Abstract:
There is provided a method that includes projecting a collimated light beam from an optical system to a plane during a first mode of operation of the optical system, and projecting a convergent light beam from the optical system to the plane during a second mode of operation of the optical system. The method further includes, (a) during the first mode of operation, controlling a trajectory of a first light bundle in a first light path in the optical system, to steer the collimated light beam through the plane at a designated incidence angle, and (b) during the second mode of operation, controlling a trajectory of a second light bundle in a second light path of the optical system, to steer the convergent light beam to a target position in the plane. There is also provided an apparatus and a system that employs the method.

Description:
GOVERNMENT LICENSE RIGHTS 
     This invention was made with government support under OD002980 awarded by the National Institute of Health. The government has certain rights in the invention. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present disclosure relates to illumination systems, and more specifically, to an illumination system that selectively provides either of a collimated light beam or a convergent light beam. The system is particularly well-suited where illumination is desired for both total internal reflection fluorescent (TIRF) microscopy and fluorescence recovery after photobleaching (FRAP) or photoactivation experiments. 
     2. Description of the Related Art 
     The approaches described in this section are approaches that could be pursued, but not necessarily approaches that have been previously conceived or pursued. Therefore, unless otherwise indicated, the approaches described in this section may not be prior art to the claims in this application and are not admitted to be prior art by inclusion in this section. 
     A fluorescence microscope is a light microscope used to study properties of organic or inorganic substances using the phenomena of fluorescence and phosphorescence instead of, or in addition to, reflection and absorption. In some cases, for example, fluorescence recovery after photobleaching (FRAP), an image plane of a microscope is best illuminated by a converging beam of light. In other cases, for example, total internal reflection fluorescence (TIRF), the image plane is best illuminated by a collimated beam of light. 
     SUMMARY OF THE INVENTION 
     There is provided a method that includes projecting a collimated light beam from an optical system to a plane during a first mode of operation of the optical system, and projecting a convergent light beam from the optical system to the plane during a second mode of operation of the optical system. The method further includes, (a) during the first mode of operation, controlling a trajectory of a first light bundle in a first light path in the optical system, to steer the collimated light beam through the plane at a designated incidence angle, and (b) during the second mode of operation, controlling a trajectory of a second light bundle in a second light path of the optical system, to steer the convergent light beam to a target position in the plane. There is also provided an apparatus and a system that employs the method. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a block diagram of an illumination system. 
         FIG. 2  is an illustration of the operation of the system of  FIG. 1  in a mode of operation that projects a collimated light beam to a plane. 
         FIG. 3  is an illustration of the operation of the system of  FIG. 1  in a mode of operation that projects a convergent light beam to a plane. 
         FIG. 4  is an illustration of an elliptical zone mirror. 
         FIG. 5  is a rough grid scan using a whole image integrated intensity as an indicator of the incidence angle of a collimated beam. 
         FIG. 6  is an illustration showing that slowly scanning galvanometers along certain trajectories could be used to identify a critical angle. 
         FIG. 7  is a block diagram of an illumination system. 
         FIG. 8  is an illustration of the operation of the system of  FIG. 7  in a mode that projects a collimated light beam to a plane. 
         FIG. 9  is an illustration of the operation of the system of  FIG. 7  in a mode that projects a convergent light beam to a plane. 
         FIG. 10  is a block diagram of a system for microscopy. 
     
    
    
     DESCRIPTION OF THE INVENTION 
     A component or a feature that is common to more than one drawing is indicated with the same reference number in each of the drawings. 
       FIG. 1  is a block diagram of an illumination system, i.e., a system  100 , that has a mode of operation that projects a collimated light beam to a plane  199 , and a mode of operation that projects a convergent light beam to plane  199 . For convenience, we refer to the mode of operation that projects the collimated light beam to plane  199  as “collimated mode”, and we refer to the mode of operation that projects the convergent light beam to plane  199  as “convergent mode.” 
