Abstract:
Illumination phase controls that provide precise and fast phase control of structured illumination patterns used in structure illumination microscopy are described. A coherent light source is used to generate a beam of coherent light that is split into at least three coherent beams of light. In one aspect, an illumination phase control is composed of at least one pair of rotatable windows to apply at least one phase shift to at least one of the beams. An objective lens is to receive the beams and focus the at least three beams to form an interference pattern. The phase control can be used to change the position of the interference pattern by changing the at least one phase shift applied to the at least one beam.

Description:
CROSS-REFERENCE TO A RELATED APPLICATION 
     This application is a filing under 35 U.S.C. 371 of international application number PCT/SE2012/050227, filed Feb. 29, 2012, published on Sep. 7, 2012 as WO 2012/118436, which claims the benefit of Provisional Application No. 61/447,707; filed Mar. 1, 2011. 
    
    
     TECHNICAL FIELD 
     This disclosure relates to fluorescence microscopy and, in particular, to systems to control the phase of excitation light used to illuminate a specimen. 
     BACKGROUND 
     Precise phase control of light sources in microscope instruments is important for various optical techniques that involve interference of separate beams as well as polarization optics. For instance, phase control is used in phase contrast microscopy, differential interference microscopy, and polarized light microscopy. In particular, precise and fast phase control is used in structured illumination microscopy, in which phase control of interfering beams is accomplished with precise changes in path lengths of separate beam paths on the order of fractions of a wavelength, which for visible light corresponds to a change in the path length of 10 nanometers or less. Typically, a piezo-electric translation device with a mounted mirror can be used to generate precise phase control. As the mirror is translated parallel to the beam path, the path of the beam is lengthened or shortened by approximately 2 times the minor travel distance. Although the position of the mirror can be changed on order of 1-2 milliseconds, the devices are expensive and generate a detectable amount of beam translation except for a 0° angle of incidence. An alternative approach is to use an electronically deformable window to modulate the phase. A voltage applied to the window changes the thickness of the window by fractions of a wavelength, which results in a change in the effective path length of the light transmitted through the window. Unlike the piezo-electric device, the deformable window transmits the beam without beam translation. However, deformable windows are considerably slower with switching times on the order of tens to hundreds of milliseconds and it is difficult to achieve extremely low optical distortion, because the window is undergoing physical deformation. For the above described reasons, engineers, scientists, and microscope manufacturers continue to seek faster systems and methods for changing the phase in the light used to illuminate a specimen. 
     SUMMARY 
     Illumination phase controls that provide precise and fast phase control of structured illumination patterns used in structure illumination microscopy are described. A coherent light source is used to generate a beam of coherent light that is split into at least three coherent beams of light. In one aspect, an illumination phase control is composed of at least one pair of rotatable windows to apply at least one phase shift to at least one of the beams. An objective lens is to receive the beams and focus the at least three beams to form an interference pattern. The phase control can be used to change the position of the interference pattern by changing the at least one phase shift applied to the at least one beam. 
    
    
     
       DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows a schematic representation of an example three-dimensional structured illumination microscopy instrument. 
         FIGS. 2A-2C  show a representation of a three-dimensional structured-illumination pattern using three coherent beams. 
         FIGS. 3A-3E  show bright lines of an interference pattern stepped through five periods of a spatial interval. 
         FIGS. 4A-4C  show top views of an objective lens and three angular positions of an interference pattern. 
         FIGS. 5A-5C  show an example representation of an interference pattern rotator used to rotate three substantially parallel beams. 
         FIG. 6  shows an isometric view of an example implementation of an interference pattern rotator. 
         FIG. 7A  shows an enlarged view of an example illumination phase control. 
         FIG. 7B  shows an isometric view of an example rotatable window. 
         FIGS. 8A-8B  show a pair of transparent plates of two rotatable windows. 
         FIG. 9  shows a snapshot of example phase shifts acquired by two beams of light transmitted through an illumination phase control. 
         FIGS. 10A-10C  show an illumination phase control reconfigured to change the position of a structure illumination pattern. 
         FIG. 11  shows an example illumination phase control implemented with a single pair of rotatable windows. 
         FIG. 12  shows an example illumination phase control implemented with two separate rotatable windows. 
     
    
    
     DETAILED DESCRIPTION 
     Various illumination phase controls (“IPCs”) are described along with a general description of three-dimensional structured illumination microscopy (“3D-SIM”). 3D-SIM achieves a factor of two improvement in lateral and axial resolution compared to conventional wide-field fluorescence microscopes used in cell biology. 3D-SIM requires no specialized fluorescent dyes or proteins, unlike certain competing super-resolution techniques. Biologists achieve high resolution with 3D-SIM, but retain convenient and familiar fluorescence labeling techniques. The illumination phase control provides for capturing multiple images of the subject by a shifting and rotating illumination pattern. Higher resolution can be achieved by solving a system of equations to restore the fine spatial detail normally blurred by diffraction. 
       FIG. 1  shows a schematic representation of an example 3D-SIM instrument  100 . There are many different types of SIM instruments and corresponding optical paths. Instrument  100  is not intended to represent the optical paths within all the different, well-known variations of instruments used in SIM microscopy, but is instead intended to illustrate the general principals of a SIM that includes an IPC. A high-intensity, substantially monochromatic beam  102  of coherent light is output from a light source  104 , such as a laser, and transmitted through a lens or a series of lenses  106  that collimate the beam  102 . The beam  102  can be output from the source  104  with a particular polarization. The beam  102  then passes through a splitter  108  that splits the beam into three separate coherent beams  110 - 112 . For example, the splitter  108  can be a one-dimensional, transmissive diffraction grading that splits the light into three divergent, co-planar (i.e., xz-plane) coherent beams  110 - 112  referred to as the 0 th , +1 st , and −1 st  order diffracted beams, respectively. The splitter  108  can be any one of a variety of different types of transmissive gratings. For example, the splitter  108  can be a one-dimensional transmissive grating composed of a transparent plate of glass with a series of substantially parallel grooves formed in one surface of the grating or the splitter  108  can be an opaque plate with a series of substantially parallel thin slits. Alternatively, the splitter  108  can be two or more beamsplitters arranged to split the beam of light output from the lens  106  into three or more separate coherent beams. The three beams  110 - 112  pass through a lens or a series of lenses  114  which reorient the beams  110 - 112  so that the beams lie in the xz-plane and the +1 st  and −1 st  order diffracted beams  111  and  112  are nearly parallel to the 0 th  order diffracted beam  110 . In the example of  FIG. 1 , the beams  111  and  112  pass through an IPC  116  that controls the phase of one or more of the beams  110 - 112 . The beams  110 - 112  then pass through an illumination pattern rotator (“IPR”)  124  and are reflected off of a dichroic mirror  126  to enter an objective lens  128 . In the example of  FIG. 1 , the beams  110 - 112  are focused within a specimen  130  so that the beams interfere with one another to generate a high-contrast, three-dimensional structured-illumination pattern within a volume of the specimen  130 . 
       FIGS. 2A-2C  show generation of a three-dimensional structured-illumination pattern using three coherent beams. As shown in  FIG. 2A , the beams  110 - 112  are transmitted into the back of the objective lens  128 . Because the beams  110 - 112  originate from a coherent light source  102 , the beams  110 - 112  have plane waves in which the phases of component waves of the beams are identical across any plane, such as plane  202 , normal to the beam direction. While the beams  110 - 112  are coherent, each beam may have a different phase displacement than the other two beams. The objective lens  128  focuses the incident beams to a focal point  204 , which changes the direction of the two non-axial beams  111  and  112 , as shown in  FIG. 2A . As a result, the three plane waves are no longer parallel with wave vectors having different directions and the three sets of plane waves intersect to form a three-dimensional pattern of bright lines, due to constructive interference, surrounded by dark regions, due to destructive interference. In other words, as shown in the example of  FIG. 2B , a stationary interference pattern  206  is located in the focal plane of the objective lens  128 . Lines  208  represent bright lines of excitation light separated by darker regions. The lattice of bright lines of the excitation light comprise the interference pattern  206  cause fluorescent emission of light from fluorophores in the specimen  130  (not shown).  FIG. 2C  shows a side view of the objective lens  128  and an end-on view of the bright lines comprising the three-dimensional interference pattern  206 . Open circles  210  represent an end-on view of the bright lines of excitation light separated by darker regions  212 . Each bright line excites fluorescence of fluorophores attached to components of the specimen  220  that intersect the bright line. Fluorophores attached to components of the specimen  220  that are located in the dark regions  212  between the bright lines  210  do not fluoresce. 
     Returning to  FIG. 1 , the objective lens  128  captures and directs a portion of the fluorescent light emitted from the fluorophores to the dichroic minor  126 . The fluorescent light passes through the dichroic mirror  126  to filter and imaging optics  132 , which filters stray excitation light and focuses the fluorescent light onto a sensor of a detector  134 . For example, the detector  134  can be a photodetector array, CCD camera, or a CMOS camera. 
