Patent Publication Number: US-2006012891-A1

Title: Illumination device for a light raster microscope with sampling in the form of a line and its use

Description:
The invention relates to an illumination device which provides an illumination beam which is essentially homogeneous in at least one cross-sectional direction, in particular for a laser scanning microscope, where an original beam which is inhomogeneous in cross-section, in particular Gaussian-shaped, is conducted to a converting unit which transmits the illumination beam.  
      In many applications an illumination beam expanded in the form of a line is used, for example, for barcode scanners or for laser scanning microscopes sampling in the form of a row. One possibility for obtaining such a beam in the form of a line consists of a fast redirection of the laser beam along a row so that indeed at each point in time only one point of the row is illuminated, but averaged over a certain period of time a row is illuminated. Another approach which is also used in the state of the art to generate illumination beams shaped in the form of a line uses cylinder optics which anisotropically expand a beam bundle in a known manner. Such a cylinder-optical design is described as mirror optics, for example, in U.S. Pat. No. 4,589,738. There a beam is first directed onto a convex mirror not described in more detail and the beams diverging there are focused by means of a cylindrical lens onto a line.  
      Cylinder optics in principle does not change the beam profile. It merely expands it in a certain direction. A Gaussian-shaped beam, as is customarily transmitted by a laser beam source or a collimator for the light guide fiber bundle, therefore remains, even after treatment with a cylinder optics, Gaussian-shaped in profile, even if the width of the Gaussian shape after the cylinder optics is no longer the same in all direction transverse to the beam propagation. This has as a consequence the fact that the beam intensity varies sharply along a row or line. In applications which are sensitive with respect to this, one is aided by the fact that the beam is first expanded with a cylinder optics, where the expansion is very much greater than the width of the row or line later required and then, by means of screens, edge areas of the row or line in which the intensity of the radiation has dropped off too sharply with respect to the center are masked. Unfortunately, this has poor efficiency with regard to the utilization of the beam intensity originally generated.  
      U.S. Pat. No. 4,862,299 discloses a lens which expands a laser beam and, in so doing, re-forms the beam profile to be not Gaussian-shaped. In this document the lens is represented in numerous forms in cross-section and it causes an expansion to approximately rectangular beam shape. For application in laser scanning microscopy the approach of U.S. Pat. No. 4,862,299 is, however, unsuitable for chromatic reasons.  
      The objective of the invention is thus to extend a microscope of the type stated initially so that there is suitability for laser scanning microscopy.  
      This objective is realized according to the invention by the fact that the converting unit comprises an aspherical, convex, or concave mirror which, at least in one sectional plane is more strongly curved in the area of the point of incidence of the original beam than in the areas removed from the point of incidence.  
      The basic principle of beam-forming in the illumination device is therefore based on performing an energy redistribution, at least in one sectional plane, by means of an aspherical mirror and converting an inhomogeneous, in particular Gaussian-distributed profile, so that in the sectional plane there is a substantially homogeneous energy redistribution. If one forms the mirror in two cross-sectional directions according to the invention aspherically, one obtains a homogenization in two sectional planes, therefore a homogenized field. Through the use of an aspherical mirror a large spectral band width for the illumination radiation can be covered, with simultaneously homogeneous illumination. According to the invention it was recognized that the reflecting aspherical surface, which is curved more strongly in one sectional plane in the area of the point of incidence of the original beam than in the areas removed from the point of incidence, a dependence on wave length in focusing and energy distribution is avoided, where at the same time the inventive concept of varying curvature of the aspherical mirror opens the possibility of a great variety of energy distributions. With the illumination device according to the invention Gaussian bundles can, for example, be re-formed in such a manner that in over 80% of the illuminated area the intensity does not fall under 80% of the maximum value. This is an essentially homogeneous distribution in the sense of the invention.  
      The variant with diaxial aspherical curvature can be used particularly advantageously for the homogenization in an intermediate plane of a wide-field microscope. Also in the case of multi-point scanning microscopes the homogeneous illumination of an intermediate image in front of the element which generates the point cloud (for example, a Nipkow disk) makes possible a uniform illumination of the sample with spatially essentially more uniform beam intensity. Also, the converting unit according to the invention makes possible the illumination of an objective pupil so that a particularly good (highly resolved) imaging is achieved since a homogeneously filled pupil permits the optical resolution to be fully exploited.  
      A form of embodiment which is particularly simple to manufacture is a mirror which is formed as a wedge and with a rounded top. Such a mirror can be produced in a simple manner from a cuboid and achieves a focal line with a homogeneous energy distribution.  
      In a variant which is mathematically particularly simple to describe, the mirror is defined by a conical constant as well as the rounding radius of the top and satisfies in (x, y, z)-coordinates with regard to the z-coordinate of the equation [y 2 /[c+(c 2 B (1+Q) y 2 ) 1/2 ], where c is the rounding radius of the top and Q is the conical constant.  
      In microscopy one would like, for an illumination in the form of a row, to distribute the radiation not only homogeneously along a longitudinal line but rather, in given cases, also to adapt the width of the line at the diameter of the entrance pupil of the following optical system. In order to achieve this, the aspherical mirror must also cause a beam expansion transverse to the direction of the line. This can be achieved in the case of the variant stated initially of a mirror in the form of a wedge with a rounded top particularly simply by the mirror surface, or at least the top, being curved along the longitudinal axis of the top.  
      The aspherical mirror with a rounded top is therefore then curved two-dimensionally, where in a first sectional direction (perpendicular to the longitudinal axis) a wedge with rounded peak, in a second sectional direction (along the top) a parabolic or spherical curvature can be present. The latter curvature then sets the height of the illuminated field, while, on the contrary, the aspherical form perpendicular to the longitudinal axis causes expansion along the field and due to the asphericity has as a consequence an energy distribution. Along the field a substantially homogeneous energy distribution is thus achieved  
      A mirror curved additionally along the top, e.g., spherically or parabolically, can be captured in a simple mathematical description as follows: F(x, y) = √{square root over ( )}(a(y)Br x ) 2 Bx 2 −r x  where r x  is the radius of curvature along the top, that is, in the aforementioned second sectional direction.  
      In order to effect an adaptation for complete illumination of an intermediate image or an entrance pupil of a following optical system in the case of the mirror curved in two directions (for example, in the first sectional direction aspherically, in the second spherically), it is expedient to dispose collecting optics behind the mirror, for example, in the form of a collecting mirror. Customarily, for the generation of a rectangular field therein, one will use a cylindrical or toroidal mirror since thus a rectangular field is obtained, as is desired for most instances of application. For other field forms the mirror form may deviate. Thus, one can, for example, also use the aspherical surfaces according to the invention for this second mirror in order to achieve a combination of homogenization of the pupil filling in a first direction (through one of the aspherical surfaces) and the intermediate image in the remaining direction (through the other aspherical surface). Also, an image error compensation can be effected by the additional aspherical surfaces. Naturally one can assign, in addition, the second aspherical surface to the collecting mirror.  
      For the form of embodiment of the aspherical mirror with spherical curvature in the second sectional plane it is thus preferred that the collecting mirror in the x-direction has a radius of curvature equal to r x +2 d, where d is the distance between the aspherical mirror and the collecting mirror. The radius of curvature r x  of the aspherical mirror in the second sectional plane then scales directly the height of the illuminated rectangular field or the profile of the illumination beam.  
      Naturally, a mirror which, according to the invention, is aspherical in both sectional directions can be used for the homogeneous illumination of the pupil. In the case of a rotationally symmetric aspherical surfaces, they then cause a homogeneously illuminated circular field. An image field illuminated homogeneously in this manner can be used for a wide-field illumination of a microscope. Also, it is possible, from the pupil illuminated in such a manner for a scanning process, e.g. multi-point scanners such as Nipkow scanners, to select and use individual areas.  
      For the illumination of the aspherical mirror it is advantageous to set the axis of symmetry of the mirror at an angle between 4 and 20 to the axis of incidence of the original beam, which is profiled, for example, to be Gaussian-shaped, since then a compact design can be obtained. The collecting mirror disposed behind, which can be formed, for example, cylindrically or toroidally, collects the radiation energy redistributed by the aspherical surfaces and compensates wave aberrations accumulating during the propagation. If such wave aberrations play no role in simple cases, a spherical lens can be used instead of the collecting mirror.  
    
    
      The invention is explained in more detail in the following, with reference to the drawings, in embodiment examples. Shown in the drawings are:  
       FIG. 1 a  schematic representation of the beam path in an illumination device for providing a rectangularly profiled illumination beam in a first sectional plane,  
       FIG. 2  the beam path in  FIG. 1  in a second sectional plane set perpendicular to the first plane,  
       FIG. 3 a  computer representation of an aspherical mirror which is used in the beam path of  FIGS. 1 and 2 ,  
       FIG. 4 a  sectional plane through an aspherical mirror of  FIG. 3  to illustrate the magnitudes characterizing this mirror,  
       FIG. 5 a  representation similar to  FIG. 4  for a mirror only forming beams in one sectional plane,  
       FIG. 6 a  representation similar to  FIG. 4  for a diaxially aspherical mirror,  
       FIG. 7  an intensity profile achieved with the beam path of  FIGS. 1 and 2  in a sectional plane,  
       FIG. 8 a  schematic representation of a laser scanning microscope with the illumination arrangement of  FIGS. 1 and 2 ,  
       FIG. 9 a  beam path for the homogenization of the illumination of an intermediate image, and  
       FIG. 10 a  beam path for the homogenization of the filling of an objective pupil. 
    
    
       FIGS. 1 and 2  show an illumination arrangement in which radiation from a radiation source S is re-formed with respect to its beam profile.  FIG. 1  is a section in a (z, x)-plane.  FIG. 2  is a section perpendicular thereto in a (z, y)-plane. The radiation source S transmits a beam which is profiled to be Gaussian-shaped in each sectional direction perpendicular to the direction of propagation. After the re-formation a beam is present in a profile plane P which illuminates essentially a rectangular field, where the intensity distribution is not Gaussian-shaped along the longitudinal field axis but rather chest-shaped.  
      For beam forming, an aspherical mirror  1  is used which expands the radiation. The expanded radiation is parallelized once more by means of a collecting mirror  2 . The aspherical mirror  1  is struck by an original beam  3  from the radiation source S, said beam having said rotationally symmetric Gaussian-shaped beam profile. The aspherical mirror  1  is curved in the section represented in  FIG. 1  according to a radius of curvature r x , in this plane therefore spherically. The aspherical component first comes to bear in the section represented in  FIG. 2  and still to be explained. Due to the sphericity of the aspherical mirror  1  along the x-axis the diverging beam transmitted from the aspherical mirror  1  is expanded while preserving the Gaussian profile. The collecting mirror  2 , which is also spherically in the sectional plane of  FIG. 1 , provides for a profiled beam  5  which also has a Gaussian profile in the profile plane P in the sectional representation of  FIG. 1 .  
      For many applications this expansion is not desired. The aspherical mirror  1  and the collecting mirror  2  are then not curved in the sectional plane represented. The dotted representation of the mirror  2  symbolizes this. Naturally, the beam bundle then does not diverge.  
       FIG. 2  shows a section perpendicular to the  FIG. 1 . In this plane the aspherical mirror  1  is formed aspherically and the original beam  3  transmitted from the radiation source S is then converted into a diverging beam  4  in a manner which redistributes energy. The aspherical mirror  1  reflects with increasing angle relative to the optical axis OA increasing beam power so that in the diverging beam  4 , seen in the sectional representation of  FIG. 2 , energy is redistributed. The collecting mirror  2  collects the diverging beam  4 , in the sectional representation of  FIG. 2  no longer Gaussian-shaped in cross-section, and parallelizes the radiation to form a profiled beam  5 . In this plane a non-equidistant distribution of the partial beams drawn in for illustration is thus shown in  FIG. 2 , in contrast to  FIG. 1 .  
      The effect of the aspherical mirror  1  shown in  FIGS. 1 and 2  in a convex mode of construction can be seen still better if one observes the mirror surface  6  represented, by way of example, in  FIG. 3 . The mirror surface  6  comprises two roof surfaces  7 ,  8  which run together in a top  9 . At the same time, the mirror surface  6  is spherically curved along the x-axis, as also becomes clear in the curvature of the top  9 . The mirror surface  9  is therefore wedge-like in a (z, y)-section (parallel to the y-axis) with rounded peak. In a section parallel to the x-axis ((z, x)-section) there is, on the contrary, a spherical curvature. In a concave aspherical mirror  1  this applies analogously.  
      The aspherical curvature in the (z, y)-plane causes the energy redistribution represented in  FIG. 2  since, due to the wedge profile rounded only in the area of the peak, increasing energy percentages are also reflected in an increasing angle to the optical axis. The spherical curvature in the (z, x)-plane causes, on the contrary, a profile-preserving expansion of the beam, as is represented in  FIG. 1 . The original rotationally symmetric Gaussian-shaped profile is thus restructured to form an approximately rectangular profile. In the case of asphericity in both sectional planes the field is homogenized in both sectional planes.  
       FIG. 4  shows a section line  12  of the mirror surface  6  in a (z, y)-section, that is, in a section along the y-axis. The section line  12  is, for illustration, entered not only in  FIG. 4  but rather also as a thicker line in  FIG. 3 . Its form is essentially determined by two geometric factors, on the one hand, by a parabola  10  which determines the form of the rounded peak of the sectional line  12 , and, on the other hand, by an asymptote  13  which defines the curve of the sectional line  13  far from the peak  11 . The parabola  10  can be defined by specifying a radius of curvature for the peak. The asymptote  13  is determined by a conical constant Q. For y-values increasing without bound, the sectional line  12  approaches the line 1/(Q*c)=y/(1−(1+Q) 1/2 ). The conical constant Q therefore determines the slope 1/(1−(1+Q)) 1/2  in the outer spherical area. The radius c determines the curvature in the area of the peak  11 . In all, the sectional line is thus defined by the equation y 2 /[c+(c 2 B (1+Q)y 2 ) 1/2 ].  
      The asphericity explained for one sectional direction can naturally also be provided in the other sectional direction. One achieves with this a homogeneous ellipsoidal or circular field, the latter in the case of a rotationally symmetric aspherical mirror  1 . Alternatively, the sphericity in the x-direction can be omitted. The aspherical mirror  1  then has for each x-coordinate the profile of the sectional line  12 .  
      The mirror surface represented in  FIG. 3  has a radius of curvature c=10 mm, a conical constant Q=−100, and a radius of curvature along the x-axis of r x =100 mm. The parameter r x  is customarily chosen to be very much larger than the diameter of the original beam  3 .  
       FIGS. 5 and 6  show representations similar to  FIG. 3 , where the mirror surface  6  of the  FIG. 5 , however, is merely curved along the y-axis and has no curvature along the x-axis. The mirror surface  6  has a roof form with a round top  9 . With this mirror surface  6  the uniform expansion of the beam represented in  FIG. 1  disappears in the (z, x)-plane. The diverging beam  4  drawn in  FIG. 1  then corresponds, with the use of the mode of construction according to  FIG. 5 , in this plane to the original beam  3 .  
      In the mode of construction shown in  FIG. 6  the mirror surface  6  is, on the contrary, not only curved aspherically along the y-axis but rather also along the x-axis. Instead of the roof surfaces  7 ,  8  of  FIG. 3 , roof surfaces  7   a ,  8   a  are thus present in the (z, y)-plane as well as  7   b ,  8   b  in the (z, x)-plane, where these roof surfaces are each aspherically curved roof surfaces in said sectional planes. The mirror surface  6  of  FIG. 6  thus has not only one sectional line  12 , but rather two sectional lines  12   a ,  12   b , each of which satisfy the connection described with the aid of  FIG. 4  and are described by the same equations. If the converted beam should, with the aid of the aspherical mirror  1 , have rotationally symmetric cross-section, the mirror surface  6  is to be chosen to be rotationally symmetric relative to the peak  30 , which in  FIG. 6  is drawn in as a point of intersection of the sectional lines  12   a ,  12   b . If one configures the mirror surface  6  with sectional lines  12   a ,  12   b , in which different conical constants Q or radii of curvature are chosen, one achieves an elliptical beam cross-section.  
      The mirror surface  6 =s profile represented in  FIGS. 3, 5 , and  6  in the (z, y)-plane causes the approximately uniform distribution of the intensity I represented as profile  14  in  FIG. 7  in the profile plane P, where the representation of  FIG. 7  shows the profile  14  along the y-axis. As is to be seen, the radiation intensity lies in 80% of the illuminated area at over 80% of the maximum value. The profile  14  is approximately chest-like, in any case very much nearer a rectangle than the Gaussian profile originally present. In the aforementioned rotationally symmetric variant the profile  14  applies for any sectional plane, the ordinate then exhibits the radius of the field.  
      The mirror surface  6  of the aspherical mirror  1  can be manufactured in the most varied ways. Thus, in a cylinder which has a radius of curvature which corresponds to the radius of curvature rx of the mirror surface in the (z, x)-plane, the profile corresponding to the sectional line  12  can be incorporated. If one wants the mirror surface  6  of  FIG. 5  which is not curved in the (z, x)-plane, that is, its radius of curvature in this sectional plane can be assumed to be infinite, the processing can be done on a cuboid or wedge which is then rounded in the area of the top corresponding to the curvature c predefined by the parabola  10 . Basically, and particularly for r x  radii less than 0 and in the mode of construction according to  FIG. 6 , re-formation techniques, in particular such as replica techniques with multiple re-formation, can be used to form the mirror surface  6  of the aspherical mirror  1 .  
      To generate the profiled beam  5 , a collecting mirror  2  is disposed behind the aspherical mirror  1 , as shown in  FIGS. 1 and 2 . This is, for example, formed as a toroidal mirror with radii of curvature r tx , r ty  and parallelizes the diverging beam  4 . In so doing, the diverging beam  4  runs out limited by the spherical curvature (in the (z, x)-plane) of the aspherical mirror  1  as well as limited by the aspherical profile according to the sectional line  12 . For collimation of the diverging beam  4  the collecting mirror  2  is thus formed as a toroidal mirror with different radii of curvature r tx , and r ty . The former divergence sets the height of the rectangular field to be illuminated by the profiled beam  5 , the latter divergence causes the expansion along the longer extension.  
      In order to be able to perform the setting of the height of the rectangular field to be illuminated particularly simply, for the toroidal mirror, the radius r tx  is chosen to be r tx +2 d, where d describes the distance between the aspherical mirror  1  and the collecting mirror  2  on the optical axis. One then obtains a beam expansion factor of r tx /r x  and thus approximately 1+2d/r x .  
      Instead of the collecting mirror  2  a corresponding achromatic toroidal lens can naturally also be used. Furthermore, to eliminate the changed bundle diameter transverse to the homogenized direction, at least one cylinder mirror can be used which is dimensioned so that it together with the radius r x  of the aspherical mirror  1  as well as the radius r tx  of the collecting mirror  2  selectively changes the focusing and the bundle diameter transverse to the homogenized direction. This cylinder mirror can be disposed before the aspherical mirror  1  or after the toroidal collecting mirror  2 . Its function can also be achieved by at least one achromatic cylinder lens.  
       FIG. 8  shows an exemplary use of the illumination arrangement in a laser scanning microscope  15  or in its illumination unit  16 . Therein the radiation onto the illumination unit  16  is redirected via a scanning head  17  as a row over a (not represented) sample and is analyzed in a detector unit  18  which is implemented in the form of embodiment of  FIG. 6  to have multiple spectral channels.  
      In detail, from a light guide fiber  19 , a beam is decoupled whose Gaussian-shaped profile is re-formed via the described combination of the aspherical mirror  1  and the collecting mirror  2  into a beam which is essentially rectangular in cross-section. The aspherical mirror  1  is implemented to be aspherical in one sectional plane, spherical in the other. By means of illumination optics  20  the beam is conducted via a principal color splitter  21  and zoom optics  22  to the scanning head  17 . There the illumination row provided in this manner is redirected transverse to the row axis over a sample. Fluorescence radiation generated on the sample in the illuminated area reaches via the scanning head  17  and the zoom optics  22  back to the principal color splitter and is transmitted there based on its spectral composition different from the illumination radiation. A secondary color splitter  23  disposed behind splits the fluorescence radiation into two spectral channels, each of which comprises a pinhole objective  24 ,  24   a  which redirects the radiation onto a CCD row  25 ,  25   a . Each pinhole objective causes in a confocal detection the selection of the depth range from which fluorescence radiation can reach the CCD row. It comprises a suitable optics with slit diaphragm which lies confocally to the focal line on the sample.  
      The use of the illumination beam bundle in the form of a line provided by means of the illumination optics makes possible a highly parallel data acquisition since, unlike in the case of a customary point-sampling laser scanning microscope, several sample points are imaged simultaneously confocally, or at least partially confocally, onto the CCD rows  25 ,  25   a . In comparison to a confocal point scanner, for the same image acquisition time, the same image dimensions, the same field of view, and the same laser power per pixel, a signal/noise ratio is realized which is improved by a factor of √{square root over ( )}n, where n denotes the number of pixels in the CCD row. A typical value for this number lies between 500 and 2,000. As a prerequisite for this, the illumination in the form of rows which is provided by the illumination unit  16 , has power n times that of the laser focus of a confocal point scanner.  
      Alternatively, the intensity of the radiation introduced on the sample can, in comparison to confocal point scanners with the same image acquisition time and the same signal/noise ratio, be reduced by a factor n if the laser power otherwise used as in customary point-scanning microscopes is distributed onto the entire field illuminated by the illumination unit  16 .  
      The combination of a line-sampling laser scanning microscope together with the illumination unit  16  therefore makes it possible, in comparison to the confocal point scanners to image, with laser scanning microscopy, weak-intensity signals of sensitive sample substances with the same surface signal/noise ratio and the same sample load faster by a factor of n, with the same image acquisition time, with a signal/noise ratio improved by a factor of √{square root over ( )}n, or with the same image acquisition time, with the same signal/noise ratio with a sample load lower by a factor of n. These advantages can, however, only be achieved with the illumination unit  16  by the use of the aspherical mirror  1  in its full extent.  
       FIGS. 9 and 10  show two possibilities of how a homogeneous illumination can be used with the aid of the converting unit.  FIG. 9  shows the use of the aspherical mirror  1  with a collecting mirror  2  disposed behind for the homogeneous filling of an intermediate image ZB which lies between the zoom optics  22  and a tubular lens TL disposed behind with following objective O. This optics TL, O disposed behind images the homogeneously illuminated intermediate image onto a sample PR so that a homogeneous wide-field illumination is achieved,  FIG. 9  shows that the described converting unit is advantageous as homogenization means in a light microscope or in a parallel scanning microscope system, for example, with a Nipkow scanner or a multi-point scanner. Here reference is made to multi-point or Nipkow arrangements in U.S. Pat. No. 6,028,306, WO 88 07695, or DE 2360197 A1, which are incorporated into the disclosure.  
      Also included are resonance scanner arrangements, as are described in Pawley, Handbook of Biological Confocal Microscopy, Plenum Press 1994, page 461 ff.  
       FIG. 10  shows an alternative use in which the converting unit serves for uniform filling of the pupil P between tubular lens TL and objective O. With this, the optical resolution of the objective O can be fully exploited. This variant is expedient in a point-scanning microscope system or in a line-scanning system (in the latter in addition to the axis in which focusing into or onto the sample occurs).