     System  100  includes a light source  105 , a light steering device  110 , lenses  115 ,  130 ,  150  and  195 , mirrors  145  and  190 , a diffuser  175 , and a light steering device  185 . 
     Light source  105  emits a light beam  107 . Light source  105  may be implemented as a laser, for example, and preferably emits light beam  107  as a collimated beam. 
     Light steering device  110  receives light beam  107  from light source  105 . Light source  105  may be coupled to light steering device  110  with a fiber optic cable (not shown) and a collimator (not shown), but it can also be directly coupled using mirrors (not shown). 
     Light steering device  110  selectively directs the light to one of two light paths generally designated as light path  112  and light path  113 , and more particularly, directs the light to light path  112  for convergent mode, and directs the light to light path  113  for collimated mode. Light steering device  110  may include, for example, a galvanometer scanning mirror or an acousto-optical deflector. 
     Light path  112  runs from light steering device  110 , through lens  115 , through lens  130 , to mirror  145 , and then to light steering device  185 . Light path  113  runs from light steering device  110 , through lens  150 , through diffuser  175 , and then to light steering device  185 . 
     Light steering device  185  receives the light via either light path  112 , from mirror  145 , or via light path  113 , from diffuser  175 , and emits the light to a downstream light path, i.e., a light path  187 . Light steering device  185  may include, for example, an elliptical zone mirror (EZM), an elliptical mirror, a round mirror, a mirror having a clear aperture, a beam combiner, a beam splitter, a beam cube, or a polarizing beam cube. Light path  187  runs from light steering device  185 , to mirror  190 , through lens  195 , and then to plane  199 . 
     In convergent mode, the light propagates via light paths  112  and  187 , and system  100  produces the convergent light beam in plane  199 . In collimated mode, the light propagates via light paths  113  and  187 , and system  100  produces the collimated light beam in plane  199 . 
     An angle at which light steering device  110  directs the light to light path  112  or to light path  113  influences an angle at which the light arrives at plane  199 . In convergent mode, by controlling a trajectory of the light into light path  112 , light steering device  110  steers the convergent light beam to a target position in plane  199 . In collimated mode, by controlling a trajectory of the light into light path  113 , light steering device  110  steers the collimated light beam through plane  199  at a designated incidence angle. 
     For two-dimensional, i.e., x-direction and y-direction, steering of the light, light steering device  110  may be implemented as an orthogonal pair of galvanometer scanning mirrors or a dual axis acousto-optical deflector. Galvanometer scanning mirrors have a response time of less than a millisecond, and can therefore be used for small beam apertures and moderate angular steps. Thus, galvanometer scanning mirrors can rapidly move light beam  107  to a specified exit angle coordinate. Depending on the exit angle specified by the light steering device  110  the system  100  can illuminate either a focused spot, i.e., the target position, within plane  199 , or can project a collimated beam through plane  199  at the designated incident angle. Also, if no other means of beam modulation is available, and if desired, light steering device  110  could be used to block light beam  107  by directing it to a beam stop (not shown). 
     System  100  can be employed as an illumination system for a microscope, where plane  199  coincides with an image plane of the microscope. Collimated mode can be employed in conjunction with processes such as (a) total internal reflection fluorescent illumination, and (b) evanescent field fluorescence recovery after photobleaching illumination. Convergent mode can be employed in conjunction with processes such as (a) fluorescence recovery after photobleaching illumination, (b) photoactivation illumination, (c) photobleaching illumination, (d) an optical tweezers operation, and (e) an optical uncaging operation. 
     Utilizing a shared source beam and a shared two-axis beam steering device, system  100  creates two different illumination conditions at a common destination plane, i.e., plane  199 : Incidence angle addressable collimated illumination and position addressable focal illumination. 
     In collimated mode, system  100  directs the collimated beam through the center of plane  199 . In this mode the angular direction coordinates of the collimated beam exiting light steering device  110  determine the incidence direction of the collimated beam intersecting plane  199 . 
