Abstract:
Various beam selectors for selectively placing one of at least two beams of light along the same output path are disclosed. In one aspect, a beam selector receives at least two substantially parallel beams of light. The beam selector includes a plate with an aperture so that when one of the at least two beams is selected for transmission, the beam selector directs only the selected beam along an output path through the aperture. The plate can also serve to block transmission of unselected beams. The output path through the aperture is the same for each of the at least two beams when each beam is selected. Beam selectors can be incorporated into fluorescence microscopy instruments to selectively place particular excitation beams along the same path through the microscope objective lens and into a specimen to excite fluorescence of fluorescent probes attached to a particular component of the specimen.

Description:
CROSS-REFERENCE TO A RELATED APPLICATION 
     This application is a filing under 35 U.S.C. 371 of international application number PCT/SE2012/050029, filed Jan. 16, 2012, published on Sep. 7, 2012 as WO 2012/118424, which claims the benefit of Provisional Application No. 61/447,709; filed Mar. 1, 2011. 
    
    
     TECHNICAL FIELD 
     This disclosure relates to fluorescence microscopy and, in particular, to systems for selecting excitation laser beams in fluorescence microscopy instruments. 
     BACKGROUND 
     In recent years, technical improvements in epifluorescence microscopy have centered on increasing the contrast between fluorescently labeled specimen components and background. As a result, many thousands of fluorescent probes have been developed to provide a means of labeling many different cellular, subcellular and molecular components of a biological specimen. In addition, the large spectral range of available fluorophores allows simultaneous imaging of different components. In order to image different components of a specimen, the different components are labeled with fluorophores that fluoresce at different wavelengths, but each fluorophore is excited with an excitation beam of a different wavelength. As a result, those working in epifluorescence microscopy have directed much attention to developing efficient and cost effective ways of superimposing the excitation beams so that the beams travel along the same path through the objective lens into the specimen. Gas-tube lasers and stacks of dichroic mirror are two systems that have been consider for producing superimposed excitation beams. Gas-tube lasers typically emit a single beam of light composed of several distinct wavelengths and a stack of dichroic mirrors can be used to combine excitation beams that emanate from different light sources.  FIG. 1  shows an example of two dichroic mirrors  102  and  104  stacked to superimpose three excitation beams of different wavelengths λ 1 , λ 2  and λ 3  represented by patterned lines  106 - 108 , respectively. Dichroic mirror  102  transmits the beam  106  and reflects the beam  107  and dichroic mirror  104  transmits the beams  106  and  107  and reflects the beam  108  to form a superimposed beam  110  composed of all three wavelengths. 
     However, typical gas-tube lasers are large, cost prohibitive, inefficient, and unstable. Gas-tube lasers also have short lifetimes and emit light over a very limited range of wavelengths. On the other hand, although the dichroic mirror-based approach is versatile, each time an excitation beam is added to the superimposed beam, a separate dichroic mirror is added to the stack which leads to substantial inefficiency as the non-negligible losses accumulate. For instance, as shown in  FIG. 1 , the beam  106  already passes through the two dichroic mirrors  102  and  104 . Addition of a third dichroic mirror to the stack to reflect a fourth wavelength and transmit the wavelengths λ 1 , λ 2  and λ 3  would further attenuate the beam  106 . In addition, neither gas-tube lasers nor dichroic mirror stacks provide switching between the different excitation beams on the sub-millisecond time scale or faster. With the dichroic mirror-based approach, it is possible to place shutters in the path of each beam input to the stack. However, each shutter adds substantial cost to the instrument and shutters are not able to achieve the desired sub-millisecond switching speeds between different excitation beams. For the above described reasons, engineers, scientists, and fluorescent microscope manufacturers continue to seek fast, efficient, and cost effective systems for placing excitation beams along the same path. 
