Abstract:
A nano-scale resolution fluorescence microscopy system and a method of obtaining an image using the nano-scale resolution microscopy system, and more particularly, a method and a microscopy system, capable of observing fluorescence probes in high resolution by radiating an irregular diffused light to have an incoherent speckle pattern that has low correlation in an adjacent space are disclosed. According to embodiments of the present invention, a diffraction limit of a fluorescence microscope may be overcome, and a super high resolution image on a nanometer scale may be obtained.

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application claims the benefit of Korean Patent Application No. 10-2011-0032783, filed on Apr. 8, 2011, in the Korean Intellectual Property Office, the disclosure of which is incorporated herein by reference. 
     BACKGROUND 
     1. Field of the Invention 
     Embodiments of the present invention relate to a nano-scale resolution microscopy system and methods of obtaining super-resolution images using the nano-scale resolution microscopy system, and more particularly, to methods and microscopy systems, capable of observing specimen at very high resolution by exploiting emission statistics of fluorescence probes excited by speckle illumination. 
     2. Description of the Related Art 
     A conventional optical microscope has a fundamental spatial resolution limit dependent on a wavelength of light and the numerical aperture of a lens. The best resolution of a conventional optical microscope corresponds to about a half of a wavelength, which refers to a diffraction limit. 
     Fluorophores, such as fluorescence probes or fluorescence proteins have been extensively designed to be specific to particular cellular functions such as signal transduction and gene expression, so fluorescence microscopy has become an invaluable tool in biology. 
     In fluorescence microscopy, fluorophores are directly attached to a region of interest within a cell or particular proteins. However, the conventional microscopes may have a limitation in overcoming the diffraction limit of the microscope optical systems, and more particularly, a limitation in resolving fluorophores which are separately less than the diffraction limit. 
     To address this problem, super-resolution far-field fluorescence nanoscopy have been extensively investigated. This super-resolution microscopy is to exploit non-linear optical phenomena to break the diffraction limit. In STED microscopy, a Gaussian shape excitation beam and a red-shifted doughnut-shaped STED beam are used to quench excited fluorophores by stimulated emission from excitation to ground state. (Klar, T. A., Jakobs, S., Dyba, M., Egner, A. &amp; Hell, S. W. Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission.  Proceedings of the National Academy of Sciences of the United States of America  97, 8206-8210 (2000).) In saturated structured illumination microscopy (SSIM), structured illumination is used to extend the spatial bandwidth of the optical system using the synthetic aperture principle. (Gustafs son, M. G. L. Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution.  Proceedings of the National Academy of Sciences of the United States of America  102, 13081 (2005).) In PALM and STORM for each imaging cycle, an optically resolvable random subset of photoswitchable fluorophores is excited to the active state and then switched off to the dark state quickly using background quenching lights. (Rust, M. J., Bates, M. &amp; Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).  Nature Methods  3, 793-796 (2006).) 
     SUMMARY 
     An aspect of the present invention provides a nano-scale resolution fluorescence microscopy using dynamic speckle illumination and array signal processing algorithms. 
     An aspect of the present invention provides a nano-scale resolution fluorescence microscopy system and methods of obtaining super-resolution images using the nano-scale resolution microscopy system, capable of overcoming the diffraction limit of a fluorescence microscope that may be useful for basic and applied scientific researches. 
     Another aspect of the present invention also provides a nano-scale resolution fluorescence microscopy system and methods of obtaining super resolution images using the nano-scale resolution microscopy system, and sensor array signal processing techniques using any type of fluorophores. 
     According to an aspect of the present invention, there is provided a nano-scale resolution fluorescence microscopy system, including: a speckle pattern generator to generate dynamic speckle illumination by passing, through a diffuser or spatial light modulator, lights from at least one coherent sources with at least one colors; an incident optical system to illuminate the generated speckle patterns on a specimen; and a fluorescence imaging optical system to detect fluorescent emission from the fluorophores excited by the speckle illumination, by passing the radiated speckle patterns and fluorescence emission sequentially through at least one excitation filter and at least one emission filter, respectively 