     In convergent mode, system  100  directs the converging beam of light to a focal spot in plane  199 . In this mode the angular direction coordinates of a collimated beam exiting light steering device  110  determine the lateral position of the focal spot within plane  199 . 
     The mode of operation is determined by selection with light steering device  110 , of the corresponding range of angular direction coordinates. The angular ranges applicable to each mode of operation are non-overlapping. Light steering device  110  is controlled electronically, and may utilize feedback and analysis to optimize the resultant illumination. 
     For convenience, below, we are using the phrase “light bundle” to designate a segment of light. 
       FIG. 2  is an illustration of the operation of system  100  in collimated mode. In collimated mode, light steering device  110  receives a light bundle from light source  105  and directs the light bundle to light path  113 . The light bundle propagates (a) from light steering device  110  to lens  150  in a collimated beam  205 , (b) through lens  150  to diffuser  175  in a convergent beam  210 , (c) through diffuser  175  to light steering device  185  in a divergent beam  215 , (d) from light steering device  185  to mirror  190  in a divergent beam  220 , (e) from mirror  190  to lens  195  in a divergent beam  225 , and (f) from lens  195  to plane  199  in a collimated beam  230 . 
     Lens  150  focuses collimated beam  205  onto diffuser  175  as convergent beam  210 . Diffuser  175  is situated at a plane  170  and rotated by a rotator  165 . Diffuser  175  creates an even illumination by collimated beam  230  in plane  199 . Lens  150  and diffuser  175  may be regarded as an optical subsystem that transforms the light bundle so that the light bundle is projected to plane  199  in collimated beam  230 . Light steering device  185  receives the light bundle in divergent beam  215 , and directs the light bundle to mirror  190  in divergent beam  220 . Mirror  190  reflects, and thus directs, the light bundle to lens  195  in divergent beam  225 . Lens  195  receives the light bundle in divergent beam  225 , and projects the light bundle, in collimated beam  230 . Light steering device  110  controls a trajectory of the light bundle in light path  113  to steer collimated beam  230  through plane  199  at a designated incident angle. 
     In collimated mode, light steering device  110  and plane  199  are located at the pupils of a telescope formed by lenses  150  and  195 . Thus, any beam being projected by light steering device  110  will be centered on the intersection of plane  199  and the optic axis. It is the approach angle with respect to the optic axis that is varied by small deflections near path  113  that are relayed by the optics to plane  199 . 
     The distance from light steering device  110  to lens  150  is equal to the focal length of lens  150 , and so, the convergent beam into plane  170  is telecentric. The distance between diffuser  175  and lens  195  equals the focal length of lens  195 , resulting in telecentric motion of the collimated beam along path  230  and a stationary intersection of the collimated beam at the center of plane  199 . 
     The collimation of collimated beam  230  can be adjusted, as indicated by an arrow  155 , by moving lens  150  and diffuser  175 , which are mounted on a common translation mount  160  for this purpose. 
     When system  100  is being employed with a microscope, it is configured such that plane  199 , i.e., the exit plane, is located coincident with a conjugate plane to the focal plane of the microscope objective lens. The objective lens focal plane is often called the specimen plane and it is the in-focus plane of a specimen that is imaged by the microscope. The conjugate plane is generally a magnification of the microscope objective focal plane. 
     Collimated beams incident on the conjugate plane are relayed through the microscope intermediate optics and the microscope objective lens and exit as collimated beams through the microscope objective lens focal plane. Thus to obtain a desired collimated beam exit direction from the microscope objective the appropriate beam direction about the collimated light path  113  is selected. 
     TIRF microscopy is used to image a thin section of a specimen by utilizing the very thin evanescent field created when an interface between a high optical index material and a low optical index material is illuminated at an incidence angle greater than the critical angle (the angle of total internal reflection). This illumination condition is called illumination at a supercritical angle of incidence or simply supercritical illumination. 
     In a typical ‘through the objective’ TIRF microscope arrangement, a specimen of interest is placed on a thin glass cover-slip that is placed in the focal plane of a microscope objective with the specimen on the surface facing away from the microscope objective. The cover-slip is illuminated with collimated light at a supercritical angle to create the thin evanescent field along the surface of the cover-slip facing away from the microscope objective. 
     To create the illumination conditions for TIRF microscopy with the system  100  coupled to a microscope, the appropriate beam direction about the collimated light path  113  is selected in order to relay a collimated beam out of the microscope objective at a supercritical angle of incidence with respect to the cover-slip. 
     To fully homogenize and eliminate artifacts from the illumination, it is better to illuminate the specimen focal plane from all sides while maintaining the same inclination angle. This is accomplished by scanning collimated beam  205  in a circular path  202  about the optic axis  113 . This collimated beam is relayed to the cover-slip as previously discussed. 
       FIG. 3  is an illustration of the operation of system  100  in convergent mode. Light steering device  110  receives a light bundle from light source  105  and directs the light bundle to light path  112 . The light bundle propagates from (a) light steering device  110  to lens  115  in a collimated beam  305 , (b) through lens  115  to a plane  125  in a convergent beam  310 , (c) from plane  125  to lens  130  in a divergent beam  315 , (d) from lens  130  to mirror  145  in a collimated beam  320 , (e) from mirror  145 , through a plane  180 , and through light steering device  185  to mirror  190  in a collimated beam  325 , (f) from mirror  190  to lens  195  in a collimated beam  330 , and (g) from lens  195  to plane  199  in a convergent beam  335 . Lens  115 , lens  130  and mirror  145  may be regarded as an optical subsystem that transforms the light bundle so that the light bundle is projected to plane  199  in convergent beam  335 . Light steering device  110  controls a trajectory of the light bundle in light path  112  to steer convergent beam  335  to a target position in plane  199 . 
     When system  100  is being used with a microscope for FRAP/photoactivation illumination, system  100  is in convergent mode and as such, light steering device  110  is set to direct light on or near light path  112 . Directions on this optic axis correspond to bleaching locations at a center of the microscope field of view. Collimated beam  305  passes through lens  115  to become convergent beam  310 , which comes to a focus at plane  125 , and is re-collimated by lens  130 . Lens  130  is on a translation mount that moves as indicated by an arrow  135 , to enable a user to focus convergent beam  335  into the image plane of the microscope. This adjustment is independent of adjustments to the light path in the collimated mode ( FIG. 2 ). Mirror  145  directs collimated beam  325  through a stationary pupil location at plane  180 . The light then passes through light steering device  185 , to mirror  190 . Collimated beam  330  is received by lens  195  as an intermediate collimated light beam, and then focused by lens  195 , as convergent beam  335 , onto plane  199 , which is located at the image plane of the microscope. 
     The pupil of light steering device  110  and the pupil of lens  115  are separated by the focal length of lens  115  such that the focus at plane  125  moves telecentricly. Similarly, plane  125  and lens  130  are separated by the focal length of lens  130 , and lens  130  and lens  195  are separated by a distance equal to the sum of those focal lengths. Thus, system  100  has telecentric performance throughout. 
     During an experiment, a beam could be directed to a series of discrete locations within a field of view in plane  199 , or it could be scanned in a raster motion to bleach an area. The position of the beam in plane  199  is dependent on an angle at which light is directed from light steering device  110  to light path  112  or light path  113 . The motion would be controlled by a computer, and in a case where light source  105  is a laser, the laser power could be switched or otherwise modulated in conjunction with this if such a capability is implemented on the laser (e.g. using an AOTM). 