     3D-SIM image data is acquired by taking a fluorescence image excited by the interference pattern, moving the interference pattern by a period perpendicular to the optical axis of the objective lens  128 , followed by taking another image, and repeating these steps for a total of n images. The splitter  112  can be translated in the x-direction, shown in  FIG. 1 , to step the interference pattern perpendicular to the optical axis of the objective lens by a period.  FIGS. 3A-3E  show an example of stepping the interference pattern  206  through five periods of a spatial interval centered about the optical axis  302  of the objective lens  128 . In the example of  FIGS. 3A-3E , the bright lines of the interference pattern  206  are directed substantially perpendicular to the z-axis, as described above with reference to  FIG. 2 . Marks labeled  1 - 5  in  FIGS. 3A-3E , represent five periods of a spatial interval centered about the optical axis  302 . Dotted line  304  identifies the center of the interference pattern  206 .  FIGS. 3A-3E  represent five discrete steps in which the interference pattern  206  is translated substantially perpendicular to the optical axis  302 . For example, in  FIG. 3A , the interference pattern  206  is in the first period denoted by “1,” and in  FIG. 3B , the interference pattern  206  is stepped to the second period denoted by “2.” At each of the five periods represented in  FIGS. 3A-3E , a fluorescence image excited by the interference pattern  206  is captured. The final step rewinds to reset the interference pattern for the next cycle. 
     The interference pattern  206  is then rotated in the xy-plane, and the n-step image process is repeated, followed by another rotation and the capture of another n images, for a total of 3n images. After the 3n images are obtained, the interference pattern  206  is moved in the z-direction and another set of 3n images is obtained.  FIGS. 4A-4C  show top views (i.e., xy-plane) of the objective lens  128  and the interference pattern  206 . In  FIG. 4A , the bright lines of the interference pattern  206  are initially angled at −60°. The interference pattern  206  is stepped through five periods of a spatial interval, as described above with reference to  FIG. 3 , with a fluorescent image captured at each step. In  FIG. 4B , the interference pattern  206  is rotated through 60° so that the bright lines are angled at 0°. The interference pattern  206  is again stepped through five periods of a spatial interval, as described above with reference to  FIG. 3 , with a fluorescent image captured at each step. In  FIG. 4C , the interference pattern  206  is finally rotated through an additional 60° so that the bright lines are angled at 60°. The interference pattern  206  is again stepped through five periods of a spatial interval, as described above with reference to  FIG. 3 , with a fluorescent image captured at each step. The interference pattern  206  is rotated 3 times in the xy-plane by 60°, with each rotation followed by the capture of 5 images for a total 15 images. These fluorescent images are used to solve a system of linear equations to recover a three-dimensional optically sectioned image with approximately double the resolution obtained by conventional wide-field microscopy. 
     The IPR  124  is used to rotate the interference pattern in the yz-plane about the optical axis  302 .  FIGS. 5A-5C  show use of the IPR  124  to rotate the beams  110 - 112  and polarization associated with each of the beams  110 - 112 . In  FIG. 5A , the IPR  124  preserves the orientation of the beams  110 - 112  so that the beams  110 - 112  travel in the xz-plane with the polarization associated with each beam oriented perpendicular to the x-axis. In  FIG. 5B , the IPR  124  rotates the beams  110 - 112  through an angle φ about the central 0 th  order diffracted beam axis (i.e., z-axis) and rotates the polarization associated with each beam through the same angle. In  FIG. 5C , the IPR  124  rotates the beams  110 - 112  through an angle θ about the central 0 th  order diffracted beam axis and rotates the polarization associated with each beam through the same angle. 