     The speckle pattern generator may generate the speckle illumination patterns, using either at least one reflection diffuser or at least one transmission diffuser or at least one spatial light modulator. 
     The speckle pattern generator may generate the speckle illumination patterns at least one color using at least one coherent source with at least one wavelengths. 
     The illumination optical system may include either diffraction limited far-field optics in transmission or reflection type or a near-field optics such as total internal reflection (TIR) optics. 
     The at least one excitation filter may excite fluorophores using the at least one color speckle patterns, and the at least one emission filter may selectively pass fluorescent emission lights in at least one color from the fluorophores. 
     At least one of the illumination optical system and the fluorescent image optical system may be disposed in a structure of at least one of a reflection microscope, a transmission microscope, and a near-field optical microscope. 
     The nano-scale resolution microscopy system may further include an image reconstruction algorithm to restore the images at high resolution using a set of sequentially measured fluorescence emission images using time dependent dynamic speckle illumination. 
     The image reconstruction algorithm may calculate data covariance matrices using the set of sequentially measured fluorescence emission images using time dependent dynamic speckle illumination. 
     The image reconstruction algorithm may obtain high resolution images by exploiting that the temporal profiles of fluorescence emission for adjacent fluorescence emissions are statistically uncorrelated due to the incoherence of speckle illumination. 
     The image reconstruction algorithm may decompose the data covariance matrix into the signal subspace and the noise subspace, and may reconstruct positions of fluorophores by exploiting the orthogonality of a model signal calculated from a fluorophore location with respect to the noise subspace. 
     The image reconstruction algorithm may reconstruct positions of fluorophores by exploiting that a model signal calculated from a fluorophore location is highly correlated with the signal subspace. 
     According to another aspect of the present invention, there is provided a method of obtaining an image using a nano-scale resolution microscopy system, the method including: at least one coherent sources in at least one color for the purpose of imaging fluorophores attached to a specimen; generating speckle illumination patterns by passing a light generated from at least one coherent sources in at least one color through at least one diffuser or spatial light modulator; radiating the generated speckle patterns to be exposed on a specimen, using at least one optical lens; and obtaining fluorescence emission images excited by the speckle patterns in at least one color, using at least one excitation filter and at least one emission filter. 
     The generating may include generating the speckle patterns in at least one color having an irregular pattern, using either a reflection type diffuser or a transmission type diffuser or spatial light modulator. 
     The at least one optical lens may correspond to a far-field optics, or a near-field optics. 
     The at least one coherent source may include lasers or other coherent light sources in at least one color. 
     The at least one excitation filter may selectively excite fluorophores using the speckle patterns with at least one colors, and the at least one emission filter may selectively pass fluorescent emission from the speckle illumination through at least one excitation filters. 
     The method may further include: sequentially obtaining florescent emission images by the fluorescent image optical system; and reconstructing images at high resolution by exploiting the signal and noise subspace structures of a data covariance matrix. 
     The reconstruction of the image at high resolution may include: generating speckle illumination pattern in at least one color; sequentially obtaining the fluorescence emission images, calculating a data covariance matrix and decomposing them into the signal and the noise subspaces; restoring high resolution images by exploiting the signal and noise subspace structure of the data covariance matrix. 
     The reconstruction of the image in high resolution may include: decomposing the data covariance matrix into the signal subspace and the noise subspace; and restoring positions of the fluorophores by exploiting the orthogonality of a model signal calculated at a fluorophore location with respect to the classified noise subspace. 
     According to still another aspect of the present invention, there is provided a nano-scale resolution microscopy system, including: an incident optical system; a fluorescent image optical system; a speckle pattern generator to generate speckle patterns in at least one color by passing, through at least one diffuser or at least one spatial light modulator, a light emitted from at least one coherent source in at least one color to detect fluorescent emission images of a specimen; and an image reconstruction algorithm execution unit to execute an image reconstruction algorithm, wherein the incident optical system and the fluorescent image optical system may include at least one optical lens disposed in a structure of at least one of a reflection microscope, a transmission microscope, and a near-field optical microscope, and the image reconstruction algorithm execution unit may execute the image reconstruction algorithm, thereby sequentially obtaining fluorescence emission images by the fluorescent image optical system, and reconstructing the images at high resolution by exploiting the statistical properties of the fluorescence emission temporal profile at every pixel excited by the speckle illumination. 
     The image reconstruction algorithm execution unit may generate speckle patterns to excite fluorophores, may calculate a data covariance matrix using sequentially obtained fluorescence emission images from a specimen excited by the speckle illumination pattern, and may reconstruct images with respect to the fluorescent probes location of the specimen based on a result of the calculation. 
     EFFECT 
     According to embodiments of the present invention, an image with respect to fluorophores in a specimen at high resolution may be obtained by obtaining a super resolution image at a nanometer scale, using time dependent speckle illumination pattern and sensor array signal processing. 
     According to embodiments of the present invention, a high power laser beam for a non-linear optical transition in fluorophores may be unnecessary. Furthermore, a nano-scale resolution microscopy system of the present invention may use not only a switchable fluorescence probes but also other non-switchable fluorescent probes and fluorescent proteins. 
     According to embodiments of the present invention, a high resolution image beyond the diffraction limit of a fluorescence microscope, that may be useful for basic and applied scientific researches, may be easily implemented by modifying a conventional fluorescence microscope to have a dynamic speckle pattern illuminator and image reconstruction software. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       These and/or other aspects, features, and advantages of the invention will become apparent and more readily appreciated from the following description of exemplary embodiments, taken in conjunction with the accompanying drawings of which: 
         FIG. 1  is a diagram illustrating a structure of a nano-scale resolution microscopy system according to an embodiment of the present invention; 
         FIG. 2  is a diagram illustrating operations of the nano-scale resolution microscopy system illustrated in  FIG. 1 ; 
         FIGS. 3A and 3B  are diagrams illustrating coherent lights passing through a transmission diffuser or a reflection diffuser in the nano-scale resolution microscopy system; 
         FIG. 4  is a diagram illustrating an example of a microscope structure that may be utilized in the nano-scale resolution microscopy system of  FIG. 1 ; 
         FIG. 5  is a diagram illustrating another example of a microscope structure that may be utilized in the nano-scale resolution microscopy system of  FIG. 1 ; 
         FIG. 6  is a diagram illustrating an example of images resulting from randomly aggregated fluorescence nano-particles measured by the nano-scale resolution microscopy system illustrated in  FIG. 1 ; 
         FIG. 7  is a profile of reconstructed images illustrating a comparison of the images resulting from fluorescence nano-particles illustrated in  FIG. 6 ; and 
         FIG. 8  is a diagram illustrating an example of images resulting from fluorescence stained actin with respect to the nano-scale resolution microscopy system illustrated in  FIG. 1 , and a conventional microscopy system; 
         FIG. 9  is a profile of reconstructed images illustrating a comparison of the images resulting from fluorescence stained actin illustrated in  FIG. 8 ; and 
         FIGS. 10A and 10B  are diagrams illustrating examples of images resulting from fluorescence stained mitochondria by the nano-scale resolution microscopy system illustrated in  FIG. 1 . 
     
    
    
     DETAILED DESCRIPTION 
     Reference will now be made in detail to exemplary embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to the like elements throughout. Exemplary embodiments are described below to explain the present invention by referring to the figures. 
     According to embodiments of the present invention, a nano-scale resolution microscopy system may include at least one coherent light source and at least one diffusor or spatial light modulator or anytime of speckle pattern generator. The nano-scale resolution microscopy system may illuminate randomized pattern such as speckle patterns generated at the diffuser to a specimen, by changing the characteristics of the diffuser, then multiple snapshots of speckle patterns can be dynamically generated on the specimen. The nano-scale resolution microscopy system may change an optical or electrical characteristic of the diffuser. In case of roughness diffuser, rotating or translating of diffuser may change optical characteristics which cause speckle patterns, and in case of spatial light modulators, different electrical signals may change different random patterns look like a speckle pattern. Also, the nano-scale resolution microscopy system may continuously obtain fluorescence signals excited by the illuminated speckle patterns. The fluorescence signals may be passed through a dichroic mirror (DM) and an emission filter, and may be measured by a camera. 
     Thus, the nano-scale resolution microscopy system may include components as illustrated in  FIG. 1 .  FIG. 1  is a diagram to describe a structure of a nano-scale resolution microscopy system according to an embodiment of the present invention. 
     Referring to  FIG. 1 , each component of the nano-scale resolution microscopy system may be shown in blocks. 
     The nano-scale resolution microscopy system may include a random pattern generator  110 , an illumination optical system  120 , a fluorescence imaging optical system  130 , and an image reconstruction algorithm  140 . 
     The random pattern generator  110  may include a diffuser and/or spatial light modulators. The random pattern generator  110  may generate speckle patterns by passing a light emitted from at least one coherent light source and random patterns by changing random patterns on modulator. The diffuser may scatter the light passed through the diffuser, and may output the light having a low spatial coherence with respect to adjacent regions. Instead of roughness diffuser, the spatial light modulators may be applied to generate random patterns by changing the electrical signal. The modulators may reflect or transmit the light from a continuous wave laser. The random pattern generator  110  may generate speckle patterns and random patterns having a low spatial coherence. 
     The illumination optical system  120  may illuminate the light passed through the diffuser to a specimen so that the speckle patterns generated by the random pattern generator  110  may be exposed on the specimen. Also, the illumination optical system  120  may overcome a diffraction limit of a fluorescence microscopy by disposing an optical lens where a size of an irregular pattern of the speckle patterns may have a diffraction limit, or by disposing a total internal reflection optical system to reduce the diffraction limit. 
     The fluorescence imaging optical system  130  may pass the speckle patterns illuminated by the illumination optical system  120 , sequentially through at least one excitation filter and at least one emission filter. The fluorescence imaging optical system  130  may acquire a fluorescence signal by measuring a fluorescence emission signal excited by the illuminated speckle patterns. 
     Here, the at least one excitation filter may apply to excite a fluorophore by the speckle patterns illuminated from the illumination optical system  120 , and the at least one emission filter may selectively pass fluorescence signals excited by the at least one excitation filter. Accordingly, the fluorescence signal excited by the speckle patterns may be selectively passed by the at least one emission filter after a fluorophore is excited by the at least one excitation filter. 
     The image reconstruction algorithm  140  may continuously obtain images measured by the fluorescence imaging optical system  130 , and may reconstruct the images in nano-scale resolution by time dependent data that may change in every pixel, based on the continuously obtained images. In this instance, the data changed depending on time may be viewed more clearly in the reconstructed images, and fluorophores of a target object may be viewed in nano-scale resolution. The image reconstruction algorithm  140  may be executed by an image reconstruction algorithm execution unit. 
     The image reconstruction algorithm may exploit a characteristic of a temporal correlation between adjacent pixels being low in a spatial domain scattered by the diffuser. 
     Particularly, when a speckle pattern S generated by the diffuser is illuminated to a specimen, a mathematical model of images obtained by the fluorescence imaging optical system  130  may be given by:
 
 I ( x,y,;t )={PSF det   *└x   f (PSF ill   *s )┘}( x,y;t )+ w ( x,y;t ).  [Equation 1]
 
     Here, (x, y) denotes coordinates of a camera, t denotes time instances for each measurement, and S and 
     x f  denote a speckle pattern and fluorescence signals. Also, PSF ill  denotes a point spread function of a condenser lens or an objective lens where the speckle pattern S may be convoluted, and PSF det  denotes a point spread function of an objective lens where fluorescence signal may be convoluted. 
     Accordingly, the speckle pattern S generated according to Equation 1 may be convoluted with the point spread function of the condenser lens or the objective lens, and may be illuminated to the specimen, and the fluorescence signal x f  emitted in response to the excitation light may be convoluted with the point spread function of the objective lens. 
     When Equation 1 is analytically expressed and point spread functions are expressed in a form of a single point spread function, Equation 2 may be derived.
 
