to solve the above-described problem, the present invention provides an automatic excitation light control device and an automatic excitation light control device for an optical microscope that can maintain the irradiation position of the excitation light that excites fluorescent molecules even if the distance to the objective lens changes due to movement of the sample area. The purpose is to provide a method for controlling excitation light.

In order to solve the above-described problem, the present invention includes: a first light source installed in the light source unit of an optical microscope and generating excitation light for exciting phosphors of a sample; a second light source that generates a guide beam to form a focus on the sample surface; a beam combining unit that matches irradiation directions of the excitation light and the guide beam; and a third beam control unit that adjusts the irradiation range and irradiation position of the excitation light according to changes in the focus position of the guide beam formed on the sample surface. It provides an automatic excitation light control device for an optical microscope.

In one embodiment, the excitation light and the guide beam may be irradiated from the opposite side of the objective lens with respect to the sample surface.

In one embodiment, the excitation light and the guide beam may be irradiated at an angle of 5 to 85 degrees with respect to the sample surface.

In one embodiment, the third beam control unit simultaneously controls the excitation light and the guide beam that passed through the beam combining unit, and maintains the focus of the guide beam at a constant position to adjust the irradiation position and irradiation range of the excitation light. It can be adjusted.

In one embodiment, it may include a first beam control unit that adjusts the irradiation position and irradiation range of the excitation light.

In one embodiment, it may include a second beam control unit that adjusts the focus and irradiation position of the guide beam.

In one embodiment, the guide beam forms a focus on the sample surface, and the excitation light can be irradiated to the entire sample surface.

In one embodiment, the guide beam may form a focus at the center of the excitation light irradiation area.

The present invention also provides a method of automatic excitation light control using an automatic excitation light control device.

In one embodiment, the automatic excitation light control method includes the steps of (a) adjusting the focus of the objective lens by observing a random sample or image for focus adjustment; (b) uniformly irradiating the excitation light to the observation area of the objective lens; (c) irradiating the guide beam to form a focus at the center of the excitation light supply area; (d) measuring the focal position of the guide beam; (e) supplying a sample to the observation area of the objective lens and then adjusting the focus of the objective lens; and (f) adjusting the third beam control unit so that the focus of the guide beam is maintained at a constant position.

In one embodiment, the guide beam may be observed through a first camera, and fluorescence generated from the sample may be observed through a second camera.

In one embodiment, the excitation light may be blocked after step (d) and re-irradiated after step (f) to excite the sample.

In one embodiment, the guide beam may be blocked when the excitation light is re-irradiated after step (f).

The automatic excitation light control device and automatic excitation light control method for an optical microscope according to the present invention improve the uneven supply of excitation light due to a change in the distance between the objective lens and the sample surface in fluorescence observation using a conventional optical microscope, thereby improving the fluorescence of the sample. Observation can be made easier.

In addition, the automatic excitation light control device and automatic excitation light control method for an optical microscope of the present invention automatically adjust the irradiation position and range of the excitation light, so that there is a change in the distance between the objective lens and the sample surface due to a change in the sample or a change in the focus of the objective lens. Even if the distance varies, the sample can be observed accurately without any manipulation.

Figure 1 shows the structure of an optical microscope including automatic excitation light control according to an embodiment of the present invention.
Figure 2 shows the structure of a conventionally used fluorescence microscope.
Figure 3 shows the structure of a fluorescence microscope that supplies excitation light in the direction of a conventionally used objective lens.
Figure 4 shows the structure of a fluorescence microscope that supplies excitation light from the top of the sample.
Figure 5 shows the change in the excitation light supply position according to the change in the position of the sample surface.
Figure 6 shows the change in the position of the excitation light according to the change in the position of the sample surface when observed from the objective lens direction.
Figure 7 shows the determined focus position of the guide beam according to an embodiment of the present invention.
Figure 8 shows the movement and shape change of the guide beam according to the change in the distance between the sample surface and the objective lens according to an embodiment of the present invention.
Figure 9 shows that pixel movement of a guide beam below the decimal point can be detected according to an embodiment of the present invention.
Figure 10 is a photograph showing the positional change and automatic alignment of excitation light according to an embodiment of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail. In describing the present invention, if it is determined that a detailed description of related known technologies may obscure the gist of the present invention, the detailed description will be omitted. Throughout the specification, singular expressions should be understood to include plural expressions, unless the context clearly indicates otherwise, and terms such as “comprise” or “have” refer to the described features, numbers, steps, operations, or components. , it is intended to specify the existence of a part or a combination thereof, but should be understood as not excluding in advance the possibility of the existence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof. . In addition, when performing a method or manufacturing method, each process forming the method may occur differently from the specified order unless a specific order is clearly stated in the context. That is, each process may occur in the same order as specified, may be performed substantially simultaneously, or may be performed in the opposite order.

