Abstract:
A confocal fluorescence microscope of the present invention consists of: a light source unit broadly comprising one or more short wavelength laser beams; a lens unit which converts parallel light, emitted from a light source, into linear light having an appropriate size; a multi-color mirror which reflects the light source and enables fluorescence to transmit so as to separate the light source and the fluorescence; a scan mirror which radiates the light source over a wide area and scatters the fluorescence over a large-area camera; a microscope unit which radiates the incident light source to a target object, collects the fluorescence emitted from the target, and outputs the collected fluorescence; and a detecting unit which removes the background of the outputted fluorescent signal and observes the outputted fluorescent signal.

Description:
FOREIGN PRIORITY CLAIM 
     This is a 35 U.S.C. §371 application of, and claims priority to, International Application No. PCT/KR2012/007190, which was filed Sep. 6, 2012, and which claims priority to KR 10-2011-0090022, filed on Sep. 6, 2011, the entirety of all of the applications are incorporated herein by reference. 
     TECHNICAL FIELD 
     The present invention relates generally to a confocal fluorescence microscope and, more particularly, to a confocal fluorescence microscope that is capable of observing a single fluorescent molecular signal in real time, and an observation method thereof. 
     BACKGROUND ART 
     Confocal fluorescence microscopy is a technology that is capable of obtaining an improved image by removing background signals emitted from parts other than the focal plane of a microscope lens using a pinhole or the like, compared to conventional fluorescence microscopy. For this reason, confocal fluorescence microscopy technology is widely used in biological research. 
     A common confocal fluorescence microscopy technology requires scanning in two axial directions in order to obtain one image because light is focused in a dot form in order to observe fluorescent light. Accordingly, the common confocal fluorescence microscopy technology is disadvantageous in that it takes an excessively long time to obtain one image and it is difficult to observe the rapid movements of biological molecules and interactions therebetween within a cell. 
     A method devised to overcome the above disadvantages is line scan confocal microscopy or spinning disk confocal microscopy. In line scan confocal microscopy, light is focused in a line form, rather than in a dot form, using a cylindrical lens. As a result, the time it takes to obtain an image can be significantly reduced because scanning only in a single axial direction is required to obtain an image. In spinning disk confocal microscopy, an image can be rapidly obtained in such a way as to divide light into several parts, form several dot-shaped focuses at the same time, bore a hole through a disk, and rotate the disk. These two methods can obtain images at a speed (of several tens of Hz) close to a real-time speed. 
     Recently, with the advancement of related technology, the fluorescence microscopy technology has been advanced to the extent that a single fluorescent molecule can be observed. As a single fluorescent molecule can be observed, the various phenomena of life that were difficult to observe have become understood, and thus the fluorescence microscopy technology is being widely used. A single fluorescent molecule can be observed only when background signals are maximally reduced because the single fluorescent molecule has a very weak fluorescent signal. For this purpose, a total internal reflection method is widely used. When total reflection is generated between a slide and a medium, an evanescent wave is generated, which excites a fluorescent light substance. In this case, background signals generated from parts other than a part where total reflection is generated can be reduced because the range of the evanescent wave is very short, that is, about 200 to 300 nm from a surface where total reflection was generated. However, phenomena occurring inside a cell cannot be observed using the total internal reflection method because only fluorescent light in a very short range near a surface of the cell can be observed using the total internal reflection method. 
     The confocal microscopy method can be used to measure fluorescent light at a deep location inside a cell and the signal of a single fluorescent molecule, unlike the total reflection microscopy method. However, a common confocal microscope may not be used to observe a rapid change within a cell because the time it takes to obtain an image is long, as described above. In particular, because of its low speed, it is difficult to apply the common confocal microscope to recent ultra-high definition imaging technology based on the determination of the location of a single fluorescent molecule. In the case of line scan confocal microscopy and spinning disk confocal microscopy used to overcome measurement speed issues, the degree to which background noise is reduced is lower than when using a dot-shaped confocal method, and a detector used for observation has low performance. Accordingly, a single fluorescent molecular signal cannot be observed using currently commercialized equipment. 
     As a result, in order to observe a single molecule fluorescent light in a thick object, such as a cell or a tissue, there is a need for 1) an ability to observe fluorescent light at a deep location, 2) a rapid measurement speed of several tens of Hz or higher, and 3) a high signal-to-noise ratio and high-performance detector for observing single molecule fluorescent light. 
     DISCLOSURE 
     Technical Problem 
     As described above, an object of the present invention is to observe a single fluorescent molecular signal in real time (at several tens of or higher or higher) in a cell or a tissue. 
     Technical Solution 
     In order to accomplish the above object, the present invention includes a light source unit configured to include one or more short-wavelength lasers, a lens unit configured to convert parallel light of a light source into linear light of an appropriate size, a polychromic mirror configured to separate the light of the light source and fluorescent light by reflecting the light of the light source and transmitting the fluorescent light, a scan mirror configured to radiate the light of the light source onto the wide area of an object and to scatter the fluorescent light over a large-area camera, a microscope unit configured to radiate the light of the light source onto the object, to condense the fluorescent light generated from the object, and to emit the fluorescent light, and a detection unit configured to remove a background from an emitted fluorescent light signal and then observe the fluorescent light signal. 
     The light source unit may include one or more short-wavelength lasers, and beams of light from the lasers are combined together using dichroic mirrors having corresponding wavelengths. 
     The lens unit may include three circular lenses and three cylindrical lenses. 
     The lens unit may be divided into a first lens unit including three cylindrical lenses and one circular lens before the polychromic mirror, and a second lens unit including two circular lenses after the polychromic mirror. 
     In the first lens unit of the lens unit, the curved surfaces of the two cylindrical lenses are in a horizontal direction and the curved surface of the one cylindrical lens is in a vertical direction, and thus the focus and size of the component of the light of the light source in a horizontal direction and the focus and size of the component of the light of the light source in a vertical direction are separately controlled, thereby converting the light of the light source into linear light when the light of the light source is radiated onto the object. 
     The two circular lenses of the second lens unit of the lens unit may function to provide the light of the light source to the microscope unit after controlling the size and focus of the light of the light source, and may also function to provide fluorescent light received from a microscope to the polychromic mirror. 
     The polychromic mirror may reflect the light of the light source, and may transmit the light of other fluorescent light wavelengths. 
     The scan mirror may scan the light of the light source, thereby allowing the light to be radiated on the wide area of the object, and, simultaneously, may inversely scan fluorescent light emitted from different parts of the object, thereby allowing beams of fluorescent light to pass through the same optical path after the scan mirror. 
     The first Galvano mirror of the scan mirror and the second Galvano mirror and camera of the detection unit may be synchronized with one another. 
     The microscope unit may include a mirror configured to function to provide the light of the light source received from the lens unit to the object lens by reflecting the light of the light source, and an object lens configured to directly radiate the light of the light source onto the object and to condense fluorescent light emitted from the object. 
     The detection unit may include an electron multiplying charge coupled device (EMCCD) camera configured to detect fluorescent light, a second Galvano mirror configured to scan the fluorescent light onto the camera, a slit configured to remove fluorescent light emitted from parts other than a focal plane, and three circular lenses configured to control the focus of the fluorescent light. 
     The width of the slit of the detection unit may be 1 airy unit (AU) in order to maximally remove a background while minimizing the loss of fluorescent signals. 
     Advantageous Effects 
     The present invention configured and operated as described above is advantageous in that a single fluorescent molecular signal can be observed using a confocal microscope method because background signals generated from parts other than an object are removed using a line scan confocal microscope method and the large-area EMCCD camera having better sensitivity than a conventional linear camera is used. 
     Furthermore, the present invention is advantageous in that observation speed (several tens of Hz) close to real-time speed can be obtained using a method of scanning inversely scanned fluorescent light onto the large-area CCD camera using the second Galvano mirror. 
     Furthermore, the present invention is advantageous in that a single fluorescent molecular signal can be observed even in a thick object because a confocal microscope method is used. 
     Furthermore, the present invention is advantageous in that an ultra-high definition image based on the determination of the position of a single fluorescent molecule can be obtained at a deeper location in a cell or a tissue using a confocal microscope method. 
    
    
     
       DESCRIPTION OF DRAWINGS 
         FIG. 1  is an overall schematic top view of a confocal fluorescence microscope according to an embodiment of the present invention; 
         FIG. 2  is a schematic diagram of a light source unit; and 
         FIGS. 3A and 3B  are diagrams illustrating the arrangement of cylindrical lenses based on the curved surfaces thereof. 
     
    
    
     DETAILED DESCRIPTION OF THE EMBODIMENTS 
     Preferred embodiments of a confocal fluorescence microscope for observing a single fluorescent molecule in real time according to the present invention will be described in detail below with reference to the accompanying drawings. 
       FIG. 1  is an overall schematic diagram of a confocal fluorescence microscope according to an embodiment of the present invention with a light source unit omitted.  FIG. 2  is a schematic diagram of a light source unit, and  FIGS. 3A and 3B  are diagrams illustrating the arrangement of cylindrical lenses based on the curved surfaces thereof.  FIG. 3A  is a top view when the cross sectionals of the cylindrical lenses are viewed from above, and  FIG. 3B  is a side view when the cross sections of the cylindrical lenses are viewed from side. In  FIGS. 2, 3A and 3B , the components illustrated in  FIG. 1  are assigned three-digit reference numerals that each include a first digit corresponding to the number of a corresponding drawing and the remaining two digits corresponding to the corresponding reference numeral of  FIG. 1 . For convenience of description, the horizontal components  3  and  303  of light are indicated by solid lines, the vertical components  4  and  304  of light are indicated by chain-clashed lines, and a fluorescent signal  5  is indicated by a dotted line. 
     The real-time linear confocal fluorescence microscope according to the present invention is characterized by including a light source unit  201  configured to include one or more short-wavelength lasers, a lens unit  6  configured to make light from the light source unit  201  linear, a first Galvano mirror  21  configured to provide light to different parts of an object, a microscope unit  2  configured to radiate received light onto an object and condense fluorescent light generated by the object, and a detection unit  3  configured to observe the fluorescent light. In particular, the technical gist of the present invention is that a large-area CCD camera  40  is used using a method in which a fluorescent signal inversely scanned by the first Galvano mirror  21  is scanned onto a large-area CCD camera using the second Galvano mirror  25  of the detection unit. 
     The light source unit  201  includes one or more short-wavelength lasers, and light emitted from the lasers is collimated into a single beam of light using dichromatic mirrors suitable for respective laser wavelengths. 
     The collimated light passes through a first cylindrical lens  10 . In this case, since the first cylindrical lens  10  has a curved surface in a horizontal direction, the horizontal component  3  of the collimated light is changed from parallel light to light that is focused, and the vertical component  4  thereof maintains parallel light. 
     The light that has passed through the first cylindrical lens  10  passes through a second cylindrical lens  11 . In this case, a curved surface of the second cylindrical lens  11  is vertical to the first cylindrical lens  10 . That is, since the curved surface of the second cylindrical lens  11  is in a vertical direction, the horizontal component  3  of the light is not influenced, and the vertical component  4  thereof is changed from the parallel light to light that is focused. 
     The light that has passed through the second cylindrical lens  11  passes through a third cylindrical lens  12 . The third cylindrical lens  12  is spaced apart from the first cylindrical lens  10  by the focal distance of the first cylindrical lens  10  plus the focal distance of the third cylindrical lens  12 . In this case, the direction of the curved surface of the third cylindrical lens  12  is the same as that of the first cylindrical lens  10 , and is vertical to that of the second cylindrical lens  11 . That is, since the curved surface of the third cylindrical lens  12  is in a horizontal direction, the vertical component  4  of the light is not influenced. The horizontal component  3  of the light becomes parallel light after passing through the third cylindrical lens  12  because the third cylindrical lens  12  is placed at a position that is spaced apart by the sum of the focal distances of the first cylindrical lens  10  and the third cylindrical lens  12 . 
     The focal distance of the third cylindrical lens  12  and the focal distance of the first cylindrical lens  10  are determined such that the focal distance of the third cylindrical lens  12  is 10 times the focal distance of the first cylindrical lens  10 . Preferably, the focal distance of the first cylindrical lens  10  and the focal distance of the third cylindrical lens  12  may be 25 mm and 250 mm, respectively. The size of the horizontal component  3  of the light is increased 10 times after the light has passed through the first cylindrical lens  10  and the third cylindrical lens  12 . The size of the increased horizontal component functions to increase the length of a line in linear focus which is generated when the increased horizontal component is subsequently radiated onto an object. 
     The light that has passed through the three cylindrical lenses passes through the first circular lens  13 . The first circular lens  13  is spaced apart from the second cylindrical lens  11  by the focal distance of the second cylindrical lens  11  plus the focal distance of the first circular lens  13 . Accordingly, the vertical component  4  of the light becomes parallel light after passing through the first circular lens  13 . Furthermore, the horizontal component  3  of the light is changed from parallel light to light that is focused. 
     Thereafter, the light is reflected from a polychromic mirror  20  and then reflected from the first Galvano mirror  21  again. The polychromic mirror  20  reflects the light of the light source, and transmits fluorescent light having other wavelengths. 
     The first Galvano mirror  21  is spaced apart from the first circular lens  13  by the focal distance of the first circular lens  13 . Accordingly, the horizontal component  3  of the light is focused on the first Galvano mirror  21 . 
     The light reflected from the first Galvano mirror  21  passes through a second circular lens  14 . The second circular lens  14  is spaced apart from the first Galvano mirror  21  by the focal distance of the second circular lens  14 . Accordingly, the horizontal component  3  of the light becomes parallel light after passing through the second circular lens  14 . The vertical component  4  of the light is changed from the parallel light into light that is focused. 
     The first circular lens  13  and the second circular lens  14  have the same focal distance as the third cylindrical lens  12 , that is, 250 mm, and thus the size of the light is not changed. 
     Thereafter, the light is reflected from a first plane mirror  22 . The first plane mirror  22  has a diameter of 2 inches, and has been devised to fully reflect the component  3  of the light in a horizontal direction that has been increased 10 times. 
     The light reflected from the first plane mirror  22  is incident on the inside of the microscope through the back of the microscope after passing through a third circular lens  15 . The third circular lens  15  is spaced apart from the second circular lens  14  by the focal distance of the second circular lens  14  plus the focal distance of the third circular lens  15 , and is also spaced apart from the object lens  19  of the microscope unit  2  by the focal distance of the object lens  19  plus the focal distance of the third circular lens  15 . The light that has passed through the third circular lens  15  is changed into light that is focused in a horizontal direction and into parallel light in a vertical direction. 
     The focal distance of the third circular lens  15  is 1.2 times the focal distance of the first circular lens  13 , for example, 300 mm. A long focal distance is used to correspond to the distance to the object lens  19 . 
     The light incident on the inside of the microscope passes through the object lens  19  after being reflected from a silver-plated mirror  23  within the microscope. 
     The silver-plated mirror  23  has been subjected to special coating processing in order to minimize the loss of light and fluorescent light. 
     The light that has passed through the object lens  19  becomes parallel light in a horizontal direction, and becomes light that is focused in a vertical direction. In this case, the focus in the vertical direction is the same as the focus of the object lens  19 . Accordingly, the light incident on an object has a linear shape. (the focus in the vertical direction and the parallel light in the horizontal direction) 
     The light that has passed through the object lens  19  is incident on an object so that the object generates fluorescent light. In this case, the fluorescent light emitted from the object is condensed by the object lens  19 , and the condensed light returns along the path through which the light was incident. 
     Only the 1-dimensional fluorescent light information of the object may be obtained based, on the linear light radiated onto the object. In order to obtain a 2-dimensional fluorescent image, the 2-dimensional fluorescent light information of the object is obtained by moving the light in a direction vertical to the linear light by changing the angle of the first Galvano mirror  21 . 
     The fluorescent light transferred externally through the silver-plated mirror  23  reaches the first Galvano mirror  21  through the third circular lens  15  and the second circular lens  14 . Reams of fluorescent light emitted from different positions of the object pass through different optical paths up to the first. Galvano mirror  21  depending on an angle of the first Galvano mirror  21 , but pass through the same optical paths after the first Galvano mirror  21  because they are inversely scanned by the first Galvano mirror  21 . 
     The inversely scanned fluorescent light passes through the polychromic mirror  20  and is then changed into light that is focused via the fourth circular lens  16  of the detection unit  3 . That is, a focus in this case and a focus at which an object is observed in the object lens  19  have a confocal characteristic. 
     A slit  30  placed at the focus of the fluorescent light functions to remove all background signals emitted from parts other than a focal plane. In this case, if the size of the slit  30  is excessively large, the ability to remove background signals is low. In contrast, if the size of the slit  30  is excessively small, it is difficult to observe a single fluorescent molecule because even a fluorescent signal to be observed is removed. For this reason, the width of the slit  30  is determined to be 1 AU corresponding to one unit of the diffraction limit of the light that is radiated onto an object. 
     The fluorescent light from which background signals have been removed by the slit  30  is changed into parallel light via a fifth circular lens  17  and then reflected from the second Galvano mirror  25 . The second Galvano mirror  25  changes its angle in synchronization with the first Galvano mirror  21 . That is, light is radiated onto different parts of an object through the first Galvano mirror  21 , and beams of fluorescent light emitted from the different parts of the object move along the same optical path, are scanned by the second Galvano mirror  25  and form fluorescent light at different parts of a large-area camera. 
     The light reflected from the second Galvano mirror  25  is focused on the large-area CCD camera  40  via a sixth circular lens  18 . 
     The focal distances of the circular lenses and cylindrical lenses of the lens unit  6  and the detection unit  3  may be changed by a user. 
     The CCD camera  40  is a back-illuminated electron multiplying charge coupled device (EMCCD) camera, and generates a 2-dimensiaonl fluorescent image of 512×512 pixels in each exposure time by being exposed during a designated exposure time. In this case, the consumption of fluorescent signals and the inconsistency between the fluorescent signals of images are minimized because the exposure time of the large-area CCD camera  40  is synchronized with the scan time of the first Galvano mirror  21  and the second Galvano mirror  25 . 
     Although the EMCCD camera has sensitivity to the extent that a single fluorescent molecule can be observed, the EMCCD camera can observe a large area at the same time but has slower observation speed than a conventional linear CCD camera. However, the conventional linear CCD camera is not sensitive enough to observe a single fluorescent molecule. In the present invention, fluorescent light is scanned again by the second Galvano mirror  25 , and the scan time of the second Galvano mirror  25  is synchronized with the exposure time of the large-area CCD camera  40 . Accordingly, even when a line scan confocal microscope is used, a large-area fluorescent image can be obtained at one time, with the result that slow observation speed can be overcome and an observation speed of 301-Iz or higher can be guaranteed. 
     Although the principle and operating method of the present invention have been described and illustrated, the present invention is not limited thereto. 
     In particular, observing a single fluorescent molecular signal in real time using a confocal microscope method is beyond significance in terms of observation, and provides technology essential to super-resolution imaging and systems biology that have recently attracted attention. Accordingly, the appropriate modifications and equivalents of the present invention should be considered to pertain to the scope of the present invention without departing from the spirit and scope of the present invention.