Abstract:
A microscope comprises an objective and a light source that produces an illumination light beam—in particular for evanescent illumination of a sample, which exhibits a focus in the plane of the objective pupil. To adjust the penetration depth, an adjustment mechanism is provided with which the spatial position of the focus within the plane of the objective pupil may be changed.

Description:
RELATED APPLICATIONS  
       [0001]     This application is a Continuation of PCT application serial number PCT/EP04/52287 filed on Sep. 23, 2004 which in turn claims priority to German application serial number DE 103 44 410.6 filed on Sep. 25, 2003 and German application serial number DE 10 2004 044 308.4 filed on Sep. 10, 2004, both of which are incorporated herein by reference in their entirety. 
     
    
     FIELD OF THE INVENTION  
       [0002]     The invention relates to a microscope with an objective and with a light source that produces an illumination light beam—in particular for evanescent illumination of a sample, which exhibits a focus in the plane of the objective.  
         [0003]     The invention further relates to an illumination module with a light source that produces an illumination beam.  
       BACKGROUND OF THE INVENTION  
       [0004]     The 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. Microscopes of this type are known as “total internal reflection fluorescent microscopes” (TIRFM).  
         [0005]     The z-resolution of TIRF microscopes is extraordinarily good because the evanescent field protrudes only about 100 nm into the sample.  
         [0006]     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.  
         [0007]     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°.  
         [0008]     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.  
         [0009]     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 outgoing light that 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.  
         [0010]     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.  
         [0011]     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 of galvanometric positioners, for example. 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.  
         [0012]     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 control 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 usually achieved by means of layered image data.  
       SUMMARY OF THE INVENTION  
       [0013]     It is a task of the present invention to disclose a microscope that enables variable adjustment of the penetration depth of illumination light, in particular for evanescent illumination of a sample.  
         [0014]     This task is solved by a microscope, wherein an adjustment mechanism is provided with which the spatial position of the focus within the plane of the objective pupil may be changed.  
         [0015]     A further task of the present invention is to disclose an illumination module for a microscope that enables illumination of a microscopic sample, in particular for evanescent sample illumination with adjustable penetration depth.  
         [0016]     The further task is solved by an illumination module, wherein the illumination module may be coupled to a microscope such that the illumination light beam in the plane of the objective pupil of the microscope exhibits a focus, and wherein the illumination module comprises an adjustment mechanism with which the spatial position of the focus within the plane of the object pupil may be changed.  
         [0017]     It has been recognized, according to the invention, that the penetration depth of an evanescent illumination field in a sample is dependent on the angle at which total reflection at the cover glass interface or at the sample holder interface occurs. This angle is directly correlated with the angle relative to the optical axis at which the illumination light beam which is provided for evanescent sample illumination exits from the objective via the front lens. This angle, in turn, is dependent upon the distance from the optical axis at which the illumination light beam passes through the rear focal plane of the objective (pupil). In order to have available a largely parallel illumination light beam for the purpose of evanescent sample illumination, the illumination light beam must exhibit a focus in the rear focal plane of the objective. Finally, the distance of the focus to the optical axis of the objective determines the aforementioned angle, and therewith the penetration depth of the evanescent field in the sample to be tested.  
         [0018]     In a preferred embodiment of the microscope according to the invention, the adjustment mechanism comprises an adjustable beam deflector that is arranged in the beam path of the illumination light beam. Preferably, the beam deflector comprises at least one galvanometric mirror. In order to position the focus at any given location within the objective pupil, the beam deflector preferably comprises two galvanometric mirrors, which cause deflection of the illumination light beam in different lateral directions (e.g., x- and y-direction). The beam deflector may also comprise rotatable or tippable prisms and/or rotatable or tippable mirrors. The use of acousto-optical or electro-optical deflection elements can also be envisioned.  
         [0019]     In a further embodiment of the invention, the adjustment mechanism comprises a light-conducting fiber which is at least partially movable. In this variant, mechanical positioners are preferably provided that enable the light outgoing end of the light-conducting fiber to be positioned precisely within the objective pupil. The illumination light beam in this further development of the invention is focused onto the light incoming end of the light-conducting fiber, conveyed through the light-conducting fiber, and de facto again exhibits a focus at the outcoupling end that is positioned within the objective pupil, because of the small diameter of customarily used light-conducting fibers.  
         [0020]     As previously explained, it is particularly important to adjust the distance of the focus of the illumination light bean in the objective pupil relative to the optical axis of the objective in order to adjust the penetration depth of the evanescent field in the sample region.  
         [0021]     It can be particularly advantageous for certain applications to drive the adjustment mechanism such that the focus describes a selectable curve path within the objective pupil plane. By so doing, particularly homogeneous illumination can, for example, be achieved. In certain experiments, it is possible with this variant to effect constant alternation in polarization direction. In a particularly preferred embodiment of the invention, the curve path is a circular path. An embodiment of the invention in which the curve path is a circular path the midpoint of which lies on the optical axis of the objective is very particularly preferred. In this variant, the penetration depth remains constant while the focus describes the circle of the curve path, whereby, however the illumination light beam that exits from the objective is continuously coupled to the cover glass or to the sample holder from various directions. It is also possible to select different coupling directions in order to compare the resultant, possibly different, images of the sample.  
         [0022]     In a very preferred variant, a compensating optic is provided in order to compensate for unevennesses in the objective pupil plane.  
         [0023]     Preferably, the microscope objective is exchangeable (e.g., objective turret), whereby a compensating optic is provided to compensate for the various pupil positions of different objectives. The distances between the front focal plane and the rear focal plane may differ from objective to objective, which may lead to problems because in order to achieve optimal evanescent sample illumination, the focus of the illumination light beam must lie more or less exactly in the objective pupil. The aforementioned compensating optic, which may, for example, be a zoom optic or several exchangeable optics arranged on a turret, compensates for these differences in distance.  
         [0024]     In a preferred embodiment of the microscope according to the invention, a light trap is provided to eliminate unused illumination light. Only a portion of the illumination light coupled to the cover glass or sample holder actually evanescently illuminates the sample. The light, which again exits from the cover glass or from the sample holder after several total reflections, many return to the microscope again and lead to imaging disturbances (as a result, for example, of scattered light). This is avoided, according to the invention, by a suitably arranged light trap.  
         [0025]     In a particularly preferred embodiment of the microscope, a switch is provided for switching between classic incident illumination and evanescent sample illumination. The switch may, for example, comprise a wing mirror.  
         [0026]     Preferably, the light cone is variable, in particular for changing the azimuth. An aperture optic such as an iris optic arranged in the intermediate image plane may be provided in order to set the diameter of the illumination light beam that exits from the microscope objective.  
         [0027]     In a particular embodiment a camera is provided for imaging. The camera may, in a particularly preferred variant, be implemented as a color camera, or as a CCD camera.  
         [0028]     Preferably, the objective exhibits a numeric aperture that is greater than 1.4, in particular greater than 1.45, in particular greater than 1.6. Preferably, the numeric aperture of the objective is 1.45 or 1.65.  
         [0029]     In a particular variant, at least the light source and the adjustment mechanism are incorporated in a single illumination module, which can preferably be coupled to a microscope and/or a microscope stand.  
         [0030]     The illumination module, according to the invention, offers the advantage that it may be coupled as a retrofit to a preexistent microscope or microscope stand. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0031]     The object of the invention is schematically represented in the diagram, and is described below on the basis of figures, wherein elements that have the same function are given the same reference numbers. They show:  
         [0032]      FIG. 1 a  microscope according to the invention;  
         [0033]      FIG. 2 a  further microscope according to the invention:  
         [0034]      FIG. 3  an illumination module that is coupled to a microscope;  
         [0035]      FIG. 4 a  microscope with an illumination module; and  
         [0036]      FIG. 5 a  further microscope according to the invention. 
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0037]      FIG. 1  shows a microscope  1  according to the invention, with an optical device  3  for manipulating a sample  5 . The optical device  3  comprises a light source  7  that emits a manipulation light beam  9 , and an adjustable beam deflector  11 . The microscope  1  comprises an objective  13  and a further light source  15  that is implemented as a laser  17 , and which produces an illumination light beam  19 . The illumination light beam  19  that is emitted from the further light source  15 , serves to evanescently illuminate the sample  5 , which is positioned on a sample holder  21 . In order to achieve evanescent sample illumination, the wing mirror  23  is tipped in the position indicated by the broken lines. By so doing, the beam path is cleared for the illumination light beam  19 . A compensating optic  25  is provided in the beam path of the illumination light beam  19  to compensate for unevennesses in the objective pupil plane  27 . The compensating optic  25  also enables compensation for different pupil positions of different objectives  13 . For this purpose, the compensating optic  25  is implemented so as to be axially movable. The illumination light beam  19  exhibits a focus  31 , which is indicated by a point, in the plane  27  of the objective pupil  29 , and which may be varied in its position in the plane  27  of the objective pupil  29  with the help of the further adjustable beam deflector  32 .  
         [0038]     Several optical elements for directing and forming the beam are arranged in the beam path of the microscope  1  There may, for example, be a first optic  33 , a second optic  35 , and an optic  25 , which produce a first intermediate image plane  37  and a second intermediate image plane  39 . The adjustable further beam deflector  32  comprises a cardanically suspended rotating mirror  41 , which is not shown. The distance of the focus  31  to the optical axis  43  of the objective  3  may be adjusted and therewith the penetration depth of the illumination light beam in the sample  5  varied with the help of the further adjustable beam deflector  31 . The detection light  45  exiting from the sample  5  passes through the objective  3  as well as through the beam splitter  47 , which directs the illumination light beam  19  through the objective  3  and through the tube optic to a detector  49 , which is implemented as a CCD camera  51 . The beam splitter  47  is implemented as a dichroic beam splitter, and is designed such that the light of the wavelength of the illumination light beam  19  is reflected, whereas light of the wavelength of the detection light  47  may pass through.  
         [0039]      FIG. 2  shows a variant in which the optical device  3  for manipulating a sample  5  produces both a manipulation light beam  9  and an illumination light beam  19 . The device  3  for manipulating a sample  5  comprises a light source  7 , a multi-linear laser  53 , from the emission light of which the portions of the desired wavelengths are selectable with an AOTF  55 . The compensating optic  57  is introduced into the beam path of the microscope  1  for the purpose of evanescently illuminating the sample  5 . The compensating optic  57  enables the illumination light beam  19  to exhibit a focus  31 , which is indicated by a point, in the plane  27  of the objective pupil  29 . For the purpose of sample manipulation, the compensating optic  57  is removed from the beam path so that the manipulation light beam  9  is focused on the sample. Both the penetration depth of the illumination light in the sample (by adjusting the position of the focus of the illumination light beam  19  in the plane  27  of the objective pupil  29 ) and sample manipulation are controlled with the beam deflector  11 .  
         [0040]      FIG. 3  shows a variant of a microscope  1  according to the invention, in which the illumination light beam  19  passes through the compensating optic  25 , which makes it possible for the illumination light beam  19  to exhibit a focus  31 , which is indicated by a point, in the plane  27  of the objective pupil  29 , while the manipulation light beam  9  is directed at the compensating optic  25  via the first deflecting mirror  59 , the second deflecting mirror  61 , the third deflecting mirror  63 , and the fourth deflecting mirror  65 .  
         [0041]      FIG. 4  shows a variant of the microscope shown in  FIG. 1 . In this variant, the optical device  3  for manipulating a sample  5  comprises an illumination pinhole aperture  67 , a detection pinhole aperture  69 , which is arranged before a multi-band detector  71 , as well as a dichroic beam splitter  77  for deflecting the further detection light  73 , which issues from the sample, into the detection beam path  75 . The microscope shown enables confocal observation of the sample  5 , whereby confocal illumination is achieved with the focus of the manipulation light beam  9 .  
         [0042]     The invention was described in relation to a particular embodiment. However, it is clear that changes and variations may be implemented without abandoning the scope of the following claims.