 I ( x,y,;t )= x   f ∫∫PSF( x−x′,y−y′;t ) s ( x′,y′;t ) dx′dy′+w ( x,y;t ).  [Equation 2]
 
     Also, when Equation 2 is expressed as a matrix, and a point spread function that may be convoluted by Equation 2 is expressed as a convolution matrix A, Equation 3 may be derived.
 
   I   ( t )= A X   ( t )+   w   )( t ).  [Equation 3]
 
     When a spatial covariance matrix R is calculated based on data  I  with respect to the changes of fluorescence signals, the structure may be given by: 
     
       
         
           
             
               
                 
                   R 
                   = 
                   
                     
                       
                         1 
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                       ⁢ 
                       
                         
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                               I 
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     Here, T denotes a total amount of time consumed for obtaining images,  I  denotes data with respect to changes of fluorophores measured at a predetermined time, R denotes a spatial covariance matrix, σ 2 I denotes a noise spatial covariance matrix, A denotes a point spread function of optical system, and P denotes a correlation of fluorophores. 
     Accordingly, based on the image reconstruction algorithm, the spatial covariance matrix may be calculated based on at least one of  I  the data with respect to changes of fluorophores signal measured at a predetermined time, R the spatial covariance matrix, σ 2 I the noise spatial covariance matrix, A the point spread function of elements, and P the correlation of fluorescence probes. 
     We may express the spatial covariance matrix R, using the correlation P and the point spread function A of elements, and may express white noise w uncorrelated to the fluorescence signal. 
     A correlation between electromagnetic fields of a randomized speckle pattern may be given by: 
     
       
         
           
             
               
                 
                   
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     Here, σ 2   o  denotes a phase change depending on a change in height of a surface of a diffuser, and x f  and x o  denote fluorescence signals and a randomized speckle pattern. Also, μ o  denotes a function with respect to a distance between two points, which may have a characteristic of converging to 1 when the distance between the two points becomes closer. R PSF  is the illumination point spread function autocorrelation function. 
     Accordingly, when the change σ 2   o  in height of the surface of the diffuser becomes greater, a function of the correlation of speckle patterns between the pixel elements may converge to a delta function, and a matrix of the correlation P between the adjacent fluorophores may correspond to a near diagonal matrix. In illumination optical system, excitation pattern for fluorescence imaging is convolved with illumination point spread function. When the matrix of the correlation P between the adjacent fluorophores may be expressed as a diagonal matrix, a performance of an estimator that may reconstruct the fluorophore may be as good as a performance of an estimator that may reconstruct a single molecule. 
     Further, according to the sensor array signal processing perspective, positions of the fluorophores may be reconstructed using a subspace method which may find a position of a target signal by classifying a spatial covariance matrix R of a measurement data into a signal subspace and a noise subspace. In particular, we may use a Multiple Signal Classification (MUSIC) method as well as other various types of subspace methods. 
     The MUSIC method may correspond to a method generally used for an antenna sensor array signal processing perspective, which may use the covariance matrix decomposition by Equation 6. 
     
       
         
           
             
               
                 
                   
                     
                       
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     The MUSIC method may perform a singular value decomposition with respect to a spatial covariance matrix R, and may classify a singular vector U into a signal subspace u s  and a noise subspace u n  based on a size of a white noise distribution σ 2 , from a distribution of singular values. Also, the MUSIC method may further use: 
     
       
         
           
             
               
                 
                   
                     P 
                     MU 
                   
                   = 
                   
                     
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     Here, p corresponds to 0≦p≦∞. According to the MUSIC method based on Equation 7, a null-spectrum of the noise subspace may be calculated based on the singular vector u n  and the point spread function A with respect to the noise subspace, and a position of a signal may be reconstructed as the peak of the spectrum in Equation 7. According to the method, the positions of the fluorophores of the target object may be reconstructed by classifying a signal of the measured images into a signal subspace and a noise subspace, and based on a principle of a target signal existing in a position where noise generated by the classified noise subspace may be minimized. 
       FIG. 2  illustrates operations of the nano-scale resolution microscopy system illustrated in  FIG. 1 , and  FIGS. 3A and 3B  illustrate coherent lights  300  and  310  passing through diffusers  301  and  311  in the nano-scale resolution microscopy system. Particularly, the diffuser  301  of  FIG. 3A  may correspond to a transmission diffuser, and the diffuser  311  of  FIG. 3B  may correspond to a reflection diffuser. 
     When light is emitted from a continuous wave laser in order to measure fluorescence signal of a target object, the random pattern generator  110  may generate speckle patterns by passing or transmitting the light emanated from the laser through a diffuser, in operation  200 . 
     Particularly referring to  FIG. 3A , the coherent light  300  of the light emitted from the continuous wave laser may be passed through the transmission diffuser  301 . 
     When the coherent light  300  is passed through the transmission diffuser  301 , the speckle patterns may be generated. When the transmission diffuser  301  diffuses and passes the coherent light  300 , the coherent light  300  passed through the transmission diffuser  301  may have incoherence according to a change in mechanical, optical, or electrical characteristics. 
     Referring to  FIG. 3B , the coherent light  310  may be reflected from the reflection diffuser  311 . When the reflection diffuser  311  diffuses and reflects the coherent light  310 , the coherent light  310  reflected from the reflection diffuser  311  may have incoherence according to a change in mechanical, optical, or electrical characteristics. 
     Consequently, the transmission diffuser  301  or the reflection diffuser  311  may continuously generate speckle patterns of the temporal coherent lights  300  and  310 . 
     In operation  210 , the illumination optical system  120  may illuminate the speckle patterns, generated in operation  200 , to be illuminated on a specimen using at least one optical component. 
     In operation  220 , the fluorescence imaging optical system  130  may excite fluorophores by passing the illuminated speckle patterns through at least one excitation filter. The fluorescence imaging optical system  130  may pass a fluorescence signal generated as a result of the excitation, through at least one emission filter. The fluorescence imaging optical system  130  may measure the fluorescence signal excited by the speckle patterns. The measured image may have fluorescence signals. 
     In operation  230 , the fluorescence imaging optical system  130  may continuously obtain images, N number of times. 
     In operation  240 , the image reconstruction algorithm  140  may reconstruct the images in nano-scale resolution by multiple measurement data dependent on time that may change in every pixel, based on the continuously obtained images. As aforementioned, the image reconstruction algorithm  140  may use a sensor array signal processing algorithm such MUSIC scheme. 
     The illumination optical system  120  and the fluorescence imaging optical system  130  may dispose the at least one optical component in a structure of at least one of a reflection microscope, a transmission microscope, and a TIR optical microscope. 
       FIG. 4  is a diagram illustrating an example of a microscope structure that may be utilized in the nano-scale resolution microscopy system of  FIG. 1 . 
     Referring to  FIG. 4 , the example may represent a structure of the fluorescence imaging optical system  130  corresponding to a transmission microscope  400 . 
     In the transmission microscope  400 , a coherent light  10  may be passed through a transmission diffuser  401 , and then speckle patterns may be generated by the transmission diffuser  401 . 
     The transmission microscope  400  may include the transmission diffuser  401 , a first optical lens  402 , a second optical lens  403 , an excitation filter  404 , a condenser lens  405 , a specimen  406 , an objective lens  407 , an emission filter  408 , an adapter lens  409 , and a charge-coupled device (CCD)/complementary metal oxide semiconductor (CMOS) camera  410 . 
     Particularly, the transmission microscope  400  may arrange the first optical lens  402 , the second optical lens  403 , and the condenser lens  405  in a row so that the generated speckle patterns may be located in a back focal length  11  of the first optical lens  402 , a back focal length  13  of the second optical lens  403  may correspond to a front focal length  12  of the first optical lens  402 , and a front focal length  14  of the second optical lens  403  may correspond to a back focal length  15  of the condenser lens  405 . 
     Also, the transmission microscope  400  may dispose the excitation filter  404 , which may filter a light wavelength to excite a fluorescence probe, between the second optical lens  403  and the condenser lens  405 . 
     Accordingly, the speckle patterns may be finally illuminated to be exposed on the specimen  406 , and an image of the light emitted as a result of the excitation of the fluorophores passed through the excitation filter  404  may be magnified by the objective lens  407 , and may be passed through the emission filter  408 . The light may be selectively passed by the emission filter  408 , and may correspond to a fluorescence signal which is a part of the light passed though the emission filter  408 . The image of the light passed through the emission filter  408  may be exposed on the CCD/CMOS camera  410  by the adapter lens  409 . 
       FIG. 5  is a diagram illustrating another example of a microscope structure that may be realized in the nano-scale resolution microscopy system of  FIG. 1 . 
     Referring to  FIG. 5 , the example may represent a structure of the fluorescence imaging optical system  130  corresponding to a reflection microscope  500 . 
     Similar to the transmission microscope  400 , the reflection microscope  500  may arrange a transmission diffuser  501 , a first optical lens  502 , a second optical lens  503 , an objective lens  505 , and a specimen  506  in a row so that a back focal length  23  of the second optical lens  503  may correspond to a front focal length  22  of the first optical lens  502 , and a front focal length  24  of the second optical lens  503  may correspond to a back focal length  25  of the objective lens  505 . 
     The reflection microscope  500  may dispose an excitation filter  504 , and a dichroic filter  507  between the second optical lens  503  and the objective lens  505 , and may also dispose an emission filter  508 , an adapter lens  509 , and a CCD/CMOS camera  510  in a path where a light may be transmitted from the dichroic filter  507 . 
     Accordingly, a coherent light  20  may be passed through the transmission diffuser  501 , and speckle patterns may be generated by the transmission diffuser  501 . Then, the generated speckle patterns may be illuminated through the first optical lens  502 , the second optical lens  503 , and the objective lens  505  to be exposed on the specimen  506 . A light emitted from excited fluorophores may be magnified by the objective lens  505 , transmitted from the dichroic filter  507 , and passed through the emission filter  508 . The light passed through the emission filter  508  may be selectively passed through by the emission filter  508 , and may correspond to a fluorescence signal which is a part of the light passed though the emission filter  408 . The image of the light passed through the emission filter  508  may be exposed on the CCD/CMOS camera  510  by the adapter lens  509 . 
       FIG. 6  is a diagram illustrating an example of resulting images of randomly aggregated fluorescence nano-particles performed by the nano-scale resolution microscopy system illustrated in  FIG. 1 , and  FIG. 7  is a graph to compare the resulting images of fluorescence nano-particles illustrated in  FIG. 6 . 
     Referring to  FIG. 6 , the reconstructed large field-of-view of randomly aggregated 50 nm nanoparticles is presented. The conventional microscope images in  610  and  640  are observed using a 100× magnification oil-immersion objective lens. Images  620  and  650  are reconstructed images, and images  630  and  660  are scanning electron microscope (SEM) images to confirm the reconstruction images by present invention. There exists an empty space surrounded by nanoparticles in white boxes in images  610 ,  620 , and  630 . Images  640 ,  650 , and  660  are the white boxes images of  610 ,  620 , and  630 , respectively. As can be seen from the dashed lines in  640  and  650 , the width of the empty space is ˜100 nm and the widths of other two fluorophores are 70 nm and 130 nm represented in  660 . As illustrated in an image  650 , present invention resolved the empty space as well as the 70 nm fluorophore probe, whereas the space is not revealed in a conventional microscope image  640 . Based on the nanoparticle experimental results, present invention achieved 70 nm lateral resolution and 3-fold resolution improvement over the diffraction limit (λ em /2NA oil ) where NA oil  and λ em  are about 1.3 and 570 nm, respectively. 
     When comparing image data  700  around a dashed line of the image  640  and image data  710  around a dashed line of the image  650 , the graph illustrated in  FIG. 7  may be represented. 
     Referring to  FIG. 7 , the horizontal axis of the graph may correspond to a length of an actual sample, and the vertical axis of the graph may correspond to a size of a normalized signal. The image data  710  around the dashed line of the image  650  may have reduced background noise and improved resolution when compared to the image data  700  of the dashed line of the image  640 . 
       FIG. 8  is a diagram to describe an example of images resulting from actin stained by fluorescence dye with respect to the nano-scale resolution microscopy system illustrated in  FIG. 1 , and a conventional microscopy system, and  FIG. 9  is a graph to compare the resulting images of actin stained by fluorescence dye illustrated in  FIG. 8 . 
     Referring to  FIG. 8 , the resulting images of actin stained by fluorescence dye may correspond to images observed using a 60× magnification objective lens, by staining of actin in a cell. Particularly, an image  800  may correspond to an image of the cell observed using a conventional fluorescence microscope, and an image  810  may correspond to an image of the cell reconstructed using the nano-scale resolution microscopy system according to embodiments of the present invention. 
     When comparing image data  900  around a dotted line A of the image  800  and image data  910  around a dotted line B of the image  810 , the graph illustrated in  FIG. 9  may be represented. 
     Referring to  FIG. 9 , the horizontal axis of the graph may correspond to a length of an actual sample, and the vertical axis of the graph may correspond to a size of a normalized signal. The image data  910  around the dotted line B of the image  810  may have reduced background noise and improved resolution when compared to the image data  900  of the dotted line A of the image  800 . 
       FIGS. 10A and 10B  are diagrams illustrating examples of images resulting from fluorescence stained mitochondria by the nano-scale resolution microscopy system illustrated in  FIG. 1 . 
     Referring to  FIGS. 10A and 10B , the images resulting from stained mitochondria may correspond to images observed using a 60× magnification objective lens, by fluorescence stained mitochondria in a cell. In particular, images  1000  and  1020  may correspond to images of the cell observed using a conventional fluorescence microscopy, and images  1010  and  1030  may correspond to images of the cell observed using the nano-scale resolution microscopy system according to embodiments of the present invention. The images  1010  and  1030  observed using the nano-scale resolution microscopy system according to embodiments of the present invention may show clearer images when compared to the images  1000  and  1020  observed using the conventional fluorescence microscopy, and accordingly improvement in resolution may be observed. 
     The above-described exemplary embodiments of the present invention may be recorded in computer-readable media including program instructions to implement various operations embodied by a computer. The media may also include, alone or in combination with the program instructions, data files, data structures, and the like. The media and program instructions may be those specially designed and constructed, or they may be of the kind well-known and available to those having skill in the computer software arts. 
     Embodiments of the present invention have provided us with new insight into current state-of-the-art nanoscopy techniques, and what performance they can achieve. For example, PALM and STORM satisfy the condition of diagonal signal covariance matrix due to the non-overlapping photo-switching fluorophores, so the sensor arrays signal processing theory guarantees that its fundamental performance limitation approaches that of single molecular target detection. A similar explanation can be given if the quantum-dot blinking statistics are totally uncorrelated. As discussed before, these approaches, however, require a new type of fluorophores to make the signal covariance matrix diagonal. Unlike these microscopy, present invention does not require photoswitchable dye because the signal source can be made to be uncorrelated by using the spatial incoherence of speckle illumination. 
     Present invention may be implemented by simply modifying a conventional epi-fluorescence optical microscopy such that a rotating diffuser and low power continuous wave laser can be used. To demonstrate the improved resolution of present invention, we imaged phantom and biological samples. The phantom image results clearly revealed that the resolution of present invention can achieve over the diffraction limit. The stained actin and mitochondria imaged by present invention reveals ultra-structures that were not visible using conventional fluorescence. 
     The resolution limiting effect of illumination optics can be further reduced by employing total internal reflection fluorescence (TIRF) optics, as was done in PALM and STORM. Furthermore, the proposed nanoscale image reconstruction algorithm can be combined with PALM and STORM to improve the resolution furthermore. 
     Although a few exemplary embodiments of the present invention have been shown and described, the present invention is not limited to the described exemplary embodiments. Instead, it would be appreciated by those skilled in the art that changes may be made to these exemplary embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.