       FIG. 6  shows an isometric view of an example IPR  600 . The IPR  600  includes a scanning mirror  602  and three separate minor clusters  604 - 606 .  FIG. 6  shows an example of scanning mirror  602  that includes a flat minor  610  attached to a rotatable shaft of a motor  612 . The motor  612  can be galvanometer, in which case the scanning minor  602  is a galvanometer mirror, or the motor  612  can be a stepper motor that divides rotation of the minor  610  into a series of rotational steps or any other kind of motor that imparts precise rotation of the mirror  610 . Alternatively, the scanning mirror can be a piezoelectric controlled minor. As shown in  FIG. 6 , the reflective surface of the mirror  610  is rotated using the motor  612  to in turn face each of the minor clusters  604 - 606 . Three parallel lines  614  represent the 0 th  and ±1 st  order diffracted beams output from a grating, such as the splitter  304  described above. As shown in  FIG. 6 , three rotational positions  1 ,  2  and  3  of the minor  610  are identified. Positions  1 ,  2  and  3  correspond to particular rotational positions of the mirror  610  with respect to minor clusters 1, 2 and 3. Each of the mirror clusters imparts a different angle of rotation to the beams  614  and matching angle of rotation in the polarization associated with the beams. The IPR  600  is operated as follows. When the minor  610  is rotated into position  1 , the beams  614  are reflected off of the mirror  610  toward mirror cluster 1  604  as represented by directional arrow  616 . Mirror cluster 1  604  rotates the beams and associated beam polarizations through a first rotational angle and reflects the beams back to the minor  610  as represented by directional arrow  617 . When the minor  610  is rotated into position  2 , the beams  614  are reflected off of the minor  610  toward minor cluster 2  605  as represented by directional arrow  618 . Minor cluster 2  605  rotates the beams through a second rotational angle and reflects the beams back to the minor  610  as represented by directional arrow  619 . When the minor  610  is rotated into position  3 , the beams  614  are reflected off of the minor  610  toward minor cluster 3  606  as represented by directional arrow  620 . Mirror cluster 3  606  rotates the beams through a third rotational angle and reflects the beams back to the minor  610  as represented by directional arrow  621 . Directional arrow  622  represents the rotated beams reflected off of the minor  610 .  FIG. 6  includes a view  624  of the reflective surface of the mirror  610  that represents co-alignment of the rotated beams reflected off of the surface of the minor  610 . Dashed lines  626 - 628  represent the three different rotations imparted on the three beams by the minor clusters  604 - 606  as viewed from the reflective surface of the minor  610 . Dashed line  626  represents the orientation of the beams produced by mirror cluster 1  604 , dashed line  627  represents the orientation of the beams produced by mirror cluster 2  605 , and dashed line  628  represents the orientation of the beams produced by mirror cluster 3  606 . The IPR  600  may also include an exit path mirror located in the path of the beam  622  to provide additional control over the direction in which the beams are output from the IPR  600 . 
       FIG. 7A  shows an enlarged view of an example implementation of an IPC  700  located between the lens  114  and the IPR  124  as shown in  FIG. 1 . The beams  110 - 112  are output from the lens  114  along substantially parallel paths, and the beams are focused by the lens  114  to focal points  701 - 703 , respectively. As shown in the example of  FIG. 7A , the system  700  is composed of four separate rotatable windows  705 - 708 . Two of the rotatable windows  705  and  706  intersect the outside beam  111  and are located on opposite sides of the focal point  702 . The other two rotatable windows  707  and  708  intersect the other outside beam  112  and are also located on opposite sides of the focal point  703 .  FIG. 7B  shows an isometric view of an example rotatable window  710  that includes a flat transparent plate  712  attached to a rotatable shaft of a motor  714 . The transparent plate  712  can be composed of glass or another suitable transparent material, and the motor  714  can be a galvanometer or a stepper motor that divides rotation of the plate  712  into a series of rotational steps or any other kind of motor that can be used to rapidly rotate the plate  712 . 
     The transparent plates of each pair of rotatable windows intersect one of the beams and when rotated change the path length of the beam, which, in turn, results in a change in the relative phase of the beams that are focused to form an interference pattern. The transparent plates of a pair of rotatable windows are rotated with the same angular magnitude but in opposite directions in order to maintain the path of the beam.  FIGS. 8A-8B  show a pair of transparent plates  801  and  802  of two rotatable windows. In the example of  FIG. 8A , when the plates  801  and  802  are parallel and oriented perpendicular to a collimated beam of light transmitted from a point A to a point B represented by a dashed-line ray  804 . The beam travels an overall distance, D, with a minimum distance, d, traveled through each of the plates  801  and  802 . In the example of  FIG. 8B , the same plates  801  and  802  are rotated in opposite directions with the same angular magnitude θ. Dashed line  806  represents the initial path the beam travels until the beam reaches the rotated plate  801 . The beam is refracted by the rotated plate  801  and placed on a path  808  substantially parallel to the initial path  806 . The beam is then refracted again by the rotated plate  802  and is placed on a path  810  that is aligned with the initial path  806 . In other words, by rotating the plates with the same angular magnitude but in opposite directions, the initial path of the beam is maintained. However, because the plates  801  and  802  are rotated, the beam is refracted. As a result, the beam travels a longer distance, d′, through each of the plates  801  and  802  (i.e., d′&gt;d) which results in a relatively larger phase shift than when the beam travels through parallel plates  801  and  802 , as shown in  FIG. 8A . In other words, as the beam travels from point A to point B through the parallel oriented plates  801  and  802  shown in  FIG. 8A , the amount by which the phase of the beam is retarded depends on the refractive index of the plates  801  and  802  and on the distance d. As a result, the beam acquires a phase shift that is proportional to 2d and the refractive index of the plates. However, when the plates  801  and  802  are rotated as shown in  FIG. 8B , the longer distance d′ the beam travels through each of the plates  801  and  802  results in an even larger phase shift. For example,  FIGS. 8A-8B  also represents snapshots of an exaggerated segment  812  of an electromagnetic wave of wavelength λ at an initial time t 0  and a later time t 1 . In  FIG. 8A , at time t 0 , a minimum  814  of the segment  812  is located at the point A, and at the later time t 1 , the same minimum  814  reaches the point B. On the other hand, in  FIG. 8B , at the initial time t 0 , the minimum of the segment  812  is located at the point A, but at the later time t 1 , the same minimum  814  is λ/4 from the point B, which corresponds to a phase shift of φ=π/2. 
