Abstract:
A device to illuminate a object, to excite its fluorescence light emission, and detect the emitted fluorescence spectrum, comprising: at least one illumination system ( 13 ), adapted to receive light from a light source ( 11 ), to select at least one wavelength bands of light spectrum of the source ( 11 ), to illuminate a object ( 15 ) with light filtered in that way ( 14 ); and a detection system ( 17 ), adapted to detect fluorescence light ( 16 ) emitted by the object ( 15 ), to select at least one wavelength bands of fluorescence, light spectrum ( 16 ), to record the spectrum of the filtered light; characterized in that said illumination system ( 13 ) comprises: at least one first dispersive element ( 41 ), at least one focusing optics ( 43 ), at least one spatial fitter of excitation ( 44 ), at least one collimating optics ( 45 ) and at least one second dispersive element ( 47 ), wherein said detection system ( 17 ) comprises: at least one dispersive element ( 81 ), at least one focusing optics ( 83 ), at least one spatial filter of detection ( 84 ), at least one imaging optics ( 85 ) and at least one light detector ( 87 ).

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention refers to the field of devices of illumination and detection of light for spectroscopic analysis. In particular the invention applies to samples where different species of fluorescent molecules are present or about which is desirable to have information about the spectrum of the emitted light of fluorescence. 
       STATE OF THE ART 
       [0002]    At the state of the art there exist many applications in which is required to illuminate a sample at various wavelengths in order to excite the emission of fluorescence by the different types of fluorescent molecules. The spectroscopic analysis of the fluorescence emitted by the sample allows to obtain information related to the number, to the spatial distribution, and to the species of fluorescent molecules. 
         [0003]    An example of such application is flow cytometry. In flow cytometry, the sample is illuminated by several lasers at the same time, in order to cause the emission of fluorescence by all the species of fluorescent markers used. To perform the analysis, fluorescent light is collected on several detectors using a combination of dichroic mirrors and chromatic filters. In this way, every detector is specific for a band of wavelengths, characteristic of only one fluorescent marker. The detection system is complicated, expensive, and poorly efficient in terms of intensity of the collected light. Furthermore the whole spectrum of the fluorescence light collected by the detectors is very limited. 
         [0004]    A further example is given by spectral confocal microscopy. In this case the sample is illuminated by only one laser at the time. The fluorescence can be analysed, as in the case of flow cytometry, by means of a combination of several detectors, dichroic mirrors and chromatic filters, or by means of a dispersive element and a multichannel detector. In any case, to perform a complete analysis of the fluorescent molecules in the sample, it is necessary in the illumination system go in succession from a laser to a different one. This implies that the images corresponding to different excitation wavelengths are acquired at different times: hence the derivable information from different fluorescent markers are not simultaneous. The switching from an excitation wavelength to a different one can be made in very short time by means of tunable acousto-optic filters, which anyway have a relevant cost. 
         [0005]    At the state of the art, polychromatic illumination and spectral detection systems which show both the following characteristics do not exist: simultaneous illumination on several wavelengths; wide spectrum and high spectral resolution detection. Aim of the present invention is the realization of an apparatus for the illumination of an object at several wavelengths at the same time and for the detection of the spectrum of the fluorescence emitted by the object with high spectral resolution and wide bandwidth. 
       SUMMARY OF THE INVENTION 
       [0006]    The present invention concerns a device able of illuminating an object on several wavelengths at the same time and of detecting the spectrum of the fluorescence emitted by the object with high spectral resolution and wide bandwidth. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0007]      FIG. 1  illustrates the working scheme of the invention. 
           [0008]      FIG. 2  illustrates the working scheme of the invention, with “backward” detection of the fluorescence light. 
           [0009]      FIG. 3  illustrates the working scheme of the invention, with a single spatial filter for the detection and for the excitation. 
           [0010]      FIG. 4  illustrates the scheme of a first preferred embodiment of the illumination system. 
           [0011]      FIG. 5  illustrates the working scheme of the spatial filters of excitation and detection. 
           [0012]      FIG. 6  illustrates the scheme of a second preferred embodiment of the illumination system. 