The technology disclosed in this specification is not limited to the implementation examples described herein and may be embodied in other forms. However, the implementation examples introduced here are provided to ensure that the disclosed content is thorough and complete and that the technical idea of the present technology can be sufficiently conveyed to those skilled in the art. In order to clearly express the components of each device in the drawing, the sizes of the components, such as width and thickness, are shown somewhat enlarged. When describing the drawing as a whole, it is described from the observer's point of view, and when an element is mentioned as being located above another element, this means that the element may be located directly above another element or that additional elements may be interposed between those elements. Includes. Additionally, those skilled in the art will be able to implement the idea of the present invention in various other forms without departing from the technical idea of the present invention. In addition, the same symbols in a plurality of drawings refer to substantially the same elements.

As used herein, the term 'and/or' includes a combination of a plurality of listed items or any of a plurality of listed items. In this specification, 'A or B' may include 'A', 'B', or 'both A and B'.

The present invention includes: a first light source installed in the light source unit of an optical microscope and generating excitation light to excite phosphors of a sample; a second light source that generates a guide beam to form a focus on the sample surface; a beam combining unit that matches irradiation directions of the excitation light and the guide beam; and a third beam control unit that adjusts the irradiation range and irradiation position of the excitation light according to changes in the focus position of the guide beam formed on the sample surface. It relates to an automatic excitation light control device for an optical microscope.

Figure 1 shows an optical microscope including an automatic excitation light control device of the present invention. Since the automatic excitation light control device and control method of the present invention can be mainly used in a fluorescence microscope that uses excitation light, it will be described based on this.

The first light source 110 is a light source for generating excitation light 111 and can generate light including a wavelength capable of exciting fluorescence contained in the sample. Generally, a short-wavelength laser is used to excite the phosphor, and separately, white light with high color rendering can also be used. White light with such high color rendering can be generated through a high color rendering light emitting device.

The high-color rendering white light-emitting device has a smooth emission spectrum distributed throughout visible light, and also emits light so that this spectrum extends to the ultraviolet region, which is commonly used as excitation light, so it has an emission pattern similar to sunlight. In the case of this high color rendering light emitting device, light is emitted by mixing various light emitters and phosphors, so excitation light of a desired spectrum can be formed using an excitation light filter. In particular, in the case of existing light sources, only light with a narrow spectrum is supplied, so if excitation light other than the spectrum supplied by the light source is required, the light source itself must be replaced. However, when a high color rendering white light-emitting device is used as described above, the light source The desired excitation light can be formed simply by replacing the excitation light filter without replacement.

Additionally, in addition to the white light-emitting device, a laser emitting monochromatic light may be used as a light source. In this case, it is preferable that the monochromatic light emitted by the laser can simultaneously excite the phosphors included in the sample. The laser can have a higher luminous intensity compared to a light source using the light emitting device and can emit monochromatic light with a narrow spectral band. Therefore, since the phosphor can be excited more brightly, this laser light source can be used if multiple types of phosphors included in the sample can use the same excitation light. However, when a wide spectrum is required to excite the phosphor, it is preferable to use the high color rendering white light-emitting device, so it is desirable to select and use an appropriate light source according to the conditions of each experiment.

A first beam control unit 112 may be installed in the first light source 110. The first beam control unit 112 can adjust the irradiation position and irradiation range of the excitation light 111 generated from the first light source 110 by combining a plurality of lenses. At this time, the first beam control unit 112 is a part that manually adjusts the position rather than automatic excitation light control, which will be described later, and is located in the observation range of the objective lens 310 before the automatic position adjustment step using the guide beam 121. This is the part that aligns the irradiation range and irradiation position of the excitation light to match.

That is, in the case of the first beam control unit 112, the initial irradiation position of the excitation light 111 is determined before observation of the sample, and the irradiation range of the excitation light 111 is limited to suit the observation range of the objective lens 310. The excitation light 111 is irradiated to an unoccupied location to prevent photobleaching from occurring.

Additionally, the first beam control unit 112 may adjust the brightness and block the excitation light 111. In the case of the excitation light 111, it is preferable to adjust the amount of light to a desired amount, and it is preferable that such brightness adjustment is performed before observation of the sample 200. Therefore, the first beam control unit 112 may include a brightness control unit for the excitation light 111. In addition, since the excitation light 111 must be irradiated only at a desired time to minimize photobleaching, photobleaching of the sample can be minimized by installing an excitation light blocking unit in the first beam control unit 112. That is, the excitation light blocking unit does not block the excitation light while aligning the excitation light, and blocks the excitation light during the sample supply and automatic excitation light control steps to minimize the time for which the excitation light is irradiated to the sample. can do.

The guide beam 121 is supplied to the center of the excitation light 111, and unlike the excitation light 111 that is irradiated over a wide area, the guide beam 121 can be supplied to form a focus on the sample surface (see Figure 1). That is, in the case of the guide beam 121, it is desirable to irradiate the sample surface 210 to have a very narrow range. This is to find the exact position by fitting the shape of the focus of the guide beam 121 using a Gaussian function or Poisson function.

Additionally, the guide beam 121 may have the same or shorter wavelength as the excitation light 111. In general, when the guide beam 121 and the excitation light 111 have the same or similar wavelength, the fluorescence of the sample can be excited by the guide beam 121, and also by using the guide beam 121. Therefore, photobleaching may occur while adjusting the position of the excitation light. However, in the case of the present invention, the guide beam 121 is irradiated only to a very narrow area (focus, 220) in the center of the excitation light as shown above, so the influence on the observation results can be minimized. In particular, when using a shorter wavelength than the excitation light 111 and having a wavelength that does not excite the phosphor of the sample, it is possible to minimize this photobleaching effect.

In addition, in order to distinguish between the guide beam 111 and the fluorescence generated from the sample 200, the guide beam 121 may have a longer wavelength than the fluorescence generated from the sample 200. In general, the excitation light 111 has a shorter wavelength than the fluorescence generated by exciting the phosphor, so when using the guide beam 121 with a longer wavelength than the fluorescence, photobleaching of the phosphor can be avoided.

The second light source 120 is used to generate the guide beam 121, and a commonly used light source may be used. The guide beam 121 may also use a light source having the same high color rendering as the excitation light 111, or a short-wavelength laser may be used as the light source. Since the description thereof is the same, it will be omitted.

A second beam control unit 122 may be installed in the second light source 120. The second beam control unit 122 is used to control the focus and position of the guide beam 121 and can independently control the guide beam 121 in the same way as the first beam control unit 112. That is, the second beam control unit 122 is also a part that performs initial alignment of the guide beam 121 regardless of automatic excitation light control, which will be described later.

At this time, the second beam control unit 122 preferably adjusts the guide beam 121 to form a focus at the center of the excitation light 111 before observing the sample 200, and also controls the photobleaching area of the sample. In order to minimize the scope of investigation, the scope of investigation can be minimized. To this end, the second beam control unit 122 may include a focus control unit, a guide beam brightness control unit, a guide beam position control unit, and a guide beam blocking unit.

The focus adjuster is for forming a focus of the guide beam 121 and allows the guide beam to form a focus on the sample surface 210. At this time, the focus of the guide beam is aligned before observing the sample, and initial focus alignment can be performed through the focus adjuster.

The guide beam brightness control unit is for controlling the brightness of the guide beam 121 and can minimize the brightness of the guide beam to minimize the photobleaching area. That is, the brightness of the guide beam can be adjusted just enough to confirm the position in automatic excitation light control, and thus photobleaching caused by the guide beam can be minimized.

The guide beam position control unit is a part that adjusts the initial position of the guide beam 121, and can generally ensure that the focus position of the guide beam 121 is located at the center of the irradiation range of the excitation light 111. However, if the fluorescence generated from the sample is concentrated in the center, it is possible to move the position of the guide beam to one side of the irradiation range of the excitation light.

The guide beam blocking unit is used to block and supply the guide beam at a desired time, and may generally be installed integrated with the guide beam brightness control unit, but may also be installed independently. In the case of the guide beam blocking unit, the guide beam can be blocked at all times when observing the fluorescence of the sample, and through this, noise formation caused by the guide beam can be minimized when observing the fluorescence of the sample.

