Abstract:
A laser-scanning microscope having an illumination-beam path and a detection-beam path and a microscope objective. A component for generating a plurality of scanning beams from at least one illumination beam is located in the illumination-beam path. A wedge-shaped, light-transmitting first component part provided in the illumination beam path generates spatially offset partial beams, the scanning beams being generated at the first component part by multiple reflections at an at least partially partially-reflecting surface. The microscope has a one-dimensional scanner for moving the scanning beams over a sample in the illumination beam path. The scanning beams have at least partially relative to one another a non-zero angle upstream of the objective in the illumination direction. The scanning beams can intersect at least partially in the objective pupil of the microscope objective. Additional compensation elements are provided for the scanning beams to compensate for a spectral dispersion and/or the beam direction.

Description:
[0001]    The present application claims priority to German Patent Application No. DE 10 2014 003 773.8 filed on Mar. 15, 2014, the disclosure of which is incorporated herein by reference in its entirety. 
       FIELD OF THE INVENTION 
       [0002]    Arrangements for beam multiplication are known from publications and patents. 
         [0003]    It is noted that citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention. 
         [0004]    An advantageous application is carried out in laser scanning microscopes (LSMs) because, in this way, the scan speed is increased compared to single-point scanning. A beam can be split either by guiding separate beam parts in different directions (geometric splitting) or by using a beam splitter and further utilizing the reflected and transmitted component of the beam (physical splitting). Examples for both principles are mentioned, e.g., in Naumann/Schröder, “Optical Components [Bauelemente der Optik]”, pages 182 ff, or in patents DE19535525A1, US20090016400A1, DE19904592C2. 
         [0005]    A beam splitting in which the partial bundles have the highest possible homogeneity over the visible spectrum and between one another is important for application in LSM. Further, the beam splitting should be very stable over the long term and insensitive to temperature. 
         [0006]    The arrangement according to DE19535525A1 has an even number of reflections per partial bundle so that the arrangement is insensitive to positional tolerances. Since the optically active surfaces are fixedly connected via the glass substrate, this arrangement is both stable and insensitive to temperature. However, it is disadvantageous that all of the partial bundles are formed parallel to one another so that no field angles can be generated. However, these field angles are required so that the objective can focus the partial beams at different locations on the sample. 
         [0007]    The arrangement according to DE19904592C2 makes it possible to generate angles between the partial bundles by tilting the mirror surfaces, but stability and insensitivity to temperature are sacrificed owing to the required separate holder of the mirrors. 
         [0008]    It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention. 
         [0009]    It is further noted that the invention does not intend to encompass within the scope of the invention any previously disclosed product, process of making the product or method of using the product, which meets the written description and enablement requirements of the USPTO (35 U.S.C. 112), such that applicant(s) reserve the right to disclaim, and hereby disclose a disclaimer of, any previously described product, method of making the product, or process of using the product. 
       SUMMARY OF THE INVENTION 
       [0010]    It is the object of the invention to implement a beam multiplier with preferably equidistant angles between the partial bundles and greater stability at the same time. 
         [0011]    In a microscope according to the invention for multi-beam scanning having an illumination beam path and a detection beam path and a microscope objective, wherein a component for generating a plurality of scanning beams from at least one illumination beam is located in the illumination beam path, a wedge-shaped, light-transmitting first component part is provided in the illumination beam path for generating spatially offset partial beams, the scanning beams being generated at this first component part by multiple reflections at an at least partially partially-reflecting surface. 
         [0012]    The microscope is configured as laser scanning microscope and has at least one one-dimensional scanner for the scanning movement of the scanning beams over a sample in the illumination beam path. 
         [0013]    The scanning beams advantageously have at least partially relative to one another an angle other than zero degrees upstream of the objective in illumination direction. 
         [0014]    The scanning beams can preferably intersect at least partially in the objective pupil of the microscope objective. Additional compensation elements can be provided at least partially for the scanning beams to compensate for a spectral dispersion and/or the beam direction. 
         [0015]    For n scanning beams, n compensation elements which are preferably configured as further wedge-shaped, light-transmitting component parts can be provided. Compensation elements can be arranged upstream and/or downstream of the first component part. 
         [0016]    With less strict requirements, partially adjustable plane plates can also be provided for adjusting an intersection point of the scanning beams or beam direction and/or beam offset thereof. 
         [0017]    The invention is further directed to a component for generating a plurality of scanning beams from at least one illumination beam, particularly in a microscope, which is wedge-shaped and light-transmitting for generating spatially offset partial beams and which generates a plurality of scanning beams by multiple reflection at an at least partially partially-reflecting surface of the component. 
         [0018]    The illumination means can advantageously be illumination lasers. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0019]      FIG. 1  shows a basic optical arrangement in a microscope with a multiple-beam light source. 
           [0020]      FIG. 2  shows a preferred embodiment example of an optical arrangement according to the invention for beam multiplication. 
           [0021]      FIG. 3  shows wedge angles β 1 -β 5  of wedge plates K 1 -K 5 . 
           [0022]      FIG. 4  shows the wedge thickness d 1 -d 5  of K 1 -K 5 . 
           [0023]      FIG. 5  shows spectral transmission of splitter layers at the output of K 2  for partial bundles ST 4 , ST 3 , and ST 2 . 
       
    
    
     DETAILED DESCRIPTION OF EMBODIMENTS 
       [0024]    It is to be understood that the figures and descriptions of the present invention have been simplified to illustrate elements that are relevant for a clear understanding of the present invention, while eliminating, for purposes of clarity, many other elements which are conventional in this art. Those of ordinary skill in the art will recognize that other elements are desirable for implementing the present invention. However, because such elements are well known in the art, and because they do not facilitate a better understanding of the present invention, a discussion of such elements is not provided herein. 
         [0025]    The present invention will now be described in detail on the basis of exemplary embodiments. 
         [0026]    First,  FIG. 1  shows a basic optical arrangement in a microscope with a multiple-beam light source. The light of a laser L proceeds to an arrangement for beam multiplication MBL as will be described in more detail in the following for the present invention. 
         [0027]    The light of the multi-beam light source MBL passes via optics, in this case a zoom ZO, to a main color splitter MBD for separating illumination light from sample light and, from there, is reflected by MDB to a sample S via a reflector R and an objective O. The sample light, particularly the fluorescent light of interest, passes back via reflector R and transmittingly through MDB to a detector group DE via detector optics DEO and a multiple-pinhole arrangement PH. 
         [0028]    The sample light which is generated on or in the sample by the illumination with a plurality of beam foci via objective O is preferably guided in detection direction through a plurality of pinholes PH, an individual pinhole being associated with each individual beam. Optionally, the detector DE can also be configured as spatially resolving area receiver. 
         [0029]      FIG. 2  shows a preferred embodiment example of an optical arrangement according to the invention for beam multiplication. 
         [0030]    The expressions wedges and wedge plates are used synonymously hereinafter. 
         [0031]    As used herein, wedge-shaped means that at least two light-active, preferably opposing lateral surfaces of the wedge plates have an angle of less than 90 degrees relative to one another. 
         [0032]    As used herein, spectrally independent means that every wavelength dispersion is ultimately compensated in an advantageous manner through the employed elements. 
         [0033]    The illumination light L proceeds into a first wedge-shaped glass body or a wedge plate K 1  and from there into a second wedge plate K 2  in which individual beams ST 1 -ST 4  are generated by multiple total reflection. 
         [0034]    While beam ST 2  undergoes no further influence after K 2 , ST 1  passes through a further wedge plate K 5 , ST 3  passes through K 3 , and ST 4  passes through K 3  and K 4 . In this way, through suitable dimensioning of wedge plates K 3 , K 4 , K 5 , the beams ST 1 -ST 4  do not run parallel to one another but rather have a converging angle relative to one another, which will be further discussed later. 
         [0035]      FIG. 2  further shows perpendiculars s relative to the respective light direction to characterize angles α 1 -α 4  of input surfaces E 1 -E 5  of K 1 -K 5  relative to s. 
         [0036]      FIG. 3  shows wedge angles β 1 -β 5  of wedge plates K 1 -K 5 . 
         [0037]      FIG. 4  shows the wedge thickness d 1 -d 5  of K 1 -K 5 . It is measured from the depicted center of the input surfaces of K 1 -K 5  perpendicular to the input surface through the respective wedge body. Exemplary measurements of the wedge plates and their position relative to one another and the material employed are compiled in the following Table T1. 
         [0000]    
       
         
               
               
               
               
               
             
           
               
                 TABLE T1 
               
               
                   
               
               
                 Wedge 
                   
                 Thickness 
                 Wedge 
                 Global tilting 
               
               
                 No. 
                 Material 
                 [mm] 
                 angle 
                 angle 
               
               
                   
               
             
             
               
                 K1 
                 N—PSK3 
                 d1: 30 
                 β1: 2.096° 
                 α1: −25.3° 
               
               
                 K2 
                 fused quartz 
                 d2: 17 
                 β2: 0.582° 
                 α2: 14.0° 
               
               
                 K3 
                 N—BaF4 
                 d3: 15 
                 β3: −0.294° 
                 α3: 24.5° 
               
               
                 K4 
                 S—NBH53 
                 d4: 15 
                 β4: −0.236° 
                 α4: 15.0° 
               
               
                 K5 
                 N—BaF4 
                 d5: 15 
                 β5: 0.266° 
                 α5: −25.1° 
               
               
                   
               
             
          
         
       
     
         [0038]    The subject matter of the invention is an arrangement of wedge plates such that three or more partial bundles which have a spectrally independent equidistant angle separation and intersect at the same point (objective pupil) preferably in a spectrally independent manner are formed at the output of this arrangement. In  FIG. 1 , the intersection point of the partial beams would lie, for example, on reflector R in front of the objective O and would fill the entrance pupil of the objective O centrically. 
         [0039]    One compensation element per partial bundle is required in order to correct the above-mentioned effects:
       a) angles between partial bundles are not equidistant,   b) more than two partial bundles do not intersect at the same point,   c) the angles of the partial bundles are wavelength-dependent due to the dispersion of the glasses.       
 
         [0043]    The degrees of freedom for an element of this type are thickness, wedge angle, tilt and material. Accordingly, it is possible to compensate for transverse chromatic aberrations through the wedge angle and for a color-dependent lateral offset of the bundles through the choice of wedge thickness and tilt. Further, through suitable choice of material, all of the bundles intersect spectrally independently at the same point (pupil). 
         [0044]    For less strict requirements with respect to the equidistance of angles of the partial bundles and spectral independence of these angles, tiltable plane parallel plates could also be used instead of wedge plates K 3 , K 4 , K 5  to compensate for the pupil position (unification in the pupil) through parallel offset and tilting of these plane parallel plates. K 1  could then even be omitted. 
         [0045]    Although the pupil position of the partial bundles should only play a subordinate part, even the plane parallel plates can be dispensed with so that only a splitter edge is required for angle separation. 
         [0046]      FIGS. 2 to 4  show a construction for four partial bundles. Wedge K 1  compensates for the chromatic aberrations of splitter wedge K 2  for partial bundle ST 2 . The effects on partial bundles ST 3  and ST 4  are compensated through wedges K 3  and K 4 . Wedge K 3  acts on both bundles for reasons of space. Partial bundle ST 1  is compensated through wedge K 5 . The advantage of the arrangement shown in  FIGS. 2 to 4  consists in that the wedges can be large because they project into the construction from the outside. This allows the required wedge angles to be fabricated in a highly precise manner. Accordingly, the positional tolerances of the parts are relaxed, which automatically results in a very thermally stable construction because the angles of the partial bundles are not affected by the positional tolerances of the wedges. They are determined exclusively through the wedge angles and mainly through the wedge angle of splitter K 2 . This easily allows angular accuracies in the seconds range, which, of course, remain stable in the subsecond range. This is very advantageous for an application in the laser scanning microscope (LSM) with multi-spot excitation and detection. 
         [0047]    K 2  can also be tilted in a limited manner for adjusting the beam spacing provided the partial beams ST 1 - 4  can still execute the subsequent passage as is shown. 
         [0048]    In order that all four of the partial bundles have the same intensity, a special coating of the four outlet regions of the splitter K 2  is required. When the spectral transmissions of all of the rest of the compensation wedges and coatings are taken into account, spectral transmission curves can be set such that exactly the same transmission values per wavelength are achieved for the four partial bundles.  FIG. 5  shows an example of an arrangement of this type. 
         [0049]    The spectral transmission of the splitter layers at the output of K 2  for partial bundles ST 4 , ST 3  and ST 2  is shown in  FIG. 5 . 
         [0050]    The depicted splitter layers control the uniform intensity at all outputs of K 2  through differing transmission/reflectivity, initially approximately 25% for ST 4 , then 33% for ST 3  and then 50% for ST 2 , and a maximum transmission is coated for partial beam ST 4 . The attenuation of partial beams ST 4 , ST 3  and ST 1  through wedges  3 ,  4 ,  5  is negligible through low transmission losses. 
         [0051]    The purpose of the relative angles is to allow the objective to focus these angles at different locations in contrast to parallel beams. The beams are preferably combined in the entrance pupil of the microscope objective and fill the latter centrically. 
         [0052]    The spread of the partial beams (angles relative to one another) is determined through the wedge angle of K 2  because the deflection at the output surface takes place at a different angle, while the tilting of K 2  determines the mutual distance of the partial beams relative to one another. This refers to the lateral partial bundle spacing at the output of K 2 , i.e., at the side which is coated in a segmented manner by different partial mirrorings. 
         [0053]    Element  2  is variably tiltable to a limited degree in the drawing provided the wedges  3 ,  4 ,  5  required for dispersion compensation are still impinged upon. 
         [0054]    A wavelength-dependent (pre-)dispersion taking place through K 1  is compensated in  2  or  3 ,  4  and  5 , respectively. 
         [0055]    Beam  3  passes through after  2  (WL compensated). 
         [0056]    The position of the wedges is constructionally fixed such that they can project in from the outside. 
         [0057]      3 ,  4 ,  5  can be configured as plane parallel plates, in which case  1  can be omitted (with relaxed requirements for dispersion compensation). Through their angle and parallel offset, they serve to ensure an intersection of all of the beams in the objective pupil. 
         [0058]    While this invention has been described in conjunction with the specific embodiments outlined above, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, the preferred embodiments of the invention as set forth above are intended to be illustrative, not limiting. Various changes may be made without departing from the spirit and scope of the inventions as defined in the following claims.