       FIG. 9  shows a snapshot of example phase shifts applied to the beams  110 - 112  based on rotating the plates of the rotatable windows  705  and  706  in opposite directions with angular magnitude α and rotating the plates of the rotatable windows  707  and  708  in opposite directions with angular magnitude β. In the example of  FIG. 9 , in order to illustrate the rotatable windows  705 - 708  creation of different relative phase shifts in the beams  111  and  112 , segments  901 - 903  of electromagnetic waves associated with the beams  110 - 112  are show as entering the IPC  700  with no phase separation, as indicated by a dashed line  904  that intersects each of segments at the same point. At a later time, when the segments  901  and  903  have passes through the rotatable windows  705 - 708  as described above with reference to  FIG. 8 , the segment  901  has acquired a relative phase shift of φ′, and the segment  903  has acquired a relative phase shift of φ″, with respect to the beam  110 . 
     An IPC, such as the IPC  700 , described above provides switching times on the order of 0.2 milliseconds. When a transparent plate is in a high-precision range of approximately 1° of normal incidence, the effective path length can be adjusted with a precision on the order of 0.1 nanometers. 
     The transparent plates of the rotatable windows are rotated into particular angles to step the interference pattern through periods along a spatial interval as described above with reference to  FIG. 3 .  FIGS. 10A-10C  show examples of three hypothetical sets of angles the transparent plates of the rotatable windows are rotated into to center the interference pattern  206  in the periods  1 ,  2  and  3  described above with reference to  FIG. 3 . In the example of  FIG. 10A , the plates of the windows  705  and  706  at 0°, and the plates of the windows  707  and  708  are rotated with maximum angles of rotation −β and β, respectively, to center the interference pattern  206  within period  1 . In the example of  FIG. 10B , the plates of the windows  705  and  706  are rotated with angles α and −α, respectively, and the plates of the windows  707  and  708  are rotated to smaller angles −γ and γ, respectively, which creates relative phase differences between the beams  110 - 112  to center the interference pattern  206  within period  2 . In the example of  FIG. 10C , the plates of the windows  705  and  706  are rotated to angles φ and −φ, respectively, and the plates of the windows  707  and  708  are rotated to 0°, which creates relative phase differences between the beams  110 - 112  to center the interference pattern  206  within period  3 . 
     IPCs are not limited to two pairs of rotatable windows located along the two outside beams, as described above with reference to  FIG. 7 . In alternative embodiments, an IPC can be implemented with three pairs of rotatable windows, where each pair of rotatable windows is located along one of the three beams. For example, the IPC  700  can be modified to include a third pair of rotatable windows that intersect the beam  110  in the same manner the rotatable windows  705 - 708  are positioned to intersect the beams  111  and  112 . Alternatively, an IPC can be implemented with as few as a single pair of rotatable windows located along any one of the beams between the lens  114  and the IPR  124 , as shown in the example of  FIG. 11  where a single pair of rotatable windows  1102  and  1104  intersects the beam  111 . Alternatively, an IPC can be implemented with one pair of rotatable windows that intersection the central beam  110  and a second pair that intersects either one of the outside beams  111  and  112 . In alternative embodiments, an IPC can be implemented with only a single rotatable window placed in each one, two or all three of the beam paths. For example, in  FIG. 12 , an IPC is composed of rotatable windows  1202  and  1204  that intersect beams  111  and  112 . Note that when only one rotatable window is disposed in a beam path to control the relative phase of the beams, as the transparent plate is rotated to steeper and steeper angles of incidence, sensitivity increases and the precision of phase control may be degraded. In addition, there is a small amount of beam translation, as described above with reference to  FIG. 8B . 
     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: