Abstract:
An optical arrangement for imaging a sample is disclosed. The optical arrangement comprises at least one first objective lens and at least one second objective lens, at least one illumination source for producing an illumination beam, detector for imaging radiation from the sample, and at least one mirror for reflecting the radiation from one of the first objective lens or the second objective lens into the detector. The at least one mirror is double-sided and dependent on the illumination beam at the other one of the first objective lens and the second objective lens.

Description:
CROSS-RELATION TO OTHER APPLICATIONS 
       [0001]    None 
       FIELD OF THE INVENTION 
       [0002]    The invention relates to an optical arrangement and a method for imaging a sample using at least two illumination beams. 
       BACKGROUND TO THE INVENTION 
       [0003]    A microscope is a scientific instrument that is used for the visualization of objects, which can be either small cells or have details that are too small to be resolved by the naked eye. 
         [0004]    There are many types of microscopes available on the market. The most common of these and the first to be invented is the so-called optical microscope, which uses light in a system of lenses to magnify images of the samples. The image from the optical micro-scope can be either viewed through an eyepiece or, more commonly nowadays, captured by a light-sensitive camera sensor to generate a so-called micrograph. There are a wide range of sensors available to catch the images. Non-limiting examples are charge-coupled devices (CCD) and scientific complementary metal-oxide semiconductor (sCMOS) based technologies, which are widely used. These sensors allow the capture and storage of digital images to the computer. Typically there is a subsequent processing of these images in the computer to obtain the desired information. 
         [0005]    The illumination sources as used in optical microscopes have been developed over the years and wide varieties of illumination sources are currently available, which can emit light or other type of radiation at different wavelengths. Optical filters can be placed between the illumination source and the sample to be imaged in order to restrict the wave-length of the radiation illuminating the sample. 
         [0006]    Modern biological microscopy uses fluorescent probes for imaging specific structures within a cell as the sample. In contrast to normal trans-illuminated light microscopy, the sample in fluorescent microscopy is illuminated through one or more objective lenses with a narrow set of light wavelengths. These narrow set of light wavelengths interact with fluorophores in the sample, which then emit light of a different wavelength. This emitted fluorescent light is detected in a detector and is used to construct the image of the sample. 
         [0007]    The use of multiple images enables a 3-dimensional reconstruction of the sample to be made. This 3-D reconstruction can be done by generating images at different positions on the sample, as the sample moves relatively to one or more objective lens. Depending on the number of detection units necessary, several detectors may be required. These detectors are quite expensive and a microscope designer will wish to reduce the number of detectors. The use of a single detector, which is moved during the imaging process, can be disadvantageous in that the movement of the detector itself can slightly effect the position of the sample, due to vibrations. Alternately the sample itself may move for other reasons whilst the detector is being placed into another position. This movement of the detector requires a precise and fast movement of a part of hardware, which is comparatively massive and in turn leads to further increase in development costs and/or in extra parts of equipment. 
         [0008]    A number of papers and patents have been published on various aspects of microscopy. For example, European patent EP 1 019 769 (Carl Zeiss, Jena) teaches a compact confocal feature microscope, which can be used as a microscope with a single objective lens or with multiple objective lenses. The microscope has separate directions of illumination and detection. The direction of detection in the objective lens is aligned inclined at a set angle in relation to the direction of illumination. 
         [0009]    Another example of a microscope is taught in the paper by Krzic al. “Multi View Light-Sheet Microscope for Rapid in tow Imaging”, Nature Methods, July 2012, vol. 9 No. 7, pages 730-733. This paper teaches a multi-view selective-plane illumination micro-scope comprising two detection and illumination objective lenses. The microscope allows in tow fluorescence imaging of the samples with subcellular solution. The fixed geometrical arrangement of the imaging branches enables multi-view data fusion in real time. 
         [0010]    Document DE 195 09 885 A1 discloses a stereo endoscope wherein illuminating light is transmitted by the light guide inserted through the elongate inserted section and is projected out of the distal end surface of the inserted section. The illuminated objects pass through the respective pupils of the two objective lens systems arranged in parallel within the distal end section of the inserted section and their images are formed on the focal surface. The respective images are transmitted to the rear side by one common relay lens system. The transmitted final images are formed respectively on the image taking surfaces of the image taking devices. The respective images are photoelectrically converted by the respective image taking devices and further processed to be signals, are displayed in the monitor and are stereo-inspected through shutter spectacles. 
         [0011]    Document U.S. Pat. No. 4,440,475 A discloses a device having a electromagnetic lens for focusing the analyzing electron beam that is provided with a central channel along the axis of the electron beam which is intended to pass through a mirror-objective having high magnification. The electromagnetic lens further comprises a lateral channel in which it is placed an auxiliary objective having low magnification. An optical illumination system, the axis of which is contained in the plane of the axes of the objectives, illuminates the sample either through the principal objective or through the auxiliary objective. An orientable mirror which is orthogonal to the plane aforesaid and placed at the intersection of the beams which form the images through the two objectives permits the use of the same observation means both for low magnification and for high magnification. 
         [0012]    In document U.S. Pat. No. 5,132,837 A is an operation microscope disclosed including a plurality of objective lenses arranged at different angles with respect to an object to be viewed and a selecting optical system having a function of selecting one of light beams from the objective lenses and enabling the object to be observed at the different angles. Accordingly, a visual field for observation of an object to be operated may be expanded. 
         [0013]    Document US 2012/044486 A1 discloses a system and a method for detecting defects on a waver. 
         [0014]    Document WO 2008/028045 A2 discloses a system and method for robust finger-print acquisition comprising combined multispectral and total-internal-reflectance biometric imaging systems. A platen has multiple facets, at least one of which has a surface adapted for placement of a purported skin site by an individual and another facet may include an optical absorber. An illumination source and an optical arrangement are disposed to illuminate the purported skin site with light from the illumination source along distinct illumination paths, including paths at angles less than the critical angle and paths at angles greater than the critical angle. Both multispectral and total-internal-reflectance illumination are received by an imaging system. The imaging system may include first and second imaging locations adapted to record images from separate illumination paths. The platen may also include non parallel exits facets 
       SUMMARY OF THE INVENTION 
       [0015]    An optical arrangement for imaging a sample is disclosed. The optical arrangement comprises at least one first objective lens and at least one second objective lens, at least one illumination source for producing an illumination beam, a detector for imaging radiation from the sample, and at least one mirror. The at least one mirror is adapted to reflecting the radiation from one of the first objective lens or the second objective lens into the detector. The position of the mirror is dependent on the illumination beam at the other one of the first objective lens and the second objective lens wherein the mirror can be double-sided. The use of the double-sided mirror enables a single detector to be used to image the sample from multiple sides and thus save on the detectors. 
         [0016]    In one aspect of the disclosure the at least one mirror is translatable or rotatable. 
         [0017]    In another aspect of the disclosure at least two mirrors are present and the reflected radiation can be directed into one of the at least two mirrors. 
         [0018]    An optical filter can be inserted in the path of the illumination beam or the path of the radiation to select only certain wavelengths of light. 
         [0019]    In a further aspect of the invention, a third objective lens can be used to collect the radiation from the sample. 
         [0020]    A method for imaging a sample is also disclosed. The method comprises illuminating the sample using a first illumination beam, detecting, at a stationary detector in a stationary position, first radiation from the sample, processing the first radiation to obtain a first data set, illuminating the sample using a second illumination beam at an angle to the first illumination beam, detecting, at the stationary detector in the stationary position, second radiation from the sample, processing the second radiation to obtain a second data set, and combining the first data set and the second data set to produce an image of the sample. 
     
    
     
       DESCRIPTION OF THE DRAWINGS 
         [0021]      FIG. 1  shows a schematic view of an optical arrangement according to this disclosure. 
           [0022]      FIG. 2 a    shows a first aspect of the optical arrangement. 
           [0023]      FIG. 2 b    shows another position of the first aspect of the optical arrangement. 
           [0024]      FIG. 3 a    shows a second aspect of the optical arrangement. 
           [0025]      FIG. 3 b    shows another position of the second aspect of the optical arrangement. 
           [0026]      FIG. 4  shows a method imaging a sample according to this disclosure. 
           [0027]      FIG. 5 a    shows a third aspect using more than two objective lenses. 
           [0028]      FIG. 5 b    shows another position of the third aspect of the optical arrangement 
           [0029]      FIG. 5 c    shows another position of the third aspect of the optical arrangement 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0030]    The invention will now be described on the basis of the drawings. 
         [0031]    It will be understood that the embodiments and aspects of the invention described herein are only examples and do not limit the protective scope of the claims in any way. The invention is defined by the claims and their equivalents. It will be understood that features of one aspect or embodiment of the invention can be combined with the feature of a different aspect or aspects and/or embodiments of the invention. 
         [0032]      FIG. 1  shows an overview of an optical arrangement  10  of this disclosure. The optical arrangement  10  has a first objective lens  30  and a second objective lens  40 . Both the first objective lens  30  and the second objective lens  40  are able to image a sample  20  and/or direct an illumination beam  60  and  60 ′ onto the sample  20 . The optical arrangement  10  shown in  FIG. 1  has two objective lenses  30  and  40 , but this is not limiting of the invention. It would be possible to have an optical arrangement with a larger number of objective lenses. 
         [0033]    The optical arrangement  10  has an illumination source  50  that produces the illumination beam  60 . The optical arrangement  10  has also a detector  70  that is able to detect radiation  80  reflected or fluoresced from the sample  20 . 
         [0034]    The sample  20  is typically a biological sample. The sample  20  is to be imaged in three dimensions. It is known that a minimum of one view is required to create a 3D stack of images. At least two views are required in order to make a multi-view image of the sample  20  in the optical arrangement of  FIG. 1 a   . The views can then be stored in a memory  110  as a first data set  120  and a second data set  130  and combined in a processor  100  in order to construct a multidimensional dataset. This multidimensional dataset can, for example, be used to create a 3D multi-view image of the sample  20 . 
         [0035]    Both of the first objective lens  30  and the second objective lens  40  can be used to illuminate the sample  20  and/or gather radiation fluoresced or reflected from the sample  20 . This will be described using a black box  95  as is illustrated in  FIG. 1 . The black box  95  outlines the manner in which the illumination beam  60  from the illumination beam source  50  can be directed either to the first objective lens  30  or, for example by use of mirrors, as an illumination beam  60 ′ to the second objective lens  40 . The black box  95  also shows that radiation from the sample  20  can be directed either through the first objective lens  30  as a radiation beam  80 ′ or from the second objective lens  40  and thence to the detector  70  as a radiation beam  80 . The optical arrangement  10  illustrated in  FIG. 1  is therefore able to create at least two images of the sample  20  from different angles in order to allow the construction of a 3D image of the sample  20 . This principle can be implemented using different mirror arrangements as described below. 
         [0036]      FIGS. 2 a  and 2 b    show a first aspect of the optical arrangement  10  in which the illumination source  50  is located to one side of the detector  70 . In  FIG. 2 a   , the illumination source  50  is shown to the left hand side of the single detector  70  and produces an illumination beam  60  arriving at the first objective lens  30  from which the illumination beam  60  is projected onto the sample  20 . Radiation from the sample  20  is imaged through the second objective lens  40  and strikes the right mirror  90   a  on the right hand side from which the radiation  80  is reflected to a central mirror  90   c  and thence into the detector  70 .  FIG. 2 a    includes further a left mirror  90   b.  It will be seen from  FIG. 2 a    that the left hand mirror  90   b  does not interrupt the passage of the illumination beam  60  from the illumination source  50  on the left hand side. 
         [0037]    The optical arrangement  10  of  FIG. 2 a    can be compared with the optical arrangement  10  shown in  FIG. 2 b   . The optical arrangement  10  of  FIG. 2 b    comprises the same elements with the same numbers as shown in  FIG. 2 a   .  FIG. 2 b    shows, however, a further illumination source  50 ′ on the right hand side which produces an illumination beam  60 ′ entering the second objective lens  40 . It will be seen that the right hand mirror  90   a  has been moved out of the path of the illumination beam  60 ′, so that this right hand mirror  90   a  does not interrupt the passage of the illumination beam  60 ′. The radiation  80 ′ from the sample  20  passes through the first objective lens  30  and is reflected by the left-hand side mirror  90   b  onto the central mirror  90   c  and thence into the detector  70 . It will be noted that the right hand mirror  90   a,  the left hand mirror  90   b  and the central mirror  90   c  have in  FIG. 2 b    been shifted to the right compared to the equivalent positions in  FIG. 2 a    in order to allow the illumination beams  60 ,  60 ′ and the radiation  80  and  80 ′ to be reflected differently. This is indicated figuratively by arrow  92 . It will be understood that the movement of the mirrors  90   a,    90   b,    90   c,  as indicated by the arrow  92 , and can be easily implemented, for example on a sliding track. 
         [0038]      FIGS. 3 a  and 3 b    show a second aspect of the invention in which a single central mirror  90   c  is moved up and down, as indicated by an arrow  94 . The optical arrangement  10  of  FIGS. 3 a  and 3 b    has otherwise the same elements as the optical arrangement shown on  FIGS. 2 a  and 2 b   . The optical arrangement  10  has, however, a single detector  70  on the left hand side and a single illumination source  50  on the right hand side. 
         [0039]      FIG. 3 a    shows a lower position of the central mirror  90   c  in which the illumination source  50  produces the illumination beam  60 ′ reflected by the central mirror  90   c  onto the right hand mirror  90   a  and thence into the second objective lens  40 , thereby illuminating the sample  20 . Radiation from the sample  20  is collected by the first objective lens  30  and reflected by the left hand mirror  90   b  onto the central mirror  90   c  and thence into the detector  70 . 
         [0040]    In the aspect shown in  FIG. 3 b    the central mirror  90   c  is moved to make way for the illumination beam  60  and the radiation  80 . In this example the illumination beam  60  is produced by the illumination source  50  and is reflected by the left hand mirror  90   b  onto the sample  20  through the first objective lens  30 . The radiation from the sample  20  is imaged through the second objective lens  40  and is reflected by the right hand mirror  90   a  into the detector  70 . Using the aspect of the invention shown in  FIGS. 3 a  and 3 b    two images of the sample  20  can be produced. 
         [0041]    The method of the invention is shown in  FIG. 4  in which an illumination beam is produced in step  200  from the illumination source  50  or  50 ′. It will be understood from  FIGS. 2 a  and 2 b    as well as  FIGS. 3 a  and 3 b    and  FIGS. 5 a - c    that there may be a single one of the illumination source  50  (as shown in  FIGS. 3 a  and 3 b    and  FIGS. 5 a - c   ) or two illumination sources  50  and  50 ′ as shown in  FIGS. 2 a  and 2 b   . There may be more illumination sources, as is the case of having more than two objectives as depicted in  FIGS. 5 a - c   . The illumination beam  50  is directed in step  205  to the sample  20  and reflected in step  206  from the sample  20 . The detection of the radiation  80  is carried out in step  210  in the detector  70 . The detector  70  can be, for example, a charge coupled device or any other detection instrument. 
         [0042]    A first image produced from the illumination beam is processed in step  220  in a processor  100  and stored in a memory  110  as a first data set  120 . 
         [0043]    A second illumination beam coming from an illumination source  50  is created in step  230  and illuminates the sample  20  from a different direction in step  235 . The second illumination beam is reflected from the sample  220  in step  238  and is detected in the same detector  70 . The image is then processed in the processor in step  250  and stored in the memory  110  as a second data set  130 . The first data set  120  and the second data set  130  forming the two images can be combined in step  260  in the processor  100  to produce the multi-view 3D image of the sample  20 . 
         [0044]      FIGS. 5 a - c    shows a third aspect of this disclosure using more than two objective lenses. The illumination beam  60 ,  60 ′ or  60 ″ is produced from the illumination source  50  and passes through one of a plurality of optical selectors  91   a,    91   b  or  91   c.  The optical selectors  91   a,    91   b  or  91   c  may be optical filters allowing passage of certain wavelengths, movable mirrors, moving shutters, or another possible optical selector device.  FIGS. 5 a - c    illustrates particular cases in which an optical filter that allows certain wavelengths to pass through is utilized for the optical selectors  91   a,    91   b,  or  91   c.  The illumination beam  60 ″ of the aspect depicted in  FIG. 5 a    passes through the optical filter  91   c.  The illumination beam  60 ″ passes through a third objective lens  31  and illuminates the sample  20 . The radiation  80  coming from sample  20  is collected by the second objective lens  40 , reflects on the optical selector  91   b,  and reflects on the mirror  90   b  towards a radiation selector  96 . The radiation selector  96  is shown as a rotating mirror in  FIGS. 5 a - c   . The radiation selector  96  can be another type of movable radiation redirecting devices. The redirected radiation  80 ,  80 ′ or  80 ″ is directed onto the detector  70 . 
         [0045]    In this aspect of the invention each one of the first, third or second objective lenses  30 ,  31  and  40  can be used for illumination or detection. It is therefore also possible to collect the radiation  80 ′ from sample  20  with the first objective lens  30 , reflecting the radiation  80 ′ on the optical selector  91   a  and on the mirror  90   a,  and subsequently causing the radiation selector  96  to redirect the radiation  80 ′ to the detector  70 , as demonstrated in  FIG. 5   c.    
         [0046]    It is also possible to illuminate the sample  20  by sending an illumination beam  60  and/or  60 ′ either through the optical selector  91   a  and/or the optical selector  91   b,  through the first objective lens  30  and/or the second objective lens  40 , and collecting the radiation  80 ″ from the third objective lens  31 , reflecting the collected radiation  80 ″ with the optical selector  91   c  and further with mirror  90   c  onto the radiation selector  96 , thus redirecting radiation  80 ″ to detector  70 , as demonstrated in  FIG. 5   b.    
       REFERENCE NUMERALS 
       [0000]    
       
           10  Optical arrangement 
           20  Sample 
           30  First objective lens 
           31  Third objective lens 
           40  Second objective lens 
           50 ,  50 ′ Illumination source 
           60 ,  60 ′,  60 ″ Illumination beam 
           70  Detector 
           80 ,  80 ′,  80 ″ Radiation 
           90   a,b,c  Mirror 
           91   a,b,c  Optical Selector 
           92  Arrow 
           94  Arrow 
           95  Black box 
           96  Radiation selector 
           100  Processor 
           110  Memory 
           120  First data set 
           130  Second data set