     SUMMARY 
     Various beam selectors for selectively placing one of at least two beams of light along the same output path from the beam selectors are disclosed. In one aspect, a beam selector receives at least two substantially parallel beams of light output from separate light sources. The beam selector includes a plate with an aperture so that when one of the at least two beams is selected for transmission, the beam selector directs only the selected beam along an output path through the aperture in the plate. The plate can also serve to block transmission of unselected beams. The output path through the aperture is the same for each of the at least two beams when each beam is selected for transmission. The beam selectors can be incorporated into fluorescence microscopy instruments to selectively place particular excitation beams along the same path through the microscope objective lens and into a specimen to excite fluorescence of fluorescent probes attached to a particular component of the specimen. 
    
    
     
       DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows an example of two dichroic mirrors stacked to superimpose three excitation beams of different wavelengths. 
         FIG. 2  shows a schematic representation of an example fluorescence microscopy instrument that includes a beam selector. 
         FIG. 3  shows a representation of an example parallel beam source. 
         FIGS. 4A-4B  show a top-plan view and an isometric view of an example beam selector, respectively. 
         FIG. 5  shows a top-plan view of the beam selector shown in  FIG. 4  in operation. 
         FIGS. 6A-6C  show example snapshots of internal paths traveled by three beams through the selector shown in  FIG. 4 . 
         FIGS. 7A-7C  show an isometric view and two top-plan views of an example beam selector. 
         FIG. 8A-8D  show an isometric view and three top-plan views of an example beam selector. 
         FIG. 9  shows a schematic representation of a beam selector combined with an example implementation of a beam launch. 
     
    
    
     DETAILED DESCRIPTION 
       FIG. 2  shows a schematic representation of an example fluorescence microscopy instrument  200  that includes a beam selector (“BS”)  202 . There are many different types of fluorescent microscopy instruments and corresponding optical paths. The instrument  200  is not intended to represent the optical paths within all the different, well-known variations of fluorescence microscopy instruments, but is instead intended to illustrate the general principals of integrating a BS into fluorescent microscopy instruments. The instrument  200  includes a parallel laser beam source  204 , the BS  202 , a first lens  206 , a dichroic mirror  208 , an objective lens  210 , a stage  212 , a second lens  214 , and a detector  216 . As shown in the example of  FIG. 2 , the beam source  204  emits N substantially parallel beams  218  of excitation light to the beam selector  202 , where N is a positive integer. Each of the beams  218 , denoted by λ 1 , λ 2 , λ 3 , . . . λ N , is a collimated, high-intensity, substantially monochromatic beam of light of a single wavelength, or light within a very narrow band of wavelengths, of the electromagnetic spectrum. A specimen  220  disposed on the stage  212  is composed of numerous different components, many of which are labeled with different fluorescent probes. Each beam output from the beam source  202  stimulates a fluorescent emission from a fluorophore used to image a particular component of the specimen  220 . The BS  202  receives the excitation beams  218  and only outputs the excitation beam selected to excite fluorescence of the fluorophore attached to the component of the specimen  220  to be imaged. For example, suppose a first component of the specimen  220  is selected for imaging. The beam selector  202  is operated to only output the excitation beam  222 , that excites the fluorophore attached to the component. The other excitation beams are blocked by the BS  202 . The lens  206  focuses the excitation beam  222  and the dichroic mirror  208  reflects the excitation beam into the back of the objective lens  210 , which, in turn, directs the excitation beam into the specimen  220 . A portion of the fluorescent light emitted from the fluorophore is captured and collimated by the objective lens  210  into a beam, represented by a shaded region  224 , that passes through the dichroic mirror  208  and is focused onto the detector  216  by the second lens  214 . The detector  216  can be a photomultiplier, photodiode, or a solid-state charged coupled device (“CCD”). When a second component of the specimen  220  is selected for imaging, the beam selector  202  switches to only output the excitation beam that excites the type of fluorophore attached to the second component. In alternative instrument configurations, the dichroic mirror  208  can be configured to transmit the excitation beam and reflect the fluorescent light, in which case the locations of the BS  202 , beam source  204  and lens  206  are switched with the lens  214  and the detector  216 . 
     The beam source  204  can be configured to output, in parallel, any suitable number of excitation beams.  FIG. 3  shows a representation of an example parallel beam source  300  that includes seven separate light sources  301 - 307  that each emit light with one of seven different wavelengths denoted by λ 1 , λ 2 , λ 3 , λ 4 , λ 5 , λ 6  and λ 7 , respectively. For example, each light source can be a laser that emits a high-intensity, substantially monochromatic beam of light in a different, very narrow band of the electromagnetic spectrum or emits light with a single wavelength. The path each beam travels through the beam source  300  is represent by a differently patterned directional arrow denoted by B 1 , B 2 , B 3 , B 4 , B 5 , B 6  and B 7 . In the example of  FIG. 3 , the beam source  300  includes seven mirrors  309 - 315  that are positioned to reflect the beams into substantially parallel paths with a desired spacing. Ideally the paths along which the beams travel are parallel or non-intersecting, but in practice, it is recognized that due to slight variations in the relative placement and orientation of the minors and light sources, the paths may be only approximately parallel or intersect at very long distances away from the beam source  300 . As a result, the paths along which the beams are output are referred to as approximately parallel. 
       FIGS. 4A-4B  show a top-plan view and an isometric view of an example beam selector  400 . The selector  400  includes a scanning mirror  402 , a first flat stationary mirror  404 , a second flat stationary mirror  406  and a plate  408  with an aperture  410 . The reflective surface of the first mirror  404  is angled toward the region between the scanning mirror  402  and second mirror  406 , and the reflective surface of the second mirror  406  is angled toward the region between the scanning mirror  402  and the first mirror  404 , and the reflective surfaces of the mirrors are substantially perpendicular to the same plane. In the example of  FIGS. 4A-4B , the scanning mirror  402  is a galvanometer mirror that includes a flat pivot mirror  412  attached to a rotatable shaft of a motor  414 , which can be a galvanometer motor or a stepper motor. Alternatively, the scanning mirror can be a piezoelectric controlled mirror. As shown in  FIGS. 4A-4B , the mirror  412  is rotated back and forth by the motor  414  through a range of angles. 
       FIG. 5  shows a top-plan view of the beam selector  400  in operation.  FIG. 5  includes a representation of the beam source  300  with the seven different substantially parallel beam paths B 1 , B 2 , B 3 , B 4 , B 5 , B 6  and B 7  directed toward the mirror  412 .  FIG. 5  shows the mirror  412  rotated into seven different positions denoted by M 1 , M 2 , M 3 , M 4 , M 5 , M 6  and M 7 . The differently patterned lines  501 - 507  represent the path each beam travels through the selector  400  when the pivot mirror  412  is rotated into one of the seven different positions. Each beam travels between the mirrors  404 ,  406  and  412  within the same plane. As shown in the example of  FIG. 5 , the stationary mirrors  404  and  406  and the pivot mirror  412  are positioned in the same plane so that each beam is output along the same path  510  through the aperture  410  in the plate  408 . In other words, when the mirror  412  is rotated into the position Mj, where j is an integer between 1 and 7, the beam Bj is output from the selector  400 , after four reflections, along the path  510 . While the mirror  412  is in the position Mj, the other beams Bk, where k is an integer between 1 and 7 and k≠j, do not exit the selector  400 . 
     As shown in  FIG. 5 , for each rotational position of the pivot mirror  412  that results in one of the beams being placed on the path  510 , the beam is reflected off of the mirror  412  a first time, the first stationary mirror  404  one time, the second stationary mirror  406  one time, and off of the mirror  412  a second time for a total of four reflections. Also, the other six beams are reflected so that they do not reach the aperture  410  in the plate  408 .  FIGS. 6A-6C  show example snapshots of internal paths of three of the seven beams (i.e., beams B 1 , B 4  and B 7 ) traveling through the selector  400  when the pivot mirror  412  is rotated into the three positions M 1 , M 4  and M 7 , respectively. In  FIG. 6A , the pivot mirror  412  is rotated into position M 1 . The beam B 1  strikes the pivot mirror  412  at a point  602  and undergoes four reflections off of the mirrors  404 ,  406  and  412  with the internal paths numbered sequentially 1, 2, 3 and 4. The 5 th  path is created by a second reflection off of the pivot mirror  412  at the point  604 , which places the beam B 1  on the path through the aperture  410  also shown in  FIG. 5  as the path  510 . As shown in  FIG. 6A , the other two beams B 4  and B 7  track different reflection paths that do not result in the beams B 4  and B 7  passing through the aperture  410 . In  FIG. 6B , the pivot mirror  412  is rotated into position M 4 . The beam B 4  strikes the pivot mirror  412  at a point  606  and undergoes four reflections off of the mirrors  404 ,  406  and  412  with the internal paths numbered sequentially 1′, 2′, 3′ and 4′. The 5 th  path is created by a second reflection off of the pivot mirror  412  at the point  608 , which places the beam B 4  on the path through the aperture  410  also shown in  FIG. 5  as the path  510 . As shown in  FIG. 6B , the other two beams B 1  and B 7  track different reflection paths that do not result in the beams B 1  and B 7  passing through the aperture  410 . In  FIG. 6C , the pivot mirror  412  is rotated into position M 7 . The beam B 7  strikes the pivot mirror  412  at a point  610  and undergoes four reflections off of the minors  404 ,  406  and  412  with the internal paths numbered sequentially 1″, 2″, 3″ and 4″. The 5″ th  path is created by a second reflection off of the pivot mirror  412  at the point  612 , which places the beam B 7  on the path through the aperture  410  also shown in  FIG. 5  as the path  510 . As shown in  FIG. 6C , the other two beams B 1  and B 4  track different reflection paths that do not result in the beams B 1  and B 4  passing through the aperture  410 . 
     The beam selector  400  is implemented with the scanning mirror  402  to provide sub-millisecond output beam selection.  FIGS. 7A-7C  show an isometric view and two top-plan views of an example beam selector  700 . The selector  700  includes a scanning mirror  702  and a plate  704  with an aperture  706 . In  FIG. 7A , the scanning mirror  702  includes a flat, fixed position mirror  708  attached to a motor  710  that translates the mirror  708  back and forth along a track  712 , as indicated by directional arrow  714 . As shown in  FIGS. 7B and 7C , the mirror  708  is oriented so that the beams B 1 , B 2 , B 3 , B 4 , B 5 , B 6  and B 7  output from the beam source  300  strike the mirror at 45° to the mirror normal. In practice, the mirror  708  can be placed at any suitable angle to reflect the beams toward the plate  704  and is not limited to a 45° angle with respect to paths of the beams. In  FIG. 7B , the mirror  708  is positioned so that all of the beams strike the mirror  708 , but only the beam B 3  is reflected off of the mirror  708  and passes through the aperture  706 , while the rest of beams are blocked by the plate  704 . In  FIG. 7C , the motor  710  has been used to translate the mirror  708  in the direction  716  so that the beam B 2  passes through the aperture  706 , while the other beams are blocked by the plate  704 . 
       FIG. 8A-8D  show an isometric view and three top-plan views of an example beam selector  800 . The selector  800  includes a transparent plate  802  attached to a motor  804  that rotates the mirror  802  back and forth, as indicated by directional arrows  806  and  807 . The transparent plate  802  can be composed of glass or a transparent plastic with a desired index of refraction, and the motor  804  can be a galvanometer motor or a stepper motor.  FIGS. 8B-8D  show the selector  800  includes an opaque plate  808  with an aperture  810 .  FIGS. 8B-8D  show how the transparent plate  802  is rotated to refract the parallel beams of light output from the beam source  300  so that one of the beams is output through the aperture  810 , while the other beams are blocked by the plate  808 . In  FIG. 8B , the transparent plate  802  is rotated so that the beam passes through the transparent plate  802  with normal incidence. In this position, the central beam B 4  passes through the aperture  810  while the other beams are blocked by the plate  808 . In the examples of  FIGS. 8C-8D , the transparent plate  802  is rotated so that the beams are refracted, as a result, the beams a shifted to that a beam other than the central beam B 4  passes through the aperture  810 . In  FIG. 8C , the transparent plate  802  is rotated to refract the beams with a beam off set that results in the beam B 5  passing through the aperture  810  while the other beams are blocked by the plate  808 . In  FIG. 8D , the transparent plate  802  is rotated farther resulting in a larger beam off set that places the beam B 6  on a path through the aperture  810  while the other beams are blocked by the plate  808 . 
     In alternative embodiments, the beam selector  400  can be combined with a beam launch that is used to control the irradiance of the beam selected.  FIG. 9  shows a schematic representation of the beam selector  400  combined with an example implementation of a beam launch  900 . In the example of  FIG. 9 , each beam is output from the parallel beam source  300  as a substantially monochromatic beam of light. The launch  900  includes the plate  408  with the circular aperture  410  and includes a lens  902 , and a single-mode optical fiber  904 . As shown in  FIG. 9 , the center of the aperture  410 , optical axis of the lens  902 , and optical axis of the fiber  904  are coincident as indicated by dot-dashed line  906 . The lens  902  is positioned along the optical axis  906  so that the focal point  908  of the lens  902  lies along the optical axis  906  and the diameter of the fiber  904  acceptance cone  910  at the lens  902  is approximately equal to the diameter of the aperture  410 . The diameter of the acceptance cone  910  is determined by the acceptance angle with the optical axis  906 , which is correlated with the numerical aperture of the fiber  904 . Light focused onto the focal point  908  lies within the acceptance cone  910  and is confined to the core  912 . In other words, the lens  902  shapes the beam of light output from the aperture  410  so that the light lies within the acceptance cone  910 . In order to achieve maximum coupling efficiency of the light into the fiber  904 , the diameter of each beam is tuned to approximately match the diameter of acceptance cone  910  of the fiber  904 . When the mirror  412  is rotated over a continuum of angles, the beam  510  is translated or shifted substantially perpendicular to the propagation direction of the beam  510 , which coincides with the optical axis  906 . As the beam  510  is translated substantially perpendicular to the optical axis  906 , a portion of the beam  510  is cut off by the plate  408  around the edge of aperture  410 , which, in turn, is used to control the irradiance of the beam that eventually enters the core  912 . The single-mode optical fiber  904  provides spatial filtering of the asymmetrical beams output from the lens  902 . For example, when the beam  510  is translated perpendicular to the optical axis  906 , the beam that reaches the core  912  has an asymmetrical irradiance distribution. Because the fiber  904  is a single-mode optical fiber, even though the beam  510  enters the fiber  904  with an asymmetrical irradiance distribution, the beam is output at the opposite end of the fiber  904  with a symmetrical irradiance distribution. 
     The foregoing description, for purposes of explanation, used specific nomenclature to provide a thorough understanding of the disclosure. However, it will be apparent to one skilled in the art that the specific details are not required in order to practice the systems and methods described herein. The foregoing descriptions of specific examples are presented for purposes of illustration and description. They are not intended to be exhaustive of or to limit this disclosure to the precise forms described. Obviously, many modifications and variations are possible in view of the above teachings. The examples are shown and described in order to best explain the principles of this disclosure and practical applications, to thereby enable others skilled in the art to best utilize this disclosure and various examples with various modifications as are suited to the particular use contemplated. It is intended that the scope of this disclosure be defined by the following claims and their equivalents: