KR20220141116A - System for multi-channel fluorescence detection having improved rotatable cylindrical filter-wheel - Google Patents

Abstract

The present invention relates to a multi-channel fluorometer with an improved rotating cylindrical filter wheel. The purpose of the present invention is to provide the multi-channel fluorometer with an improved rotating cylindrical filter wheel, which optimizes arrangement and minimizes the number of components to maximize miniaturization of the fluorometer and economic efficiency. More specifically, the purpose of the present invention is to provide the multi-channel fluorometer with an improved rotating cylindrical filter wheel, which optimizes an optical path design to remove a dichroic beam splitter which has been usually necessarily used when measuring fluorescent materials and optimizes the arrangement and structures of band path filters to more minimize the number of the components when measuring the various types of the fluorescent materials.

Description

{System for multi-channel fluorescence detection having improved rotatable cylindrical filter-wheel}

The present invention relates to a multi-channel fluorescence meter equipped with a rotating cylindrical filter wheel, and more particularly, to a fluorescence meter for measuring fluorescence generated from biochemical substances, which can smoothly measure various types of fluorescence and at the same time the device It relates to a multi-channel fluorimeter equipped with a rotating cylindrical filter wheel, which enables to achieve miniaturization and economical improvement.

In order to measure fluorescence generated from a biochemical, excitation light is provided to give energy to the fluorescent material to generate fluorescence. Because the wavelength band of excitation light with high excitation efficiency exists separately according to various fluorescent materials, in order to smoothly measure a specific fluorescent material, in order to maximize the amount of generated fluorescence of the corresponding fluorescent material, excitation in a wavelength band suitable for the corresponding fluorescent material light will be used. According to this principle, several types of excitation light are used for multiple fluorescence detection, and these excitation lights should not influence each other on fluorescence detection. Accordingly, the multi-channel fluorescence measuring apparatus is formed to supply excitation light in different wavelength bands suitable for various types of fluorescent materials for each fluorescent material. In order to realize such a function, a white light source including light of all wavelength bands is usually used as a basic light source, but a plurality of bandpass filters that selectively pass only light in a wavelength band suitable for each fluorescent material are installed on the filter wheel Then, by rotating the filter wheel, a band-pass filter suitable for the fluorescent material to be measured is selected to generate excitation light. Also, in the case of the light receiving unit that detects fluorescence, a plurality of band-pass filters that pass only a specific wavelength are installed on the filter wheel, and the amount of fluorescence is measured by rotating the filter wheel to select a band-pass filter suitable for the fluorescent material to be detected. . In this process, a dichroic beam splitter is typically used to minimize background interference caused by excitation light.

US Patent Registration No. 6818437 ("Instrument for monitoring polymerase chain reaction of DNA", November 16, 2004, hereinafter 'Prior Document 1') discloses a real-time gene amplification apparatus, wherein a multi-channel fluorescence measurement apparatus is used. In the multi-channel fluorescence measuring device disclosed in Prior Document 1, as described above, the three types of excitation light band pass filter, fluorescence band pass filter, and dichroic beam splitter are configured as one module, and the wavelength band of the fluorescent material is It is configured to use a separate module according to That is, 5 modules are required to detect 5 types of fluorescent substances. That is, in the case of the device of Prior Document 1, in order to detect more types of fluorescent materials, modules composed of three types of components must be provided as many as the number of fluorescent materials, so that the volume of the device itself becomes excessively large.

In addition, the apparatus of Prior Document 1 also has the following problems. In general, a container (hereinafter, a plate) having 8, 96, or 384 wells is used to contain a plurality of samples when detecting a fluorescent substance. and, accordingly, the problem of an increase in the volume of the device occurs again. In addition, since a surface light source is used, there is also a problem of measurement error due to the difference in brightness between the periphery and the center.

In addition, as in Prior Document 1, an expensive and high-sensitivity image sensor (CCD or CMOS sensor) used in a camera or the like must be used to measure fluorescence generated by a surface light source. complicated and expensive. In addition, since it is necessary to separate the image sensor from the fluorescent material by a certain distance or more for fluorescence measurement, there is a disadvantage in that it is difficult to miniaturize it and it is necessary to use an expensive optical lens with a large size.

U.S. Patent Registration No. 8835118 ("Systems and methods for fluorescence detection with a movable detection module", September 16, 2014, hereinafter referred to as 'Prior Document 2') discloses that a fluorescence measurement module performs each well on a plate for fluorescence measurement of multiple samples. A configuration configured to measure fluorescence while moving each time is disclosed. Although the device of Prior Document 2 has the advantage that the device can be configured with a relatively inexpensive excitation light source and light receiving sensor, only one fluorescent material can be measured at a time and must be moved after measurement, so for example, 96 In the case of a plate composed of wells, 96 measurements have to be performed while moving 96 times, which has a major disadvantage in that the measurement time is excessively extended and the measurement efficiency is extremely reduced. In addition, due to the increase in the measurement time, when the reaction proceeds in the sample, there is also a problem that a deviation may occur due to a difference in the degree of reaction between the sample measured at the beginning of the measurement and the sample measured later.

In addition, the apparatus of Prior Document 2 also requires a measurement channel including two bandpass filters, a dichroic beam splitter, an LED light source, and a PD light receiving sensor in order to detect fluorescence in a specific wavelength band. In order to do this, a large number of measurement channels are required, which increases the volume and cost of the device excessively.

