Abstract:
A Z-axis focusing mechanism having a scanner and a prism whose front and rear faces are not normal to the direction of travel of a light beam prior to the light beam impinging upon the front face of the scanner. The scanner and the prism are oriented with respect to one another such that when a light beam is scanned onto the front face of the prism, a scanned beam having varying Z-axis focus points at distinct lateral locations exits the rear face of the scanner. Alternatively, the rear face of the prism may be normal to the light beam as it passes through the prism, which generates a retro-reflected, scanned light beam that exits the front of the prism

Description:
BACKGROUND 
       [0001]    Confocal, multiphoton, and sectioning microscopes are capable of producing 3-dimensional (3D) images of a volumetric specimen. These images require that the device acquire a series of optical sections from a range of focusing depths. That is, a series of optical sections in the Z-plane. In most systems, focusing in the Z-plane is performed by mechanically moving the specimen relative to the objective lens, or vice-versa. Moving either the microscope stage or the objective lens has two major drawbacks. First, scan speeds in the Z-plane accomplished by mechanical manipulations are slow; second, the scanning movements of the stage in the Z-plane can (and do) disturb the specimen during the imaging process. Focusing deeper into the sample by manipulating the objective lens yields a slow Z-axis scan speed because the objective lens is relatively massive. Once set in motion, it takes a relatively long time period for the objective to settle prior to the next data points being acquired. Manipulating the sample itself is also problematic because the samples are often living cells, and the area of interest to be imaged is a very small portion of the cell. Moving the specimen can disturb the intra-cellular structures that are in the process of being imaged. This results in sub-par micrographic images. Thus, there remains an unmet need for an alternative focusing method that does not require mechanical movement of either the specimen or the objective lens. See, for example, Botcherby et al. (Jul. 15, 2007) “Aberration-free optical refocusing in high numerical aperture microscopy” 32(14):2007-2009, incorporated herein by reference. 
       SUMMARY 
       [0002]    A first version of the device is directed to a Z-axis focusing mechanism comprising at least one scanner. The scanner is dimensioned, configured, and operationally disposed to scan a light beam through a prism. The prism has a front face proximal to the scanner and a rear face distal to the scanner. The prism is operationally situated so that the light beam exiting the scanner impinges upon the front face of the scanner. The prism itself is shaped and operationally situated in the light beam at an angle such that the front and rear face of the prism are not normal to the direction of travel of the light beam prior to the light beam impinging upon the front face of the prism. The scanner and the prism are oriented with respect to one another such that when the light beam is scanned onto the front face of the prism, a scanned beam having varying Z-axis focus points at distinct lateral locations exits the rear face of the scanner. The Z-axis focusing mechanism may optionally further comprise at least one lens and a descanner, wherein the lens is dimensioned, configured, and operationally disposed to collimate the scanned beam exiting the rear face of the prism, thereby yielding a collimated beam. The collimated beam is directed to the descanner, wherein the descanner is dimensioned and configured to bring the collimated beam to focal points at different Z-axis positions. This first version of the device may also include at least one lens operationally disposed such that the scanned beam exiting the rear face of the scanner passes through the at least one lens. 
         [0003]    Another version of the device is a a Z-axis focusing mechanism that includes a scanner as described earlier. Likewise, the prism has a front face proximal to the scanner and a rear face distal to the scanner such that the light beam exiting the scanner impinges upon the front face of the scanner. Also, the prism is disposed in the light beam at an angle such that the front and rear face of the prism are not normal to the direction of travel of the light beam prior to the light beam impinging upon the front face of the prism. But in this second version of the device, the rear face of the prism is oriented with respect to the front face of the prism such that light passing through the prism impacts the rear face of the prism at normal and is retro-reflected onto itself. The scanner and the prism are oriented with respect to one another such that when the light beam is scanned through the front face of the prism, a retro-reflected scanned beam having varying Z-axis focus points exits the front face of the prism. Here, the device may further comprise at least one lens operationally disposed between the scanner and the front face of the prism. 
         [0004]    Another version of the Z-axis focusing mechanism comprises a scanner as defined previously and an array comprising a plurality of lenses defining at least one plane disposed in the light beam at an angle such that the plane defined by the array of lenses is not normal to the direction of travel of the light beam. In this version of the device, the scanner and the array are oriented with respect to one another such that when the light beam is scanned through the array, a scanned beam having varying Z-axis focus points is generated. This can be accomplished using offset lens all having the same focal length, or using lenses in the array having more than one focal length. 
         [0005]    In yet another version of the device, the Z-axis focusing mechanism comprises a scanner and an array comprising a plurality of lenses and a corresponding plurality of lenses defining at least one radial path disposed in the light beam. Here, the scanner and the array are oriented with respect to one another such that when the light beam is scanned through the array, the scanned light beam passes through the plurality of lenses, is retro-reflected from the plurality of mirrors to generate a retro-reflected, scanned light beam. The retro-reflected, scanned light beam passes back through the corresponding plurality of lenses, whereby a plurality of discrete light beams having varying Z-axis focus points is generated. 
         [0006]    The phrase “operationally disposed” when describing the relative arrangement between two named elements defines how the two element structurally and functionally interact and does not exclude intervening elements between the two named elements. Thus, for example, “a scanner ‘operationally disposed’ to scan a light beam through a prism” does not exclude the presence of additional unnamed elements, such as lenses, mirrors, and the like, that the light beam may pass through or reflect from during its passage from the scanner to the prism. 
         [0007]    Numerical ranges as used herein are intended to include every number and subset of numbers contained within that range, whether specifically disclosed or not. Further, these numerical ranges should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 2 to 8, from 3 to 7, 5, 6, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth. 
         [0008]    All references to singular characteristics or limitations of the present invention shall include the corresponding plural characteristic or limitation, and vice-versa, unless otherwise specified or clearly implied to the contrary by the context in which the reference is made. 
         [0009]    The methods of the present invention can comprise, consist of, or consist essentially of the essential elements and limitations of the method described herein, as well as any additional or optional ingredients, components, or limitations described herein or otherwise useful in optics in general and microscopy in particular. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0010]    In each of  FIGS. 1-5 , the Z-axis is in the horizontal direction. In  FIG. 6 , the Z-axis is in the vertical direction. 
           [0011]      FIG. 1  is a schematic diagram of a first version of the Z-focusing mechanism. 
           [0012]      FIG. 2  is a schematic diagram of a second version of the z-axis focusing mechanism. 
           [0013]      FIG. 3  is a third version of the Z-axis focusing mechanism. 
           [0014]      FIG. 4  is a fourth version of the z-axis focusing mechanism. 
           [0015]      FIG. 5  is a fifth version of the Z-axis focusing mechanism. 
           [0016]      FIG. 6  is a schematic diagram of the Z-axis focusing mechanism shown in  FIG. 2  incorporated into a multiphoton micrcoscope. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0017]    At the heart of the device is the realization that arranging a prism or other optical deflector within a beam path in which the lateral displacement can vary results in a varying optical distance of refraction or retro-reflection in confocal microscopy and the like. The necessary result of this varied optical distance is a correspondingly varied focal plane on the return beam. Thus, by judiciously placing an appropriately shaped prism at a pre-selected angle (or range of angles) within the beam path of a confocal microscope, the incoming beam can be scanned through the prism at various lateral displacements, thereby yielding a different Z-axis focal plane depending on the displacement of the ray as it enters/exits the prism. Regarding the individual components illustrated in the drawing figures, all of the lenses, mirrors, scanners, prisms, galvanometric controllers, detectors, etc., are of conventional design. Their assembly into the Z-axis focus mechanism described and claimed herein is, however, novel and inventive. 
         [0018]    Thus, a first version of the device is depicted in  FIG. 1 . A light source (not shown) directs a beam of light at scanner  14 . In the discussion that follows, “scanner” (or “descanner” as the context dictates) is used for sake of brevity and clarity only. There are many different means to deflect a light path in a controlled fashion, such as galvanometer-controlled mirrors, acousto-optical deflectors, polygonal scanners, diffraction gratings, microelectromechanical systems (MEMS), reflective and transmissive spatial light modulators, and the like. All of these devices, as well as those developed in the future for scanning a beam of light are encompassed by the word “scanner” or “descanner” and can be used in place of the galvanometer-controlled mirrors explicitly depicted in the drawing figures. The scanner  14  generates a scanned beam of light  38  which can be focused via lens  20  to yield a focused, scanned beam  36  which is then directed through prism  12 . The incoming beams of light  36  are thus refracted through the prism to yield separate light beams  50 ,  52 , and  54 , all of which exit the prism  12  at distinct lateral locations, thus yielding corresponding beams  40 ,  42 , and  44 . These beams are then collimated via lens  18  and pass onto an optional descanner  16  to yield corresponding beams  30 ,  32 , and  34 . These beams,  30 ,  32 , and  34  pass through lens  21  (seen just below the descanner  16  in  FIG. 1 ) which brings beams  30 ,  32 , and  34  to focal points at different depths (i.e., at different Z-axis points) within the sample. By judiciously programming scanners/descanners  14  and  16 , and adjusting the angle of the prism  12  accordingly, the Z-axis dimension can be scanned quickly without moving the objective lens or the specimen itself. In short, the Z-plane can be scanned purely by optical means, in the absence of any mechanical movement of the specimen or the objective lens. 
         [0019]    An alternate version of the Z-axis focusing mechanism is shown in  FIG. 2 . Here, a prism  12  is arranged at such an angle that the back reflective face of the prism is at an angle normal to the internal ray, and is thus reflected back upon itself. This is shown in beams  30 ,  32 , and  34 . Because of the angle of the prism  12 , the return beams  40 ,  42 , and  44  reach focus at different points in the Z-axis. (Again, recall that in the figures the horizontal direction is the Z-axis.) Thus as shown in  FIG. 2  a light source is scanned through scanner and directed through lens  20  to prism  12 . The resulting rays  30 ,  32 , and  34  strike the back wall of the prism at normal and are back-reflected through the prism and exit as beams  40 ,  42 , and  44 , each of which have a different focal point in the Z-axis dues to the angle of prism  12 . Thus, beams  40 ,  42 , and  44  can be used (via an optical relay; see  FIG. 6 ), to scan a sample in the Z-axis (for example, at points  38  and  10 ) via lens  18 . This is possible because the point of focus in the Z-plane is different for each of beams  40 ,  42 , and  44  due to the angle of tilt of the prism  12 . 
         [0020]      FIG. 3  depicts another version of the device which is similar to the version shown in  FIG. 1 , but omits the descanner  16  which is depicted in  FIG. 1 . Thus, as shown in  FIG. 3 , a scanner  14  sends a beam light  38  through the lens  20  to yield incoming scanned beam  36  which impinges on prism  12 . The prism generates refracted beams  50 ,  52 , and  54 , which exit prism  12  at different lateral displacements to yield corresponding rays  40 ,  42 , and  44 . These beams are passed through lenses  22  and  24  to yield corresponding rays  30 ,  32 , and  34 , each of which has a different focal point in the Z-axis. (Again, the Z-axis is in the horizontal plain in  FIGS. 1-5 .) In this fashion, the Z-axis can be scanned quickly by raster scanning the scanner  14 . 
         [0021]      FIG. 4  depicts another version of the Z-axis focusing mechanism which, rather than using a prism, uses a series of micro-lenses  30 ′,  32 ′, and  34 ′ to yield differing focal points within the Z-axis. As shown in  FIG. 4 , a light source (not shown) is used to generate a light beam  30  which is scanned through scanner  14 , to yield the scanned beams  40 ,  42 , and  44 . Lens  18  is employed to direct the scanned beams to a 2D array of micro-lenses having differing focal lengths or an array of micro-lenses having the same focal length, but offset from one another in the Z-axis. As shown in  FIG. 4 , beams  40 ,  42 , and  44  exit lens  18  as corresponding beams  30 ,  32 , and  34 , and are directed to corresponding offset micro-lenses  30 ′,  32 ′, and  34 ′. Because the lenses  30 ′,  32 ′, and  34 ′ are offset, or have different focal lengths, or a combination of the two, the beams  30 ,  32 , and  34  retro-reflected from mirror  60  have different focal points  10  in the Z-axis. In this fashion, the Z-axis can be scanned optically by controlling the scanner  14  to sweep the retro-reflected beams  30 ,  32 , and  34  through the sample. 
         [0022]      FIG. 5  depicts yet another version of the Z-axis focusing mechanism. Here, a series of mirrors  24  and  26  (analogous to mirror  60  in  FIG. 4 ), and ancillary lenses  18  and  20 , are arranged in a radial pattern. The mirrors  24  and  26  are positioned to retro-reflect back onto the scanned incoming beam  30 . Using a scanner (not shown) the beam  30  can be scanned across the array of mirrors  24  and  26  to yield multiple Z-axis focus points  10  quickly. The multiple Z-axis focal points are then relayed to an objective lens (no shown) to scan the sample in the Z-axis. 
         [0023]      FIG. 6  depicts the Z-axis scanner shown in  FIG. 2 , integrated into a multiphoton microscope. Thus, in the same fashion as in  FIG. 2 , a prism  12  is arranged at such an angle that the back reflective face of the prism is at an angle normal to the internal ray, and the internal ray is thus reflected back upon itself. This is shown in beams  30 ,  32 , and  34 . Because of the angle of the prism  12 , the return beams  40 ,  42 , and  44  reach focus at different points in the Z-axis. Thus as shown in  FIG. 6  a light source (shown by the hollow arrow at the center of the figure, entering scanner  14 ) is scanned through scanner  14  and directed through lens  20  to prism  12 . The resulting rays  30 ,  32 , and  34  strike the back wall of the prism at normal and are back-reflected through the prism and exit as beams  40 ,  42 , and  44 , each of which have a different focal point in the Z-axis dues to the angle of prism  12 . Beams  40 ,  42 , and  44  are then directed via an optical relay in a microscope to image a sample. 
         [0024]    As shown in  FIG. 6 , the scanner  14  provides lateral scanning to address the prism  12  at different thicknesses. The optical relay at F 1 -B and F 2 -B and transfers the first galvanometer pair plane from scanner  14  to a second galvanometer pair plane as shown by the XY galvanometer pair depicted in the upper left corner of  FIG. 6 . The scanned beam is passed through a convention scan lens to propagate the initial varied Z-axis focus positions as shown by F 1 -C, F 2 -C and F 3 -C in  FIG. 6 . The remainder of  FIG. 6 , in the bottom left corner of the figure, is a conventional multiphoton microscope that includes a conventional tube lens, dichroic beamsplitter, objective lens, collector lens, and photon detector for detecting light emitted from the sample. The varied Z-axis focus positions are shown in the magnified view of the objective lens shown at the lower center of  FIG. 6 . The varied focus positions in the Z-axis are labeled F 1 -D, F 2 -D, and F 3 -D.