           [0013]      FIG. 7  illustrates the scheme of a third preferred embodiment of the illumination system. 
           [0014]      FIG. 8  illustrates the scheme of a first preferred embodiment of the detection system. 
           [0015]      FIG. 9  illustrates the scheme of a second preferred embodiment of the detection system. 
           [0016]      FIG. 10  illustrates the scheme of a third preferred embodiment of the detection system. 
           [0017]      FIG. 11  illustrates the scheme of the combined system of illumination and detection, with a single spatial filter. 
           [0018]      FIG. 12  illustrates the scheme of a first preferred embodiment of the invention. 
           [0019]      FIG. 13  illustrates the scheme of a second preferred embodiment of the invention. 
           [0020]      FIG. 14  illustrates the scheme of a third preferred embodiment of the invention, only concerning the illumination system. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0021]    The present invention concerns an optical apparatus to illuminate an object and to detect the fluorescence light emitted by the object. The spectrum of excitation light is composed of several bands of wavelengths. As a consequence, it is possible to excite in the object different types fluorescent molecules at the same time. The detection system records the spectrum of the light emitted by the object, after elimination of the spectral components of excitation light. 
         [0022]    With reference to  FIG. 1 , the device disclosed by the present invention is composed of an illumination system  13  and of a detection system  17 . The illumination system  13  has the function to: take the light  12  from an external polychromatic light source  11 ; select inside the spectrum of light  12  one or more bands of wavelengths; send this filtered light  14  to the object  15  under examination, in order to excite its emission of fluorescence. The detection system  17  has the function to collect the fluorescence light  16  emitted by the object  15  and to record its spectrum on a detector, after elimination of the spectral components of excitation light  14 . With reference to  FIG. 2 , the detection system  17  can be conveniently placed in order to collect, by means of a beamsplitter  21 , the fluorescence light  16  emitted backward by the object  15 . With reference to  FIG. 3 , the illumination system  13  and the detection system  17  can be combined in order to have some common optical element, in particular a single spatial filter  31 . 
         [0023]      FIG. 4  illustrates the illumination system  13 , which is composed of: a first dispersive element  41 ; a focalisation optic  43 ; an excitation spatial filter  44 ; a collimation optic  45 ; a second dispersive element  47 . The first dispersive element  41 , constituted by a prism or by a diffraction grating, takes the light  12  coming from a polychromatic source, and disperses its spectral components  42  at different angles. The focalisation optic  43 , constituted by one or more lenses, by one or more concave mirrors, or by their combination, focalises every spectral component  42  in a precise point of the excitation spatial filter  44 . The excitation spatial filter  44  has the function to select one or more bands of the spectrum of the incident light. The collimation optic  45 , constituted by one or more lenses, by one or more concave mirrors, or by their combination, takes the spectral components selected by the excitation spatial filter  44  and collimates them on the second dispersive element  47 . The second dispersive element  47 , constituted by a prism or by a diffraction grating, operates in a symmetric and opposite manner with respect to the first dispersive element  41 , recomposing in a single beam  14  the spectral components  46 . 
         [0024]    The excitation spatial filter  44  is constituted by a mask which selects by means of transmission or reflection one or more bands of the spectrum of the incident light  12  and extinguishes the other spectral components.  FIG. 5  illustrates the structure of the excitation spatial filter  44 : it is composed of a series of selection bands  54  alternated with extinction bands  53 . In the case that such a filters works in transmission, the selection bands  54  of the mask are transparent and transmit a series of spectral bands  52  of the spectrum  51  of the light source  11 . Extinction bands  53  are opaque and extinguish the other spectral components. The transmission mask can be realized by means of: a thin plate of a transparent material, treated in a way that the extinction bands  53  are opaque; a thin opaque plate, with holes along the selection bands  54 ; a liquid crystal spatial modulator. In the case that the excitation spatial filter  44  works in reflection, the selection bands  54  are reflective. The reflection mask can be realized by means of: a plate treated in a way that only the selection bands  54  are reflective; a liquid crystal spatial modulator; a micro-mirrors digital device, which reflects to the collimation optic  45  the selected spectral components  52 , and disperses in other directions the spectral components to extinguish. 
