Abstract:
The invention relates to a lens ( 1 ) for total internal reflection microscopy. The lens ( 1 ) is characterized in that a light source ( 5, 7 ) and/or at least one deviating means ( 31, 3 ) which deviates the light from a light source ( 5, 7 ) into the lens and/or the emitting end ( 35 ) of an illuminating optical fiber ( 37 ) is arranged in the region of the rear focal plane ( 3 ) of the lens ( 1 ).

Description:
RELATED APPLICATIONS 
       [0001]    This application is a Continuation of PCT application Ser. No. PCT/EP2004/052295 filed on Sep. 23, 2004, which in turn claims priority to German Patent Application Nos. DE 103 44 410.6 filed on Sep. 25, 2003 and DE 10 2004 044 274.6 filed on Sep. 10, 2004, all of which are incorporated herein by reference in their entirety. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The invention relates to an objective for total internal reflection microscopy. 
         [0003]    The invention further relates to a microscope for total internal reflection microscopy with a light source and with an objective. 
       BACKGROUND OF THE INVENTION 
       [0004]    A microscope with evanescent illumination of a sample is known from US 2002/0097489 A1. The microscope comprises a white light source, the light of which is coupled for the purpose of evanescent illumination via a slit aperture through the microscope objective onto a sample holder, which holds a sample. The illumination light propagates itself in the sample holder by means of total internal reflection, whereby the illumination of the sample occurs only in the region of the evanescent field that protrudes from the sample holder. 
         [0005]    Microscopes of this type are known as “total internal reflection fluorescent microscopes” (TIRFM). 
         [0006]    The z-resolution of TIRF microscopes is extraordinarily good because the evanescent field protrudes only about 100 nm into the sample. 
         [0007]    A high-aperture objective specifically for TIRF application is known from DE 101 08 796 A1. The objective comprises a first lens with positive refractive power and a second lens with negative refractive power, whereby the focal distance ratio between the two lenses is in the −0.4 and −0.1 range, and the total refractive power is greater than zero. The objective further comprises two positive lenses, the diameter ratio to focal length of which is greater than 0.3 and less than 0.6. The objective further comprises a negative lens and a collecting lens, whereby the negative lens faces the front group, and the focal distance ratio of the negative lens to the collector lens is between −0.5 and −2. 
         [0008]    An incident illumination device for TIRF microscopy is known from DE 102 17 098 A1. The incident illumination device comprises an illumination source that emits a polarized illumination beam when in operation, which propagates at an angle to the optical axis and a deflector that deflects the illumination light beam and couples it parallel to the optical axis in the objective. Provision is made in this incident illumination device for the illumination light beam emitted by the illumination source to exhibit a phase difference in the s- and p-polarization directions, and for the deflection arrangement to reflect the illumination light beam x times, whereby x=(n×180°−d)/60°. 
         [0009]    A microscope for total internal reflection microscopy (TIRM) is known from DE 101 43 481 A1. The microscope exhibits a microscope housing and an objective. The illumination light emitted by an illumination device can be coupled via an adapter that can be inserted into the microscope housing. 
         [0010]    A microscope with an optical illumination system that enables simple switching between evanescent illumination and reflective illumination is known from US 2004/0001253 A1. The illumination system comprises a laser light source, the light of which is coupled in an optical fiber. Furthermore, an outcoupling optic is provided that focuses the light that exits from the fiber onto a rear focal point of the microscope objective. The optical fiber is movable along a plane that is perpendicular to the optical axis of the microscope objective. 
         [0011]    A device for coupling light in a microscope is known from DE 102 29 935 A1. Here, a laser light is directed onto a sample in the illuminated field diaphragm plane by a laser light fiber coupling, which is implemented as a slide. The invention is particularly suitable for the TIRF method. 
         [0012]    In scanning microscopy, a sample is illuminated with a light beam to observe the detection light emitted by the sample as reflection- or fluorescent light. The focus of an illumination light beam is moved on an object plane with the help of a movable beam deflector, generally by tipping two mirrors, whereby the axes of deflection are usually positioned perpendicular to each other, so that one mirror deflects in the x-direction and the other in the y-direction. The mirrors are tipped with the help, for example, of galvanometric positioners. The power of the light coming from the object is measured dependent on the position of the scanning beam. Generally, the positioners are provided with sensors to determine the actual position of the mirrors. In confocal scanning microscopy in particular, an object is scanned in three dimensions with the focus of a light beam. 
         [0013]    A confocal scanning microscope generally comprises a light source, a focusing optic with which the light from the source is focused on a pinhole aperture—the so-called excitation aperture—, a beam splitter, a beam deflector to direct the beam, a microscope optic, a detection aperture, and detectors to detect the detection light or fluorescent light. The illumination light is coupled via a beam splitter. The fluorescent light or reflected light emitted by the object returns to the beam splitter via the beam deflector, passes through it, and is subsequently focused onto the detection aperture, behind which are located the detectors. This arrangement of detectors is called a descan arrangement. Detection light that does not originate directly from the focal region takes another light path and does not pass through the detection aperture so that pixel information is obtained, which is converted into a three-dimensional image by sequential scanning of the object with the focus of the illumination light beam. A 3-dimensional image is generally achieved by layered imaging. 
         [0014]    The systems known from the state-of-the-art have the disadvantage that, in part, very costly and space-consuming optics are needed in the beam path of the microscope in order to couple the TIRF illumination light. This has a disadvantageous effect, particularly in the detection beam path, and often leads to a loss in the power of the detection light. 
       SUMMARY OF THE INVENTION 
       [0015]    The task of the present invention is to disclose an objective for use in total internal reflection microscopy that enables particularly efficient coupling of the TIRF illumination light in the beam path of the microscope. 
         [0016]    This task is solved by an objective wherein a light source and/or at least one deflector that deflects the light from a light source into the objective and/or the outgoing end of the illumination conducting fiber is arranged in the area of the rear focal plane of the objective. 
         [0017]    A further task of the present invention is to disclose a microscope in which coupling of the TIRF illumination light is as efficient and low-loss as possible, and which simultaneously makes additional optics in the beam path of the microscope largely superfluous. 
         [0018]    The further task is solved by a microscope wherein the light source and/or at least one deflector that deflects the light source into the objective and/or the outgoing end of the illumination conducting fiber that is supplied by the light source is arranged in the area of the rear focal plane of the objective. 
         [0019]    The invention has the advantage that no beam splitters are necessary in the beam path of the microscope to couple the TIRF illumination light. This results in higher fluorescence output—because no signal loss is caused. In addition, high time resolution may be achieved because no optical components, in particular no beam splitters, need to be switched. 
         [0020]    In a preferred embodiment, the light source and/or at least the deflector and/or the outgoing end of the illumination light fiber is arranged directly in the rear focal plane of the objective. 
         [0021]    Almost any customary light source may be used as a light source in microscopy. Preferably, the light source comprises at least one laser, which may, for example, be implemented as a semiconductor laser and/or at least one LED (light-emitting diode). In a particular embodiment the light source can comprise several single-light sources; the single-light sources are preferably arranged in the form of a ring. The ring of single-light sources can, for example, be arranged in the housing of the objective itself, or outside of the housing of the objective, whereby openings are provided for light coupling. If the arrangement of single-light sources is outside of the housing of the objective, a deflector is preferably arranged in the rear focal plane or in the area of the rear focal plane of the objective, which deflects the light from the single-light sources such that it exits the front lens of the objective at the desired angle. 
         [0022]    In a particularly preferred embodiment, the light source comprises several single-light sources that emit illumination light of different wavelengths. The single-light sources can preferably be switched on and off independently of each other. 
         [0023]    In a preferred embodiment of the microscope according to the invention, a detector, in particular a multiband detector or a spectrometer, is provided, which detects synchronously when the light source and/or the single-light sources are switched on. This variant is particularly advantageous for use with samples that, for example, comprise several fluorescent dyes because, according to the invention, this enables very rapid spectral testing of the sample. 
         [0024]    It is possible, according to the invention, to select any desired excitation light wavelengths, or to select different combinations of excitation wavelengths, and correspondingly to switch the single-light sources simultaneously or sequentially. 
         [0025]    In one variant of the objective according to the invention or of the microscope according to the invention, an optic is provided, which focuses the light from the light source onto the deflector. 
         [0026]    In a preferred embodiment, the deflector is formed by the at least partial coating (present in any case) of an objective element such as a lens. The coating may be partially reflective or completely reflective. In a particularly preferred variant, the coating is implemented as a dielectric reflective coating, whereby the coating is implemented such that the detection light can largely pass through it unimpeded. 
         [0027]    Preferably, the light from the light source exits the objective after passing through the objective at least at an adjustable angle to the optical axis. This angle is extremely important for evanescent penetration depth in the sample. Preferably, the position of the light source and/or of at least one deflector and/or of the outgoing end of the light-conducting fiber is adjustable within the focal plane. The position of the light source or of the deflector or of the outgoing end directly influences the angle to the optical axis at which the light from the light source exits the objective after passing through the objective. Mechanical positioners such as are customary in microscopy may be used to adjust the position of the light source or of the deflector or of the outgoing end. In a particular variant, the angle for illumination light of different wavelengths is different in each case. 
         [0028]    In a particular embodiment of the invention, the microscope comprises a scanning microscope, in particular a confocal scanning microscope. A microscope that is implemented as a confocal scanning microscope is particularly suitable for conducting experiments based on photoactivation or caged compound release, and therefore for observations at the TIRF plane. Time resolution is particularly great with such an arrangement because, for example, sample stimulation and camera observation may be implemented simultaneously. 
         [0029]    The microscope according to the invention is suitable in particular for conducting biological experiments, in particular under photoactivation and/or caged compound release and/or FRAP (fluorescence recovery after photobleaching). 
         [0030]    Preferably, bandpass filters and/or cut-off filters to which each particular emission bandwidth of the fluorescence signal is tuned are arranged before the detector, which may, for example, be implemented as a camera. A dispersive element that produces spectral splitting, from which the wavelength portion to be detected can be blocked, may be provided for color selection. The detector may also be implemented as a color detector, for example as a color camera. In the same way, it is possible for a dispersive element to divide the detection light among several detectors in order to achieve spectral detection. 
         [0031]    The above and other features of the invention including various novel details of construction and combinations of parts, and other advantages, will now be more particularly described with reference to the accompanying drawings and pointed out in the claims. It will be understood that the particular method and device embodying the invention are shown by way of illustration and not as a limitation of the invention. The principles and features of this invention may be employed in various and numerous embodiments without departing from the scope of the invention. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0032]    In the accompanying drawings, reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale; emphasis has instead been placed upon illustrating the principles of the invention. Of the drawings: 
           [0033]      FIG. 1  shows an objective according to the invention; 
           [0034]      FIG. 2  shows a further objective according to the invention; 
           [0035]      FIG. 3  shows a further objective according to the invention; 
           [0036]      FIG. 4  shows a microscope according to the invention; and 
           [0037]      FIG. 5  shows a further microscope according to the invention. 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
       [0038]      FIG. 1  shows an objective  1  according to the invention that can be used for total internal reflection microscopy. A light source  5 , which is formed out of a first single-light source  7  and a second single-light light source  9 , is arranged in the rear focal plane  3  of the objective  1 . The first single-light source  7  comprises a light emitting diode (LED), while the second single-light source comprises a semiconductor laser. The objective comprises a front lens  15  and a further lens  17 . The illumination light  11  and illumination light  13 , respectively, exit the microscope objective at an angle β and at an angle α, respectively, to the optical axis  19 . The angle is adjustable by moving the single-light source  7  and by moving the single-light source  9 , respectively, within the rear focal plane. For this purpose, the distance of the single-light source  7  and of the further single-light source  9 , respectively, to the optical axis is changed with a mechanical positioner, which is not shown. The objective  1  exhibits an objective housing  21 . 
         [0039]      FIG. 2  shows a further objective  1  according to invention with an objective housing  21  that exhibits a first opening  23  and a further opening  25 . The objective exhibits single-light sources  27 ,  29  that are arranged in the form of a ring, and that belong to a light source. The light source  5  is arranged outside the objective housing  21 , and the light from the light source  5  is coupled via the first opening  23  and via the second opening  25 , as well as via further openings in the objective housing  21 , which are not shown. For coupling, deflectors  31 ,  33 , which deflect the light from the light source  5  into the further beam path of the objective  1 , are provided in the area of the rear focal plane  3  of the objective  1 . 
         [0040]      FIG. 3  shows a further objective  1  according to the invention with an objective housing  21 , behind the rear focal plane of which is arranged the outgoing end  35  of an illumination light-conducting fiber  37 . The illumination light  39  from a light source  5 , which is implemented as a laser  41 , is coupled in the illumination light-conducting fiber  37  with the help of a coupling optic  43 . The distance of the outgoing end  35  of the illumination light-conducting fiber  37  to the optical axis  19  of the objective  1  is mechanically adjustable. For this purpose, the outgoing end of the illumination light-conducting fiber is movably arranged within the rear focal plane  3  of the objective  1 . The illumination light  45  that exits the illumination light-conducting fiber  37  exits the objective  21  at an angle α to the optical axis  19 . This angle is adjustable by adjusting the position of the outgoing end  35  of the illumination light-conducting fiber  37  within the rear focal plane  3  of the objective  1 . 
         [0041]      FIG. 4  shows a microscope according to the invention with an objective  1  as described in  FIG. 2 . The illumination light  47  emitted by the single-light source  27  evanescently illuminates the sample  51 , which is arranged on the cover glass  49 . The detection light  53  that exits from the sample passes through the microscope objective  1  to the tube lens  55 , and is then deflected by the mirror  57  and via the optic  59  to the detector  61 , which is implemented as a CCD camera  63 . A filter wheel  65  with several different single filters from which to select the detection region is arranged before the detector  61 . 
         [0042]      FIG. 5  shows a further microscope according to the invention, which is implemented similarly to the microscope represented in  FIG. 4 . In addition, a confocal scanner  67  is provided, the scanning light beam  69  of which is coupled in the beam path of the microscope after passing through the scanning lens  71  via the beam splitter  73 . The scanning light beam  69 , which is indicated by broken lines in the figure, passes through the tube lens and the microscope objective to the sample  51 . The scanning light beam  69  can, for example, be used to excite the sample in addition to the evanescent field, and/or to manipulate the sample. Confocal imaging of the sample may be accomplished simultaneous to imaging the sample with the detector  61 , whereby the confocal scanner  67  receives detection light  75  (broken lines) exiting from the sample, which passes along the reverse light path as the scanning beam  69  reaches the confocal scanner  67 . 
         [0043]    While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.