The beam combining unit 130 is used to match the irradiation directions of the excitation light 111 and the guide beam 111. In the case of the present invention, as seen above, the focus of the guide beam 121 is formed at the center of the excitation light 111. At this time, if the irradiation directions of the guide beam 121 and the excitation light 111 do not match, when the distance between the sample surface 210 and the objective lens 310, which will be described later, changes, the guide beam 121 and the The amount of movement of the excitation light 111 may vary. Therefore, it is desirable to make the excitation light 111 and the guide beam 121 move as one by matching the irradiation direction. 130), the first light source 110 and the second light source 120 form an angle of 90° and may supply the excitation light 111 and the guide beam 121 (see also FIG. 1). In order to match the irradiation directions of the light 111 and the guide beam 121, a dichroic mirror can be used when the wavelengths of the excitation light and the guide beam are different, and the wavelengths of the excitation light and the guide beam are similar. or if they match, a neutral density filter can be used.

The dichroic mirror is a type of mirror that reflects light in a specific wavelength band and transmits light in the remaining wavelength band. In the case of the present invention, the dichroic mirror reflects one wavelength of the excitation light and the guide beam. Selective reflection and transmission can be achieved using wing mirrors.

In addition, the neutral density filter is a type of mirror that reflects part of the incident light and transmits part of it. When used, it reflects and transmits part of the excitation light and the guide beam to match the irradiation direction. You can do it.

The third beam control unit 140 is a part that simultaneously controls the excitation light 111 and the guide beam 121 that passed through the beam combining unit 130, and is specifically formed on the sample surface 210. The irradiation range and irradiation position of the excitation light can be adjusted according to changes in the position of the focus 220 of the guide beam 121.

Looking at this in detail, in the case of the sample surface 210, the distance from the objective lens 310 must be kept constant, but the distance and position may vary due to movement of the sample, change in the sample, and changes in the curvature of the cover glass and slide glass. can change. At this time, in the case of a commonly used fluorescence microscope, the excitation light 111 is supplied from a certain position, so the irradiation position and irradiation range of the excitation light may change due to the above movement (see FIG. 5).

In this case, it is common for the focus of the objective lens 310 to change, but since the objective lens 310 has a focus control device, the focus can be adjusted. However, the excitation light is adjusted so that the sample 200 is located in the center. Problems may arise due to changes in the survey location. In the case of the present invention, as seen above, the focus 220 of the guide beam 121 is located in the center of the excitation light 111, so simply adjusting the guide beam 220 so that it is located in the center is sufficient to control the excitation light 111. Light 111 can be irradiated at an accurate location.

Therefore, in the case of the third beam adjusting unit 140, the guide beam 121 and the excitation light 111 that passed through the beam combining unit 130 are simultaneously adjusted to irradiate the excitation light 111 at an accurate location. In this case, by maintaining a constant position of the guide beam 121 based on the guide beam 121, the excitation light can be accurately irradiated to the desired location.

In addition, since the change in distance between the sample surface 210 and the objective lens 310 as described above may also affect the irradiation range of the excitation light, in the case of the present invention, it is also possible to keep the irradiation range of the excitation light constant. Accordingly, it is possible to prevent photobleaching from occurring when the excitation light is supplied to an undesired range.

Additionally, in order to accurately measure the position of the guide beam, the shape of the guide beam focus can be fitted using a Gaussian function or Poisson function. In this way, by fitting the focus of the guide beam using a Gaussian function or Poisson function, the center point of the guide beam focus can be found to the pixel unit below the decimal point, and the third beam control unit is used to maintain this center point at a constant position. The excitation light can be accurately controlled by adjusting the irradiation position of the guide beam.

As shown below in FIG. 9, the brightness of each pixel of the measured image is calculated using a two-dimensional Gaussian function. You can find the center point of the guide beam focus in units of pixels below the decimal point by entering x ₀ and y _o to minimize the error by the least square method. Additionally, if the position of the excitation light does not need to be controlled very precisely, this fitting can be omitted and the position of the brightest pixel of the guide beam can be used as a reference.

The excitation light 111 and the guide beam 121 may be irradiated from the opposite side of the objective lens 310 with respect to the sample surface 210 (see FIGS. 1 and 4). In the case of a commonly used fluorescence microscope, the excitation light is irradiated to the sample surface in the same direction as the objective lens (see Figures 2 and 3). In this case, auto-fluorescence, or noise, is generated in the objective lens by the excitation light, and this noise lowers the signal-to-noise ratio, making accurate fluorescence observation difficult. In the case of the present invention, in order to minimize the occurrence of such noise, the noise caused by the excitation light can be minimized by irradiating the excitation light 111 from the upper part of the sample surface.

In addition, when the excitation light is directly incident on the objective lens during fluorescence observation, observation of fluorescence may be difficult for the same reason. That is, when the excitation light is irradiated in the direction of the objective lens, auto-fluorescence is generated in the objective lens due to the excitation light, which lowers the signal-to-noise ratio, making accurate observation difficult. Therefore, it is preferable that the excitation light 111 and the guide beam 121 used in the present invention are irradiated at an angle of 5 to 85° with the sample surface 210 (see FIG. 4). Through this, the excitation light 111 can excite the phosphor of the sample, but most of the excitation light 111 is irradiated to the outside of the objective lens, thereby greatly increasing the signal-to-noise ratio.

However, in this case, the guide beam 121 may also be difficult to observe in the direction of the objective lens 310, but unlike the excitation light 111, the guide beam 121 is concentrated in a narrow area to form a focus, Since some of the guide beam may be incident in the direction of the objective lens due to scattering from the sample surface 210, it is possible for the guide beam to be observed through the objective lens 310.

At this time, if the angle is less than 5°, it is difficult to avoid interference with components for mounting the sample, and if the angle exceeds 85°, the excitation light is incident on the objective lens, which may lower the signal-to-noise ratio. In addition, as the angle decreases, the excitation light takes the form of an elongated ellipse with a large ratio of the long axis to the short axis. A pair of cylindrical lenses is added to the first beam control unit 112 or the second beam control unit 140. By arranging the light to reduce the long axis or increase the short axis of the incident light, the excitation light can be made in the form of a circle.

The present invention also relates to a method of automatic excitation light control using an automatic excitation light control device.

Hereinafter, the present invention will be described in detail through an automatic excitation light control method.

The automatic excitation light control method includes the steps of (a) adjusting the focus of the objective lens by observing a random sample or image for focus adjustment; (b) uniformly irradiating the excitation light to the observation area of the objective lens; (c) irradiating the guide beam to form a focus at the center of the excitation light supply area; (d) measuring the focal position of the guide beam; (e) supplying a sample to the observation area of the objective lens and then adjusting the focus of the objective lens; and (f) adjusting the third beam control unit so that the focus of the guide beam is maintained at a constant position.

Step (a) is a step of adjusting the focus of the objective lens 310 by observing a random sample or image for focus adjustment. At this time, it is desirable to use the same or similar sample as the sample requiring observation, and adjust the objective lens 310 to align the initial distance between the objective lens 310 and the sample surface 210. You can adjust the focus to obtain a clear image. Additionally, in addition to the above arbitrary samples, images for focus adjustment can be used, and in the case of these images for focus adjustment, grid patterns, numbers, or letters can be used to easily adjust focus. In the case of these images, changes in focus can be intuitively detected, allowing users to easily adjust focus and initially align the objective lens.

The step (b) is a step of uniformly radiating the excitation light 111 to the observation area of the objective lens 310. At this stage, the irradiation angle, brightness, irradiation range, etc. of the excitation light 111 can be adjusted, and such adjustment can be performed through the first beam adjuster 112. Also, at this stage, it is desirable to adjust the irradiation angle of the excitation light 111 so that the excitation light 111 is as close to a circular shape as possible and is not directly irradiated to the objective lens 310. In addition, since the irradiation angle of the guide beam, which will be described later, can be integrated with the excitation light, it is desirable to adjust the irradiation angle of the excitation light at the same time.

The step (c) is a step of irradiating the guide beam 121 to form a focus 220 at the center of the excitation light supply area. In this step, the focus 220 of the guide beam 121 can be adjusted to be located at the center of the excitation light 111 while the excitation light 111 is being irradiated. The brightness can be adjusted to achieve appropriate brightness.

As described above, initial alignment of the excitation light and the guide beam may be completed through steps (a), (b), and (c). When this step is completed, the initial alignment of the observed sample is completed, and automatic adjustment and observation, which will be described later, can be performed based on the completed initial alignment.

Step (d) is a step of measuring the focus position of the guide beam (see FIG. 7). In the case of the guide beam 121, a focus 220 may be formed on the sample surface 210 to be located at the center of the excitation light 111 and have a constant size. Therefore, the movement of the sample surface 210 can be detected by measuring the position of the focus 220 of the guide beam 121, and in order to detect this movement, the first focus can be measured and used as a reference point. .

In addition, it is preferable that the excitation light 111 is blocked after step (d) is completed. In the subsequent step, a sample combined with a phosphor is supplied, and if the excitation light is continuously irradiated, the phosphor may be exposed for a long period of time and photobleaching may occur. Therefore, after the initial alignment and initial focus are measured as described above, it is desirable to block the excitation light to prevent photobleaching.

The step (e) is a step of supplying the sample 200 to the observation area of the objective lens 310 and then adjusting the focus of the objective lens 310. At this stage, a sample for observation can be supplied, and the focus of the objective lens can be finely adjusted to facilitate observation of the sample. However, at this stage, as seen above, a height difference may occur due to a change in the sample surface, and the irradiation position of the excitation light may change accordingly, so an automatic excitation light control step, which will be described later, is required.

Step (f) is a step of adjusting the third beam control unit 140 so that the focus of the guide beam is maintained at a constant position. When the sample 200 is supplied in step (f), a height difference may occur due to a difference in the sample surface 210. At this time, the change in the sample surface as described above can change the irradiation position of the excitation light 111 (see Figure 6) and also change the focal position of the guide beam (see Figure 8). Therefore, by moving the guide beam 121 to the focal position measured in step (d) (see FIG. 7), the excitation light can also be irradiated at the correct position.

The position of the guide beam can be adjusted through the third beam control unit 140. In the case of the third beam control unit 140, the guide beam and the excitation light that pass through the beam combining unit 130 Since can be adjusted at the same time, the excitation light can be aligned to an accurate position simply by adjusting the position of the guide beam as described above.

After step (f) is completed as above, the fluorescence of the sample can be observed. For this purpose, the excitation light blocked after step (d) can be re-irradiated, and it is also possible to block the guide beam.

In addition, the guide beam and the fluorescence may be photographed and observed through one camera, but preferably, they can be observed through two cameras 330 and 340, respectively (see FIG. 1). To this end, a dichroic mirror 320 is installed at the rear end of the objective lens 310, that is, on the opposite side of the sample based on the objective lens, to separate the fluorescence generated from the sample and the guide beam. In addition, since the guide beam forms powerful light focused in a narrow range, if the guide beam cannot be completely separated through the dichroic mirror, a dichroic filter is additionally installed in front of the fluorescence observation camera 330. Can be installed.

Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings so that those skilled in the art can easily implement them. Additionally, in describing the present invention, if it is determined that a detailed description of a related known function or known configuration may unnecessarily obscure the gist of the present invention, the detailed description will be omitted. Additionally, some features presented in the drawings are enlarged, reduced, or simplified for ease of explanation, and the drawings and their components are not necessarily drawn to appropriate scale. However, those skilled in the art will easily understand these details.

Example 1

As shown in Figure 1, an automatic excitation light control device was installed at the light source. Afterwards, the alignment sample was inserted into the area where the sample was placed, and the focus of the objective lens was aligned so that the alignment sample was clearly observed. After the alignment of the objective lens was completed, the excitation light was irradiated to the observation area of the objective lens using the first light source and the first beam control unit, and the excitation light was adjusted so that it was not directly irradiated to the objective lens. At this time, the angle at which the excitation light was incident on the sample surface was measured to be 43 degrees.

The second beam control unit was adjusted so that the guide beam could be focused at the center of the excitation light irradiation area formed on the sample surface. At this time, the guide beam used a laser with a peak wavelength of 450 nm.

In order to check the position coordinates of the guide beam, the guide beam was fitted with a Gaussian function, and this was continuously performed to display the coordinates (Y) of the center point of the guide beam.

Excitation light was irradiated to the sample, the sample was observed with a sample observation camera, and the position of the objective lens was finely adjusted to show the sample most clearly. At this time, the position of the guide beam (initial Y coordinate) was determined to be 200 (yellow line). .

After the alignment using the second beam controller was completed, the sample surface was moved up and down to check the change in the guide beam when the distance from the objective lens changed.

As shown in FIG. 8, it was confirmed that the center of the guide beam moves by about 10 pixels every time the distance moves by 1 ㎛ (in FIG. 8, Y is the amount of change in the distance between the sample surface and the objective lens), through which the guide It was confirmed that when the coordinates of the beam were maintained at 200, the distance between the sample surface and the objective lens could also be maintained.

In addition, as shown in Figure 9, in the case of the present invention, fitting using a Gaussian function is performed, so it is possible to check the pixel range below the decimal point rather than an integer multiple of the pixel, which means that more precise control is possible when using the guide beam of the present invention. it means. Since the center of the guide beam moves by about 10 pixels every time the distance moves by 1 ㎛, it can be seen that when the center of the guide beam moves by 1 pixel, the distance between the sample surface and the objective lens moves by 100 nm, and 0.1 pixel is 10 nm. , it can be seen that the 0.01 pixel has moved by 1 nm. In this way, very precise control was possible by fitting with a Gaussian function.

Example 2

Using the results of Example 1, it was confirmed whether the movement of excitation light could be aligned. As shown in Figure 10, when the sample surface was moved up and down (i.e., when the distance between the sample surface and the objective lens was changed), it was confirmed that the excitation light was moved upward or downward, but the position of the guide beam was changed. When moved to the initial position, it was confirmed that the excitation light was also aligned at the center.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

110: first light source
111: excitation light
112: first beam control unit
120: second light source
121: guide beam
122: second beam control unit
130: Beam coupling part
140: third beam control unit
200: sample
210: Sample side
220: focus
310: Objective lens
320: dichroic mirror
330: Fluorescence observation camera
340: Guide beam observation camera

Claims (13)

It is installed in the light source part of the optical microscope,
a first light source that generates excitation light to excite phosphors in the sample;
a second light source that generates a guide beam to form a focus on the sample surface;
a beam combining unit that matches irradiation directions of the excitation light and the guide beam; and
a third beam control unit that adjusts the irradiation range and irradiation position of the excitation light according to changes in the focal position of the guide beam formed on the sample surface;
Automatic excitation light control device for an optical microscope comprising a.
According to paragraph 1,
An automatic excitation light control device for an optical microscope in which the excitation light and the guide beam are irradiated from the opposite side of the objective lens with respect to the sample surface.
According to paragraph 2,
An automatic excitation light control device for an optical microscope in which the excitation light and the guide beam are irradiated at an angle of 5 to 85° with the sample surface.
According to paragraph 1,
The third beam control unit simultaneously controls the excitation light and the guide beam that passed through the beam combining unit,
An automatic excitation light control device for an optical microscope that controls the irradiation position and irradiation range of the excitation light by maintaining the focus of the guide beam at a constant position.
According to paragraph 1,
An automatic excitation light control device for an optical microscope including a first beam control unit that adjusts the irradiation position and irradiation range of the excitation light.
According to paragraph 1,
An automatic excitation light control device for an optical microscope including a second beam control unit that adjusts the focus and irradiation position of the guide beam.
According to paragraph 1,
The guide beam forms a focus at the sample plane,
An automatic excitation light control device for an optical microscope in which the excitation light is irradiated to the entire area of the sample surface.
In clause 7,
An automatic excitation light control device for an optical microscope wherein the guide beam forms a focus at the center of the excitation light irradiation area.
A method of automatically controlling excitation light using the automatic excitation light adjustment device of any one of claims 1 to 8.
According to clause 9,
The automatic excitation light control method is,
(a) adjusting the focus of the objective lens by observing a random sample or image for focus adjustment;
(b) uniformly irradiating the excitation light to the observation area of the objective lens;
(c) irradiating the guide beam to form a focus at the center of the excitation light supply area;
(d) measuring the focal position of the guide beam;
(e) supplying a sample to the observation area of the objective lens and then adjusting the focus of the objective lens; and
(f) adjusting the third beam control unit so that the focus of the guide beam is maintained at a constant position;
Automatic excitation light control method including.
According to clause 10,
The guide beam is observed through a first camera,
An automatic excitation light control method in which fluorescence generated from the sample is observed through a second camera.
According to clause 10,
The method of automatically controlling the excitation light, wherein the excitation light is blocked after step (d) and re-irradiated after step (f) to excite the sample.
According to clause 12,
The guide beam is blocked when the excitation light is re-irradiated after step (f).