         [0025]      FIGS. 4 ,  6 ,  7  illustrate three possible embodiments of the illumination system  13 . The  FIG. 4  exemplify the illumination system  13  in the case that the excitation spatial filter  44  works in transmission, the dispersive elements  41  and  47  are constituted by prisms, and the focalisation  43  and collimation  45  optics are constituted by lenses.  FIG. 6  exemplify the illumination system  13  in the case that the excitation spatial filter  44  works in transmission, the dispersive elements  41  and  47  are constituted by diffraction gratings, and focalisation optics  43  and collimation optics  45  are constituted by concave mirrors.  FIG. 7  exemplify the illumination system  13  in the case that the excitation spatial filter  44  works in reflection, the dispersive elements  41  and  47  are constituted by prisms, and focalisation optics  43  and collimation optics  45  are constituted by lenses. 
         [0026]    As illustrated in  FIG. 8 , the detection system  17  is composed of: a dispersive element  81 ; a focalisation optic  83 ; a detection spatial filter  84 ; an imaging system  85 ; a detector of light  87 . The dispersive element  81 , constituted by a prism or by a diffraction grating, takes the fluorescence light  16  coming from the object  15 , and disperses its spectral components  82  at different angles. The focalisation optic  83 , constituted by one or more lenses, by one or more concave mirrors or by their combination, focalises each spectral component  82  in a definite point on the detection spatial filter  84 . The detection spatial filter  84  has the function to select one or more bands in the spectrum of fluorescence light  16 . The imaging system  85 , constituted by one or more lenses, by one or more concave mirrors or by their combination, takes the spectral components selected by the detection spatial filter  84  and focalises them on the detector  87 , realizing on it an image of the detection spatial filter  84 . The detector of light  87  is a multichannel detector, and can be constituted by a multi-anode photomultiplier tube, by an array of photodiodes, or by a CCD. Every channel of the detector takes the light of a band of the fluorescence spectrum selected by the detection spatial filter  84 . In this way it is possible to reconstruct the fluorescence emission spectrum  16  of the object  15 . 
         [0027]    Similarly to the excitation spatial filter  44 , the detection spatial filter  84  is constituted by a mask which selects by means of transmission or reflection one or more bands of the spectrum of the fluorescence light  16  and extinguishes the other spectral components.  FIG. 5  illustrates the band structure of the detection spatial filter  84 , complementary to that of the excitation spatial filter  44 : the selection bands  55  for the detection are placed in correspondence with the extinction bands  53  for the excitation, vice versa the extinction bands  56  for the detection are placed in correspondence with the selection bands  54  for the excitation. Since the excitation light  14  contains the spectral components  52  corresponding to the extinction bands  56  for the detection, the fraction of excitation light eventually collected by the detection system  17  is extinguished by the detection spatial filter  84  and hence is not recorded by the detector  87 . 
         [0028]    In the case that the detection spatial filter  84  works in transmission, the selection bands  55  of the mask are transparent and transmit a series of spectral bands of the fluorescence light  16  emitted by the object  15 . The extinction bands  56  are opaque and extinguish the other spectral components. The transmission mask can be realized with: a thin plate of transparent material, treated in a way that the extinction bands  56  are opaque; a thin opaque plate with holes along the selection bands  55 ; a liquid crystal spatial modulator. 
         [0029]    In the case that the detection spatial filter  84  works in reflection the selection bands  55  are reflective. The reflection mask can be realized by means of: a plate treated in a way that only the selection bands  55  are reflective; a liquid crystal spatial modulator; a micro-mirrors digital device, which reflects to the imaging system  85  the selected spectral components, and disperses in other directions the spectral components to extinguish. 
         [0030]      FIGS. 8 ,  9 ,  10  illustrate three possible embodiments of the detection system  17 .  FIG. 8  exemplify the detection system  17  in the case that the detection spatial filter  84  works in transmission, the dispersive element  81  is constituted by a prism, the focalisation optic  83  is constituted by a lens, and the imaging system  85  is constituted by a couple of lenses.  FIG. 9  exemplify the detection system  17  in the case that the detection spatial filter  84  works in transmission, the dispersive element  81  is constituted by a diffraction grating, the focalisation optic  83  is constituted by a concave mirror, and the imaging system  85  is constituted by a couple of concave mirrors.  FIG. 10  exemplify the detection system  17  in the case that the detection spatial filter  84  works in reflection, the dispersive element  81  is constituted by a prism, the focalisation optic  83  is constituted by a lens and the imaging system  85  is constituted by a couple of lenses. 
         [0031]      FIG. 11  illustrates the possibility to realize an excitation and detection filter by means of a single element  31 , constituted by a mask where the selection bands  54  of the excitation light are transparent, and the selection bands  55  of the fluorescence light are reflective. The filter  31  is tilted in order to reflect the fluorescence light to the imaging system  85 , which is part of the detection system  17 . 
         [0032]      FIG. 12  illustrates the first preferred embodiment of the device according to the present invention, that is multispectral confocal microscope. In this embodiment, that follow the working scheme of the  FIG. 2 , the excitation light  14  is directed from illumination system  13  by polarizer beamsplitter  21  on the scanning system  121 , which shall the scan of object  15  in the object plane. The light  12  that comes from source  11  is conveniently polarized in order to be reflected by polarizer beamsplitter  21 . A optics system  122  shall to couple the excitation beam with objective of microscope  123 , that focalize the excitation light on a point of object  15 . The fluorescence light emitted from object  15  is collected by means of same objective  123 , go trough the optics  122  and the scanning system  121 , and is partially transmitted by polarizer beamsplitter  21 . The detection system  17  collect the reflected fraction of the fluorescence light  16 . To obtain the confocality of the apparatus, conveniently can be placed a pinhole along the path between the polarizer beamsplitter  21  and the detection system  17 , otherwise a slit in the plane of the spatial filter of detection  84 , otherwise a slit in the plane of the detector  87 . This embodiment is different from the state of art of the multispectral confocal microscopes because the excitation of the fluorescence happens at the same time on several wavelength, without the need to shift from wavelength to wavelength of the excitation in turn. The image of object  15  is acquired by detector  87  point by point. For each point of the image the detector  87  store the fluorescence emission spectrum  16 . The multispectral illumination allow to excite in the object  15  at the same time different type of fluorescent molecules; the recording of the spectrum of the images allow to differentiate the distribution of the different fluorescent molecules in the object  15 . 
         [0033]      FIG. 13  illustrates the second preferred embodiment of the device according to the present invention, that is flow cytometry apparatus. In this embodiment, that follow the working scheme of the  FIG. 1 , the excitation light  14  is directed from illumination system  13  on the focusing optics  131 , that focuses in the flow cell  132 . The emitted fluorescence light  16  from cells that flow in the flow cell  132  is collected by appropriate optics  133  and sent at detection system  17 . 
         [0034]    This embodiment is different from the state of art of the flow cytometry apparatus because the excitation of fluorescence does not require a complex system of lasers and dichroic mirrors. Moreover the detection system does not require use numerous dichroic mirrors, chromatic filters, and dedicated detectors at specific wavelength. This embodiment allow to excite the fluorescence on several wavelengths at same time with a unique source and in a flexible way: the wavelengths used for the excitation is selected by spatial filter of excitation  44 , and can be easy changed replacing the spatial filters or using programmable spatial filters (liquid crystal spatial modulator or digital micromirro device). 
         [0035]      FIG. 14  illustrates a third preferred embodiment of the device according to the present invention, only for the part concerning the illumination system  13 . In such realization, the polychromatic light source is constituted by several lasers  141 , whose beams are superimposed by means of dichroic mirrors  142 . In the illumination system  13  the excitation spatial filter  44  is constituted by liquid crystal spatial modulator or by a micro-mirrors digital device. In this way the excitation spatial filter  44  is programmable, that is it is possible to choose every time which bands of wavelengths are selected from the filter. By means of the control electronics of the excitation spatial filter  44 , it is possible to fast select which laser beams  141  are selected and illuminate the object  15 . The advantage of the present realization with respect to the use of a tunable acousto-optic filter is that several laser at the same time can be sent to the object  15 .