In addition, in order to measure the fluorescence of different wavelength bands in one well, it is necessary to perform measurement with one measurement channel optimized for a specific wavelength band, then move and change to another measurement channel optimized for another specific wavelength band and measure again. However, in this process, as described above, the risk of measurement error due to the difference in response degree due to the time delay also increases.

In order to solve this problem, the present applicant has applied for and registered Korean Patent Registration No. 1953864 ("Multi-channel Fluorometer with Rotating Cylindrical Filter Wheel", 2019.02.25, hereinafter 'Prior Document 3'). In Prior Document 3, the optical path design is optimized to remove the dichroic beam splitter, which has been always used, and the arrangement of bandpass filters and the arrangement of the bandpass filters to further minimize the number of components in measuring various types of fluorescent materials By introducing a rotating cylindrical filter wheel that optimizes the structure, it is possible to obtain a great effect of maximizing the miniaturization and economy of the fluorescence meter.

However, when the device of Prior Document 3 was actually manufactured and used, it was found that there was still some noise caused by the scattered light of the excitation light source, and there was room for improvement.

1. US Patent Registration No. 6818437 (“Instrument for monitoring polymerase chain reaction of DNA”, 2004.11.16) 2. US Patent Registration No. 8835118 (“Systems and methods for fluorescence detection with a movable detection module”, 2014.09.16.) 3. Korean Patent Registration No. 1953864 (“Multi-channel Fluorometer with Rotating Cylindrical Filter Wheel”, 2019.02.25.)

Accordingly, the present invention has been devised to solve the problems of the prior art as described above, and an object of the present invention is to maximize the miniaturization and economic feasibility of a fluorescence meter by minimizing the number of components and optimizing the arrangement. , It is to provide a multi-channel fluorometer equipped with a rotating cylindrical filter wheel. More specifically, it is an object of the present invention to optimize an optical path design so that a dichroic beam splitter, which has been normally used for measuring fluorescent materials, can be removed, and the number of components in measuring various types of fluorescent materials. An object of the present invention is to provide a multi-channel fluorimeter equipped with a rotating cylindrical filter wheel, which optimizes the arrangement and structure of bandpass filters to further minimize .

In order to achieve the above object, the multi-channel fluorescence meter having a rotating cylindrical filter wheel of the present invention includes a light source for irradiating excitation light traveling in one horizontal direction, the light source and the other horizontally and vertically downwardly spaced apart from the light source. a main body comprising a light receiving unit sensing fluorescence that has progressed horizontally, and an upper frame portion 351 and a lower frame portion 352 fixing upper and lower ends of the light source unit and the light receiving unit, respectively; A polarization unit configured to irradiate only a partial polarization component of the excitation light propagating from the light source to the sample by changing the optical path vertically downward, and change only the other partial polarization component of the fluorescence generated from the sample to the other horizontally to irradiate the light receiving unit. (331); The band-pass filter 313 for excitation light and the band-pass filter 313 for excitation light, which are formed rotatably in a cylindrical shape surrounding the polarization part 331 and pass only a partial wavelength band of the excitation light that have been propagated from the light source part, are horizontal. A filter set is provided that is spaced apart from the other side and vertically downward and includes a band-pass filter 323 for fluorescence that passes only a partial wavelength band of fluorescence that has progressed from the sample. a cylindrical filter wheel 332; It is made to include a fluorescence measuring unit 300 including a, and as the cylindrical filter wheel 332 is rotated, it may be possible to change the wavelength band selection of excitation light and fluorescence.

At this time, the light source unit is provided fixed to the light source frame unit 353 and the light source frame unit 353 detachably coupled to one side of the upper frame unit 351 and the lower frame unit 352 to emit excitation light. and a light source 311 for generating and a first lens 312 for collimating the excitation light propagating from the light source 311 , wherein the light receiving unit includes the upper frame part 351 and the lower frame part 352 . ), a sensor frame part 354 detachably coupled to the other side, a light receiving sensor 321 fixed to the sensor frame part 354 for sensing fluorescence, and a second lens ( 322), wherein the polarizing part 331 is disposed at the lowermost end on the optical path formed vertically downward by the polarizing part 331 and includes a third lens 326 for condensing light to the sample. It may be made and is coupled to the lower frame portion 352 . In this case, the light source 311 may be a white light emitting diode (LED) or a laser diode. In addition, the light receiving sensor 321 may be formed of at least one selected from a photodiode, a photo multiplier tube (PMT), a multi-pixel photon counter (MPPC), a CCD, and a CMOS sensor.

In addition, the light source frame portion 353 may include a light source side cover 353a formed to surround a portion of the outer side of the cylindrical filter wheel 332 . In addition, the light source frame portion 353 may include a light-receiving side cover 353a formed to surround a portion of the outer side of the cylindrical filter wheel 332 .

In addition, the polarization unit 332 includes a first polarizer 314 that passes only a partial polarization component of the excitation light propagating from the light source unit, and changes the optical path vertically downward by reflecting the light traveling from the first polarizer 314 . A polarization beam splitter 325 that is spaced apart from the surface mirror 315 and vertically downward of the surface mirror 315 and passes the same type of polarization component as the first polarizer 314 and reflects a different type of polarization component; , the first polarizer 314 may include a second polarizer 324 that passes through the polarization component and the polarization component different from the polarization component passing through, and passes the light reflected from the polarization beam splitter 325 . .

At this time, the multi-channel fluorescence meter converts the excitation light from the light source unit to have a preselected wavelength band and polarization component by sequentially passing through the band-pass filter 313 for excitation light and the first polarizer 314 . is reflected by the surface mirror 315, the optical path is changed vertically downward and irradiated to the sample, and the fluorescence generated from the sample is reflected by the polarization beam splitter 325 and the first polarizer 314 passes is converted to have a polarization component and a different type of polarization component, the optical path is changed to the other horizontally, and the second polarizer 324 and the fluorescence band pass filter 323 sequentially pass through a preselected wavelength band and polarization component An optical path may be formed so as to be converted to have an irradiated light to the light receiving unit.

Also, the cylindrical filter wheel 332 may include a filter wheel motor 301 for rotating the cylindrical filter wheel 332 .

In addition, the multi-channel fluorescence meter may include: a moving unit for moving the fluorescence measuring unit 300 in a first axis included in a horizontal plane and a second axis included in a horizontal plane and perpendicular to the first axis; It may be made to further include.

At this time, the moving part is formed in a caterpillar shape driven by a first shaft driving unit 101 made of a rotary motor, a first shaft guide unit 102 extending in the first axis direction, and the first shaft driving unit 101 . A first shaft transfer part 103, a first shaft body part 104 guided by the first shaft guide part 102 and connected to the first shaft transfer part 103 to be movable in the first axis direction. , a second shaft driving unit 201 made of a rotary motor, a second shaft guide 202 extending in the second axis direction, and a second shaft transfer unit in the form of a caterpillar driven by the second shaft driving unit 201 ( 203), is guided by the second shaft guide part 202 and is connected to the second shaft transfer part 203 to include a second shaft body part 204 that is formed to be movable in the second shaft direction, , one end of the second shaft guide part 202 is fixed to the first shaft body part 104 , and the fluorescence measuring part 300 is fixed to the second shaft body part 204 , so that the fluorescence The measurement unit 300 may be formed to be movable in the first axis and the second axis direction.

According to the present invention, the number of components used in multi-channel fluorescence measurement is minimized and the number of components used in multi-channel fluorescence measurement is minimized through a configuration in which a single light source and a light-receiving unit are used, but a filter wheel composed only of band-pass filters suitable for excitation light and fluorescence in various wavelength bands rotates. This has the effect of optimizing the layout.

Accordingly, there is a great effect of maximizing the economic feasibility in manufacturing, and also has the effect of greatly relieving the constraints of optical design by not using a dichroic beam splitter that is commonly used in conventional fluorescence measurement devices. .

In addition, according to the present invention, by further optimizing the arrangement of the light source and the light receiving sensor and forming a cover around them, the problem of generating noise due to the effect of unwanted light scattered by the excitation light source on the light receiving sensor is solved, It has a great effect in that it has much less noise and can obtain high-accuracy and high-precision measurement values.

In addition, according to the present invention, as described above, by maximizing the miniaturization of the fluorescence meter, since its own weight is small, stable scanning at a much faster speed than in the prior art for a large number of measurement objects such as 96-well and 384-well plates is possible. can also be obtained

1 is an external view of a fluorescence measurement unit constituting a multi-channel fluorescence meter of the present invention.
2 is a cross-sectional view of a fluorescence measuring unit;
3 is an exploded perspective view of a part of a light source side of a fluorescence measuring unit;
4 is an exploded perspective view of a part of a light-receiving side of a fluorescence measuring unit;
5 is a comparison of the improved optical path of the prior art and the present invention.
6 is another embodiment of the multi-channel fluorometer of the present invention having a scan function.

Hereinafter, a multi-channel fluorometer equipped with a rotating cylindrical filter wheel according to the present invention having the configuration as described above will be described in detail with reference to the accompanying drawings.

As described above, a dichroic beam splitter is essential to separate the excitation light and fluorescence because the optical path is designed so that the optical axes of the excitation light and the fluorescence coincide with the conventional sample. However, in the present invention, excitation light and fluorescence in all wavelength bands and excitation light and fluorescence in all wavelength bands with a single beam splitter regardless of wavelength by having only polarization components on a plane perpendicular to each other by using the polarization characteristic of light. Introduces a configuration that allows the separation of the dichroic beamsplitter, thereby removing the dichroic beamsplitter from the configuration. In addition, in the present invention, by installing only bandpass filters suitable for excitation light and fluorescence in each wavelength band on a rotating cylindrical filter wheel, it is possible to measure fluorescence in various wavelength bands with only one light source and one detector. While detecting, the number of parts can be minimized compared to the conventional one.

The overall configuration of the fluorescence measurement unit and the detailed configuration of each part

1 is an external view of a fluorescence measurement unit constituting a multi-channel fluorescence meter of the present invention, and FIG. 2 is a cross-sectional view of the fluorescence measurement unit. In addition, FIG. 3 is an exploded perspective view of a part of the light source side of the fluorescence measuring unit, and FIG. 4 is an exploded perspective view of a part of the light receiving side of the fluorescence measuring unit. The multi-channel fluorescence meter of the present invention basically includes the fluorescence measurement unit 300 shown in FIGS. 1 to 4 , and the fluorescence measurement unit 300 irradiates the sample with excitation light, and It performs a function of sensing the fluorescence generated by the First, a detailed configuration of the fluorescence measuring unit 300 will be described with reference to FIGS. 1 to 4 .

As shown in FIGS. 1 and 2 , the fluorescence measuring unit 300 largely includes a main body, a polarizing unit 331 , and a cylindrical filter wheel 332 .

The main body includes a light source unit for irradiating excitation light traveling in one horizontal direction, and a light receiving unit arranged to be spaced apart from the light source unit in the other horizontally and vertically downwardly and senses fluorescence traveling in the horizontal direction. The upper frame part 351 and the lower frame part 352 fix the upper and lower ends of the light source part and the light receiving part, respectively, to firmly fix the positions of the light source part and the light receiving part.

A preferred embodiment of the configuration of the body part will be described in detail with reference to FIGS. 3 and 4 . First, as shown in FIG. 3 , the light source unit may include a light source frame unit 353 , a light source 311 , and a first lens 312 . Also, as shown in FIG. 4 , the light receiving unit may include a sensor frame unit 354 , a light receiving sensor 321 , and a second lens 322 . In addition, as shown in FIGS. 3 and 4 , the upper frame part 351 and the lower frame part 352 are combined with the light source frame part 353 and the sensor frame part 354 to synthesize various parts. A third lens 326 for condensing light to the sample is further provided here, but serves to support.

First, among the detailed configurations of the body part, the light source 311 , the first lens 312 , the light receiving sensor 321 , the second lens 322 , and the third component that irradiate light to the sample or sense the light emitted from the sample. The lens 326 will be described as follows.

The light source 311 is fixed to the light source frame portion 353 that is detachably coupled to one side of the upper frame portion 351 and the lower frame portion 352 to generate excitation light. In this case, the light source 311 may be a white light emitting diode (LED) or a laser diode. The first lens 312 serves to collimating the excitation light propagating from the light source 311, that is, to make it into parallel light. That is, the light generated from the light source 311 passes through the first lens 312 to travel in one horizontal direction.

The light receiving sensor 321 is fixed to the sensor frame 354 that is detachably coupled to the other side of the upper frame 351 and the lower frame 352 to sense fluorescence. In this case, the light receiving sensor 321 may be a photo diode, a photo multiplier tube (PMT), a multi-pixel photon counter (MPPC), a CCD, or a CMOS sensor. The second lens 322 serves to image the fluorescence that has progressed in the sample. Although it will be described in more detail later, the light path of the fluorescence generated from the sample is controlled by various optical components (to be described later), and finally proceeds in the form of parallel light traveling from one horizontal direction. As this type of fluorescence passes through the second lens 322 , an image is formed on the light receiving sensor 321 (image formation), and accordingly, the light receiving sensor 321 can sense the fluorescence.

The third lens 326 is disposed at the lowermost end of the optical path formed vertically downward by the polarizer 331 and serves to condense light to the sample, that is, to irradiate the excitation light to the sample. Of course, the third lens also serves to collect the fluorescence generated from the sample by irradiating the excitation light and advance it to the polarizing part vertically upward.

Next, among the detailed configurations of the body part, the upper frame part 351, the lower frame part 352, etc., which are parts that stably fix the above-described light irradiation and sensing parts to their positions or prevent external influences, will be described below. same as

The upper frame part 351 and the lower frame part 352 basically serve to fix the upper and lower ends of the light source part and the light receiving part, respectively, in addition, the light source frame part 353 and the sensor frame part 354, the Various components constituting the fluorescence measuring unit 300, such as the polarizing unit 331 and the cylindrical filter wheel 332, serve as a base to which they are coupled and fixed. In addition, the fluorescence measurement unit 300 has a side cover 1 (355) that surrounds the side surfaces of various parts constituting the fluorescence measurement unit 300 to block external light so that external light does not unnecessarily affect the measurement. and a side cover 2 356 are further provided to be fixed to the upper frame part 351 and the lower frame part 352 .

The light source frame part 353 is detachably coupled to one side of the upper frame part 351 and the lower frame part 352, so that the light source 311, the first lens 312, etc. This allows for a stable, fixed placement. In addition, it is preferable that the light source frame portion 353 includes a light source side cover 353a formed to surround a portion of the outer side of the cylindrical filter wheel 332 . The light source side cover 353a is provided to properly block the scattered light of the excitation light emitted from the light source 311 , thereby preventing the excitation light from unnecessarily affecting the light receiving sensor 321 . .

The sensor frame part 354 is detachably coupled to the other side of the upper frame part 351 and the lower frame part 352 so that the light receiving sensor 321, the second lens 322, etc. This allows it to be stably fixed in place. In addition, it is preferable that the sensor frame part 354 includes a light-receiving side cover 354a formed to surround a part of the outer side of the cylindrical filter wheel 332 . Since the light-receiving side cover 354a is provided, it is possible to better prevent unwanted influence of the scattered light or external light by the excitation light on the light-receiving sensor 321 .

The polarizer 331 separates polarization components with respect to excitation light and fluorescence and changes respective optical paths. As shown in FIG. 2, the cylindrical filter wheel 332 has a cylindrical shape surrounding the polarizer 331, and allows only light of a predetermined wavelength band for excitation light and fluorescence to pass therethrough. As the cylindrical filter wheel 332 is rotated, it is possible to change the wavelength band selection of excitation light and fluorescence. Each part will be described in more detail as follows.

The polarization unit 331 irradiates a sample by changing only a partial polarization component of the excitation light propagating from the light source unit to a vertical downward direction, and changes the optical path to the other horizontally only some other polarization components of the fluorescence generated in the sample. It is irradiated with the light receiving unit. In this case, as described above, since the light receiving part is disposed to be spaced apart from the other side of the light source part horizontally and vertically downward, the horizontal optical path of the fluorescence is vertically downward from the horizontal optical path of the excitation light. The polarizing part 332 is also formed so that the light source part is present on an optical path in the horizontal direction of the excitation light and the light receiving part is present on the optical path in the horizontal direction of the fluorescence. The polarization unit 332 not only adjusts the optical path as described above, but also serves to appropriately adjust the polarization component. In order to realize this function, the polarizer 332 includes a first polarizer 314 , a surface mirror 315 , a polarization beam splitter 325 , and a second polarizer 324 as shown in FIGS. 1 and 2 . may be included.

The first polarizer 314 transmits only a partial polarization component of the excitation light that has progressed from the light source unit. For example, the first polarizer 314 may be formed to pass only the s-wave polarization component.

The surface mirror 315 reflects the light traveling from the first polarizer 314 to change the optical path vertically downward.

The polarization beam splitter 325 is vertically spaced apart from the surface mirror 315 and passes a polarization component of the same type as that of the first polarizer 314 and reflects a different type of polarization component. For example, if the first polarizer 314 is configured to pass only the s-wave polarization component as described above, then the polarization beam splitter 325 passes the s-wave polarization component and reflects the p-wave polarization component. It should be done according to

The second polarizer 324 is formed to pass a polarization component different from the polarization component that the first polarizer 314 passes through, and passes the light reflected from the polarization beam splitter 325 . For example, if the first polarizer 314 is configured to pass only the s-wave polarization component as described above, the second polarizer 324 only needs to pass the p-wave polarization component.

The cylindrical filter wheel 332 is rotatably formed in a cylindrical shape surrounding the polarizing part 331 as described above, and as the cylindrical filter wheel 332 is rotated, the wavelength band selection of excitation light and fluorescence is changed. made possible

More specifically, the cylindrical filter wheel 332 has a band-pass filter 313 for excitation light that passes only a partial wavelength band of the excitation light traveling from the light source unit and a band-pass filter 313 for excitation light vertically downward. A filter set is provided which is spaced apart and includes a band-pass filter 323 for fluorescence that passes only a partial wavelength band of fluorescence that has progressed from the sample. At this time, the plurality of the filter sets are formed to be arranged around the side of the cylinder, as shown in FIGS. 1 and 2 . As described above, in order to maximally excite fluorescence from a specific fluorescent material, it is necessary to irradiate excitation light of a specific wavelength band suitable therefor. In addition, the generated fluorescence is limited to another specific wavelength band. Since the physical properties of the fluorescent material to be detected, that is, the wavelength band of excitation light best for excitation of the fluorescent material and the wavelength band of fluorescence generated from the fluorescent material are known in advance, the band-pass filter for excitation light 313 and Each wavelength band of the fluorescence band-pass filter 323 may be appropriately determined accordingly.

1 illustrates that the six filter sets are exemplarily arranged around the side of the cylinder, of course, the present invention is not limited thereto. That is, the number of filter sets may be changed and determined as desired according to the number of fluorescent materials to be measured.

In addition, the cylindrical filter wheel 332 is preferably provided with a filter wheel motor 301 for rotating the cylindrical filter wheel 332 so as to easily and accurately rotate the cylindrical filter wheel 332 .

Detailed optical path of excitation light and fluorescence in the fluorescence measurement unit

A process in which the fluorescent material is detected by the fluorescence measuring unit 300 as described above will be described in detail. That is, the optical path in the state in which each part shown in the external view of FIG. 1 and the exploded perspective views of FIGS. 3 and 5 are all combined and configured as shown in FIG. 2 will be described as follows. .

First, the arrangement of each part will be described. As shown in FIG. 2 , in a state in which each component constituting the fluorescence measuring unit 300 is all combined, the light source unit (consisting of the light source 311 and the first lens 312 ) - (the light receiving unit) The light receiving part (consisting of the sensor 312 and the second lens 322) is disposed to be spaced apart from each other in a horizontal direction and a vertical direction. In addition, the surface mirror 315 - the polarization beam splitter 325 - the third lens 326 - the sample is also arranged to be spaced apart in parallel in the vertical direction.

At this time, as also shown in FIG. 2 , in a state in which the cylindrical filter wheel 332 is covered on the polarizing part 331 , the light source 311 - the first lens 312) - the excitation of the light source part One selected one of the bandpass filters for light 313 - the first polarizer 314 - the surface mirror 315 is spaced apart from each other in a horizontal direction. Similarly, the light receiving sensor 321 - the second lens 322) of the light receiving unit - a selected one of the fluorescence band pass filter 323 - the second polarizer 324 - the polarization beam splitter 325 They are spaced apart from each other in the horizontal direction.

In such an arrangement state, the optical path of the excitation light will first be described.

The excitation light proceeding from the light source unit sequentially passes through the bandpass filter 313 for excitation light and the first polarizer 314 and is converted to have a preselected wavelength band and polarization component. As described above, the wavelength band through which the fluorescent material passes may be determined in advance, and the polarization component through which the first polarizer 314 passes may be appropriately determined from either an s-wave or a p-wave. For ease of understanding, it is assumed that the band-pass filter 313a for excitation light in FIG. 2 passes the A-A' wavelength band and the first polarizer 314 passes the s-wave polarization component. do.

As described above, the excitation light adjusted to have only the preselected wavelength band and polarization component is reflected by the surface mirror 315, the optical path is changed vertically downward, and is irradiated to the sample. According to the above example, the excitation light reflected by the surface mirror 315 and proceeding vertically is light having only the A-A' wavelength band and the s-wave polarization component. Since the polarization beam splitter 325 is configured to pass a polarization component of the same type as that of the first polarizer 314 and reflect a polarization component of a different type, the light passes through the polarization beam splitter 325 without any change. do.

When the excitation light is focused by the third lens 326 and irradiated to the sample, fluorescence is generated in the sample. Fluorescence generated from the sample is collected by the third lens 326 and proceeds vertically upward. At this time, it is assumed that the fluorescence generated from the sample has a B-B' wavelength band. In addition, this fluorescence has both s-wave and p-wave polarization components.

At this point, the optical path of fluorescence is now described.

The fluorescence generated from the sample has all polarization components as described above. At this time, since the polarization beam splitter 325 is configured to pass a polarization component of the same type as that of the first polarizer 314 and reflect a different type of polarization component, according to the previous example, the polarization beam splitter 325 is s The wave-polarized component is passed through, but the p-wave-polarized component is reflected.

The fluorescence reflected by the polarization beam splitter 325 is converted to have a polarization component different from the polarization component that the first polarizer 314 passes through (that is, a p-wave polarization component according to the above example), and the other horizontally The light path is changed to

The second polarizer 324 and the fluorescence band pass filter 323 for fluorescence reflected by the polarization beam splitter 325 and traveling in the other horizontal direction with only the p-wave polarization component are shown in FIG. 2 . It is converted to have a preselected wavelength band and polarization component by sequentially passing through. Since the second polarizer 324 is formed to pass a polarization component different from the polarization component that the first polarizer 314 passes through, according to the above example, the second polarizer 324 has only a p-wave polarization component. pass through In addition, since the wavelength band of fluorescence generated from the fluorescent material is known in advance, it is assumed that the band-pass filter 323a for fluorescence in FIG. 2 passes the B-B' wavelength band. By passing the fluorescence band-pass filter 323a, light in a noise wavelength band that may be generated may be blocked.

At this point, the fluorescence irradiated to the light receiving unit after passing through the second polarizer 324 and the fluorescence band-pass filter 323 sequentially is light having only the B-B' wavelength band and the p-wave polarization component. That is, the fluorescence irradiated to the light receiving unit is in a state in which the influence of unnecessary noise, such as excitation light having an s-wave polarization component and light outside the B-B' wavelength band, is completely removed, and accurate detection by the light receiving unit is possible.

In the present invention, a method of separating excitation light and fluorescence using a polarization component is used. The advantages obtained by this will be described in more detail as follows.

Conventionally, when the optical axes of excitation light and fluorescence coincide with respect to the sample to be measured, a dichroic beam splitter that selectively passes or reflects light according to a wavelength band is used for excitation light/fluorescence separation. / A total of three optical components, a bandpass filter for fluorescence and a dichroic beam splitter, had to be configured as a single module. In addition, in order to view multiple types of fluorescence, it was necessary to install a plurality of the module and measure the fluorescence while rotating the module, or to measure the fluorescence by separately installing a light source and a lens for each filter set. Therefore, there was a problem of mechanical complexity, difficulty in miniaturization, and an increase in manufacturing cost during product design. In addition, in order to change the fluorescence to be used, there was a problem that not only the bandpass filter but also the dichroic beamsplitter had to be exchanged, which became a limitation in using various fluorescent materials.

However, in the present invention, the use of a dichroic beam splitter is excluded for excitation light/fluorescence separation, and a polarizer and a polarization beam splitter are used to perform excitation light/fluorescence separation using a polarization component. That is, in the present invention, only some polarization components of excitation light/other polarization components of fluorescence are used for fluorescence generation/sensing. In this case, since the characteristics of passing or reflecting light in the polarizers and the polarization beam splitter are independent of the wavelength band, the polarizers and the polarization beam splitter can be commonly used for light of various wavelength bands.

In the present invention, only a single light source, a light receiving sensor, and a polarization beam splitter are used, respectively, but an optical path design and component arrangement to generate fluorescence using band pass filters that allow only light of a desired wavelength band to pass through an appropriate position on the optical path is made up of In this case, as described above, since the polarization beam splitter can be commonly used in all wavelength bands, fluorescence generation and sensing can be smoothly performed while only these bandpass filters are changed. That is, unlike the conventional need for multiple sets of [bandpass filter for excitation light + dichroic beamsplitter + bandpass filter for fluorescence] for multi-channel fluorescence measurement, in the present invention, a single polarizing beam splitter is It is only necessary to provide a plurality of sets of [band-pass filter for light + band-pass filter for fluorescence].

In addition, in the present invention, by arranging the light source unit and the light receiving unit spaced apart in the horizontal direction to face each other, and forming the light source side cover 353a and the light receiving side cover 354a respectively, the scattered light generated by the excitation light source during fluorescence sensing is reduced. By ensuring that the impact is minimized, the sensing accuracy is further improved. 5 shows a conventional light path and an improved light path in the present invention, respectively. As shown in the upper side view of FIG. 5 , the band-pass filter 313 for excitation light passes only light in a specific wavelength band and reflects light in the other wavelength band. Among the reflected excitation light sources, light in a wavelength band corresponding to fluorescence is also included, which is reflected by a lens and a device, and the scattered light is incident on a light receiving sensor to cause noise. In particular, in the related art, since the light source and the light receiving sensor are disposed in the same direction, they are more affected by the scattered light, and it is difficult to completely remove the noise. However, as shown in the lower view of FIG. 5 , in the present invention, the light source and the light receiving sensor are disposed in opposite directions, so that the influence from the light source in the light receiving sensor can be minimized. In addition, as described above (although it is not shown well in the drawing in FIG. 5), the light source side cover 353a and the light receiving side cover 354a are used to prevent light that can cause noise, such as scattered light by the excitation light source, from entering the lens as much as possible. By blocking, noise generated by the influence of the above-described scattered excitation light on the light receiving sensor can be smoothly removed.

As described above, in the present invention, by optimizing the optical path design and component arrangement as described above, the dichroic beam splitter, which is the cause of price increase and volume increase, is eliminated, and only a cylindrical filter wheel composed only of bandpass filters is used to view various types of fluorescence. It is configured to be rotated. Accordingly, all of the conventional problems (increase in price, increase in volume, difficulty in replacement, etc.) as described above are all fundamentally solved, so that the device can be made much smaller and multi-channel fluorescence measurement is possible economically and effectively. In addition, by further optimizing the arrangement of the light source and the light receiving sensor and forming a cover around them, the problem of generating noise by avoiding unwanted scattered light from entering the light receiving sensor is solved, much less noise and more accurate than the conventional one. and high-precision measurement values can be obtained.

Configuration of a multi-channel fluorometer that scans with a fluorescence measurement unit

6 shows another embodiment of the multi-channel fluorometer of the present invention having a scan function. The multi-channel fluorescence measuring device of the present invention includes the fluorescence measuring unit 300, and as described above, the fluorescence measuring unit 300 can be made much smaller than the conventional one by optimizing the optical path design and component arrangement. Accordingly, it is much easier to perform a detection operation on a plurality of samples while moving the fluorescence measuring unit 300 .

In general, the sample in such an experimental work is contained in each well of a plate 400 having a plurality of wells arranged in rows and columns on a horizontal plane, and these plates are 96-well, 384-well, etc. It is commercialized as a standardized product. Accordingly, in the multi-channel fluorescence meter of the present invention, as shown in FIG. 6 , the fluorescence measurement unit 300 includes a first axis included in a horizontal plane and a second axis included in the horizontal plane and perpendicular to the first axis. By further comprising a moving unit moving in the direction, it is possible to realize a fast and effective detection operation for a plurality of samples while moving the fluorescence measuring unit 300 on a horizontal plane.

A detailed configuration of an embodiment of the moving unit shown in FIG. 6 will be described as follows. The moving unit may include parts responsible for movement in the first axial direction and parts responsible for movement in the second axial direction.

First, the parts responsible for movement in the first axis direction are, according to the embodiment of FIG. 4 , a first axis driving unit 101 made of a rotation motor, a first axis guide unit 102 extending in the first axis direction, A first shaft transfer unit 103 formed in a caterpillar shape driven by the first axis driving unit 101 , guided by the first axis guide unit 102 and connected to the first axis transfer unit 103 , the first The first shaft body portion 104 is formed to be movable in the axial direction.

In addition, the parts responsible for movement in the second axis direction are, according to the embodiment of FIG. 4 , a second axis driving unit 201 made of a rotary motor, a second axis guide unit 202 extending in the second axis direction, A second shaft transport unit 203 formed in a caterpillar shape driven by the second shaft driving unit 201, guided by the second shaft guide unit 202, and connected to the second shaft transport unit 203, the second The second shaft body portion 204 is formed to be movable in the axial direction.

When the moving part is configured in this way, one end of the second shaft guide part 202 is fixed to the first shaft body part 104 , and the fluorescence measuring part ( By fixing 300 , the fluorescence measuring unit 300 may be formed to be able to move smoothly in the first and second axis directions.

Of course, the configuration of the moving unit is only an example, and any configuration may be employed as long as it can effectively move the fluorescence measuring unit 300 according to the positions of a plurality of wells arranged in rows and columns on a horizontal plane. It is, of course, free to do so.

The present invention is not limited to the above-described embodiments, and the scope of application is varied, and anyone with ordinary knowledge in the field to which the present invention pertains without departing from the gist of the present invention as claimed in the claims It goes without saying that various modifications are possible.

101: first axis driving unit 102: first axis guide unit
103: first axis transfer unit 104: first axis body portion
201: second axis driving unit 202: second axis guide unit
203: second axis transfer unit 204: second axis body portion
300: fluorescence measurement unit 301: filter wheel motor
311: light source 312: first lens
313: band pass filter for excitation light
314: first polarizer 315: surface mirror (Surface Mirror)
321: light receiving sensor 322: second lens
323: band pass filter for fluorescence
324: second polarizer 325: Polarizing Beam Splitter (PBS)
326: third lens
331: polarizer 332: cylindrical filter wheel
351: upper frame portion 352: lower frame portion
353: light source frame portion 353a: light source side cover
354: sensor frame part 354a: light receiving side cover
355: side cover 1 356: side cover 2
400: plate

Claims (11)

A light source unit for irradiating excitation light traveling in one horizontal direction, a light receiving unit arranged to be spaced apart from the light source unit in the other horizontal and vertically downward directions, and a light receiving unit sensing fluorescence progressing in the horizontal one side, and an upper frame fixing the light source unit and upper and lower ends of the light receiving unit, respectively a body portion including a portion 351 and a lower frame portion 352;
A polarization unit configured to irradiate only a partial polarization component of the excitation light propagating from the light source to the sample by changing the optical path vertically downward, and change only the other partial polarization component of the fluorescence generated from the sample to the other horizontally to irradiate the light receiving unit. (331);
The band-pass filter 313 for excitation light and the band-pass filter 313 for excitation light, which are formed rotatably in a cylindrical shape surrounding the polarization part 331 and pass only a partial wavelength band of the excitation light that have been propagated from the light source part, are horizontal. A filter set is provided that is spaced apart from the other side and vertically downward and includes a band-pass filter 323 for fluorescence that passes only a partial wavelength band of fluorescence that has progressed from the sample. a cylindrical filter wheel 332;
It is made to include a fluorescence measurement unit 300 comprising a,
A multi-channel fluorescence meter with a rotating cylindrical filter wheel, characterized in that it is possible to change the wavelength band of excitation light and fluorescence as the cylindrical filter wheel (332) is rotated.
The method of claim 1,
The light source unit includes a light source frame unit 353 detachably coupled to one side of the upper frame unit 351 and the lower frame unit 352 , and is fixed to the light source frame unit 353 to generate excitation light. It consists of a light source 311 and a first lens 312 for collimating the excitation light proceeding from the light source 311,
The light receiving unit includes a sensor frame unit 354 detachably coupled to the other side of the upper frame unit 351 and the lower frame unit 352 , and a light receiving unit fixed to the sensor frame unit 354 to sense fluorescence. It is made including a sensor 321 and a second lens 322 for imaging the fluorescence that has progressed in the sample,
The polarizing part 331 is disposed at the lowermost end of the optical path formed vertically downward by the polarizing part 331 and a third lens 326 for condensing light to the sample.
A multi-channel fluorescence meter having a rotating cylindrical filter wheel, characterized in that it comprises a.
According to claim 2, wherein the light source (311),
A multi-channel fluorometer equipped with a rotating cylindrical filter wheel, characterized in that it is a white LED (Light Emitting Diode) or a laser diode.
According to claim 2, wherein the light receiving sensor 321,
A multi-channel fluorometer with a rotating cylindrical filter wheel, characterized in that it consists of at least one selected from a photodiode, a photo multiplier tube (PMT), a multi-pixel photon counter (MPPC), a CCD, and a CMOS sensor.
According to claim 2, The light source frame portion 353,
A multi-channel fluorescence meter with a rotating cylindrical filter wheel, characterized in that it includes a light source side cover (353a) formed to surround a part of the outer side of the cylindrical filter wheel (332).
According to claim 2, wherein the sensor frame portion 354,
A multi-channel fluorescence meter with a rotating cylindrical filter wheel, characterized in that it includes a light-receiving side cover (354a) formed to surround a part of the outer side of the cylindrical filter wheel (332).
According to claim 1, wherein the polarizer 332,
a first polarizer 314 that passes only a partial polarization component of the excitation light proceeding from the light source unit;
a surface mirror 315 for changing the optical path vertically downward by reflecting the light traveling from the first polarizer 314;
A polarization beam splitter 325 disposed vertically downward of the surface mirror 315 and passing a polarization component of the same type as that of the first polarizer 314 and reflecting a polarization component of a different type;
A second polarizer 324 that is formed to pass a polarization component different from the polarization component that the first polarizer 314 passes through, and passes the light reflected from the polarization beam splitter 325 .
A multi-channel fluorescence meter having a rotating cylindrical filter wheel, characterized in that it comprises a.
The method of claim 7, wherein the multi-channel fluorometer,
The excitation light proceeding from the light source unit sequentially passes through the bandpass filter 313 and the first polarizer 314 for excitation light and is converted to have a preselected wavelength band and a polarization component, and is applied to the surface mirror 315. The light path is changed vertically downward by being reflected and irradiated to the sample,
The fluorescence generated from the sample is reflected by the polarization beam splitter 325 and is converted to have a polarization component different from the polarization component that the first polarizer 314 passes through, and the optical path is changed to the other horizontally, and the second Sequentially passing through the polarizer 324 and the fluorescence band-pass filter 323, converted to have a preselected wavelength band and polarization component and irradiated to the light-receiving unit
A multi-channel fluorescence meter with a rotating cylindrical filter wheel, characterized in that an optical path is formed.
According to claim 1, wherein the cylindrical filter wheel (332),
A multi-channel fluorescence meter with a rotating cylindrical filter wheel, characterized in that a filter wheel motor (301) for rotating the cylindrical filter wheel (332) is provided.
According to claim 1, wherein the multi-channel fluorometer,
a moving unit for moving the fluorescence measuring unit 300 in a first axis included in a horizontal plane and a second axis included in a horizontal plane and perpendicular to the first axis;
A multi-channel fluorescence meter with a rotating cylindrical filter wheel, characterized in that it further comprises a.
11. The method of claim 10, wherein the moving unit,
A first shaft driving unit 101 made of a rotary motor, a first shaft guide unit 102 extending in the first axis direction, and a first shaft transport unit 103 having a caterpillar shape driven by the first shaft driving unit 101 . ), a first shaft body portion 104 guided by the first shaft guide portion 102 and connected to the first shaft transfer portion 103 to be movable in the first axis direction;
A second shaft driving unit 201 made of a rotary motor, a second shaft guide 202 extending in the second axis direction, and a second shaft transfer unit 203 having a caterpillar shape driven by the second shaft driving unit 201 ), a second shaft body part 204 guided by the second shaft guide part 202 and connected to the second shaft transport part 203 to be movable in the second shaft direction.
is made, including
One end of the second shaft guide part 202 is fixed to the first shaft body part 104, and the fluorescence measuring part 300 is fixed to the second shaft body part 204,
A multi-channel fluorescence meter having a rotating cylindrical filter wheel, characterized in that the fluorescence measuring unit (300) is formed to be movable in first and second axis directions.

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