     As noted above, light steering device  185  may include an elliptical zone mirror (EZM).  FIG. 4  is an illustration of an EZM  400  having a mirrored surface  405  and a clear aperture, i.e., an aperture  410 , for a circular beam incident at  45  degrees. Consider a case of light steering device  185  being implemented with EZM  400 . In the collimated mode of system  100  (see  FIG. 2 ), the light bundle in divergent beam  215  is reflected by mirrored surface  405 , and thereafter continues in divergent beam  220 . In the convergent mode of system  100  (see  FIG. 3 ), the light bundle in collimated beam  325  propagates from mirror  145 , and passes through aperture  410  to mirror  190 . 
     Referring again to  FIG. 2 , consider a case where steering device  185  is implemented with EZM  400 , and system  100  is in collimated mode and being used for TIRF illumination. Note that aperture  410  does not affect the TIRF illumination since it is located near plane  170 . Plane  170  is a conjugate plane to the entrance pupil of the objective lens. The TIRF illumination conditions require that the light be focused at the periphery of the objective lens aperture. Aperture  410  is sized so that divergent beam  215  does not strike aperture  410 , but instead strikes mirrored surface  405 . 
     Referring again to  FIG. 3 , consider a case where steering device  185  is implemented with EZM  400 , and system  100  is in convergent mode and being used for FRAP illumination. Mirrored surface  405  has a minimal effect on the FRAP performance. Mirrored surface  405  is located near plane  180  at which collimated beam  325  is both on-axis and stationary. Aperture  410  limits the numerical aperture of the FRAP focus somewhat, but since in the experiments, regions are normally bleached, diffraction limited performance is not required. 
     Optical Variations 
     It should be noted that although a telecentric optical arrangement results in the optimal illumination conditions, it is not an absolute necessity. Practical implementations of the system will by necessity be imperfectly telecentric to accommodate the movement of optics for focusing, collimation adjustment, as well as the physical constraint of having two axis of light steering which may not occur at precisely the same location along the beam path. 
     Rapid switching of the illumination can be accomplished simply by directing the light bundle from light steering device  110  to a location other than the collimated beam path  113  or the converging beam path  112 . The light could be directed to a beam dump placed at an intermediate position. 
     Diffuser  175  is used to increase the area of illuminate in the collimated mode. It should be noted that diffuser  175  is not an essential component to system  100  and most practical implementations will not require it. 
     EXAMPLE 
     Consider an example of a 60× objective with numerical aperture 1.45. Such a lens has the following parameters: 
     Beam acceptance aperture=8.7 mm. (assuming a 200 mm tube length) 
     Oil immersion index (n3)=1.518 
     Tissue index (n1)=1.38 
     Critical angle for TIRF=63.6 degrees (based on n1 and n3) 
     Critical diameter at objective pupil (aperture)=8.16 mm 
     For such a lens the diameter of aperture  410  could be 5 mm. This would allow a 3 mm FRAP beam some scanning room (since EZM  400  is not located exactly at the pupil at plane  170 ). The TIRF beams have some room for divergence after the focal point, and can still illuminate at sub-critical (&lt;8.16 mm), critical (8.16 mm) and supercritical (&gt;8.16) positions. These relationships assume that lens  195  has equal focal length to the tube lens of the microscope such that the magnification of the combined relay telescope is 1. Otherwise, a scaling factor should be applied. 
     Calibration of Galvanometer Positions 
     This discussion assumes that a CCD or other camera is being used to monitor an experiment and that the images from that camera are available for analysis. 
     Galvo and CCD Parameters
         Galvanometer position coordinates: (G x , G y )   CCD camera position coordinates: (C x , C y )       

     FRAP Transform Coordinates:
         Galvo Origin Position: (G x0 , G y0 )   Galvo position derivatives: (G x dC x , G y dC x ), (G x dC y , G y dC y )       

     Transforms from CCD camera coordinates to Galvo coordinates for FRAP
 
 G   x   =G   x0 +( G   x   dC   x   ,G   x   dC   y )·( C   x   ,C   y )
 
 G   y   =G   y0 +( G   x   dC   x   ,G   y   dC   y )·( C   x   ,C   y )
 
     TIRF Transform Coordinates:
         Galvo coordinates for TIRF axis: (T x0  T y0 )       

     Automated Calibration of FRAP Galvanometer Positions 
     A simple linear transform can be used to convert from camera coordinates (C x , C y ) to galvanometer coordinates (G x , G y ). The calibration is well suited to automation or by a simple user driven procedure. 
     Using image analysis to provide feedback of the focused spot position on the CCD camera the spot could be directed to the origin position (probably the center of the CCD or the corner) and those coordinates recorded as (G x0 , G y0 ). Next the galvo could be driven (with feedback) to a position located horizontally with respect to the origin (dC y =0) to calculate the X-axis position derivatives (G x dC x , G y dC x ). Similarly, using a position located vertically with respect to the origin (dC x =0) the Y-axis position derivatives (G x dC y , G y dC y ) could be determined. 
     This technique would require a specimen to be in focus and that the focused spot to be visible. The image analysis used to locate the spot could use the intensity maxima, or possibly a two dimensional centroid calculation. 
     Automated Calibration of TIRF Galvanometer Positions 
     The TIRF galvo calibration is complicated by the fact that the beam is not focused in the specimen focal plane and as such cannot be located directly. The most useful feedback mechanism would be overall image brightness. This is based on the observation that the due to the deeper illumination penetration, specimens are illuminated brighter under epi-illumination conditions than under TIRF illumination. This is particularly true for aqueous fluorescent liquid specimens that have even fluorescence from the cover slip surface outward. Another useful specimen would be a glass cover slip uniformly coated with quantum dots or another thin fluorescent coating. Using such a calibration specimen, a calibration algorithm can be automated. 
     The total integrated image intensity is used as feedback. This is appropriate for all galvanometer locations near the TIRF light path since the diffuser  175  fills the entire field of view. If a raster scan was made of all the galvanometer locations near the TIRF light path axis, the resultant image would be a bright filled circle with a sharp drop off to darker values outside a radius corresponding to the critical illumination angle. The center of the circle would correspond to the galvanometer coordinates of the TIRF light path axis (T x0  T y0 ). 
     The total integrated image intensity can be measured either by numerically summing the pixels of an image recorded by a camera, or by placing a light detector near a conjugate plane to the microscope objective pupil to directly measure total specimen fluorescence across a wide field. 
       FIG. 5  illustrates how measuring the whole field&#39;s integrated intensity at points on a coarse grid of galvanometer locations can be used to indicate which galvanometer locations generate sub-critical, critical and supercritical illumination conditions. 
     Once the center of the circle is located roughly it can be more accurately located by sampling along trajectories radiating outward from the center. These trajectories can be used to identify the critical angle location (G x , G y ) precisely either using a threshold measure or by locating the position with maximum slope. With this information very accurate TIRF imaging trajectories can be designed. These would take the form of circles centered on the TIRF light path axis (T x0  T y0 ) with different radii depending on the desired depth penetration, or even epi-illumination. 
       FIG. 6  is an illustration showing that slowly scanning the galvanometers along the trajectories shown (solid lines) away from the rough center (cross hair) while measuring the whole field integrated image intensity, could be used to identify the set of galvo positions resulting in illumination through the microscope objective at the critical angle (circle shown with dotted line). 
       FIG. 7  is a block diagram of an illumination system, i.e., system  700 , that, similarly to system  100  that has a collimated mode of operation that projects a collimated light beam to a plane  755 , and a convergent mode of operation that projects a convergent light beam to plane  755 . System  700  includes a light steering device  710 , lenses  720 ,  730  and  740 , a mirror  725 , and a light steering device  745 . 
     Light steering device  710  is similar in functionality to light steering device  110 , and can be implemented similarly to light steering device  110 . Light steering device  745  is similar in functionality to light steering device  185 , and can be implemented similarly to light steering device  185 , but is shown in system  700  as being a beam cube. 
     In the collimated mode of operation of system  700 , light steering device  710  receives a light bundle via a light path  705 , and directs the light bundle to a light path  715 . Light path  715  runs from light steering device  710 , through lens  720 , to mirror  725 , through lens  730  to light steering device  745 . Light steering device  745  receives the light bundle via light path  715  and directs the light bundle to a downstream light path, i.e., a light path  750 . 
     In the convergent mode of operation of system  700 , light steering device  710  receives a light bundle via light path  705 , and directs the light bundle to a light path  735 . Light path  735  runs from light steering device  710 , through lens  740 , to light steering device  745 . Light steering device  745  receives the light bundle via light path  735 , and directs the light bundle to light path  750 . 
     Lens  740  is moveable, along a portion of light path  735 , to adjust focus. Lens  730  is moveable, along a portion of light path  715 , to adjust collimation. 
       FIG. 8  is an illustration of the operation of system  700  in collimated mode. Light steering device  710  receives a light bundle in a collimated beam  805 , and directs the light bundle to lens  720  in a collimated beam  810 . Lens  720  receives the light bundle in collimated beam  810 , and directs it, in a convergent beam  815 , to a plane  820 . From plane  820 , the light bundle propagates to mirror  725  in a divergent beam  825 . The light bundle propagates from mirror  725  to lens  730  in a divergent beam  830 . Lens  730  receives the light bundle in divergent beam  830 , and directs it to light steering device  745  in a collimated beam  835 . Light steering device  745  receives the light bundle in collimated beam  835 , and directs it to plane  755  in a collimated beam  840 . Lens  720 , mirror  725  and lens  730  may be regarded as an optical subsystem that transforms the light bundle so that the light bundle is projected to plane  755  in collimated beam  840 . Light steering device  710  controls a trajectory of the light bundle in light path  715  to steer collimated beam  840  through plane  755  at a designated incident angle  845 . 
       FIG. 9  is an illustration of the operation of system  700  in convergent mode. Light steering device  710  receives a light bundle in collimated beam  805 , and directs the light bundle to lens  740  in a collimated beam  905 . The light bundle propagates from lens  740  to light steering device  745  in a convergent beam  910 , and through light steering device  745  to plane  755  in a convergent beam  915 . Lens  740  may be regarded as an optical subsystem that transforms the light bundle so that the light bundle is projected to plane  755  in convergent beam  915 . Light steering device  710  controls a trajectory of the light bundle in light path  735  to steer convergent beam  915  to a target position (for example, at an xy coordinate indicated in  FIG. 9  by an offset  920 ) in plane  755 . 
       FIG. 10  is a block diagram of a system  1000  for microscopy. System  1000  includes a light source  1015 , an optical system  1020 , a microscope  1035 , a camera  1030 , a computer  1005 , and driver electronics  1010 . 
     Light source  1015 , e.g., a laser, emits light. Optical system  1020  receives the light from light source  1015 , modifies the light and illuminates a plane  1025  with either of a collimated light beam or a convergent light beam. Optical system  1020  may be implemented, for example, by either of system  100  or system  700 . Plane  1025  coincides with an image plane of microscope  1035 . Microscope  1035  produces an image of a specimen situated in plane  1025 . Camera  1030  is an imaging apparatus that converts the image from microscope  1035  into an image in a digital data format that is processed by computer  1005 . Computer  1005  evaluates the image, and provides a signal to driver electronics  1010 , which in turn controls optical system  1020 . 
     Computer  1005  evaluates a characteristic of the image, for example, a physical feature in the image, or a quality of the image that is indicative of its focus. Based on the characteristic, computer  1005  performs calculations for, ultimately, controlling the illumination in plane  1025 . For example, in a case where system  1000  is being employed to illuminate plane  1025  with a collimated light beam, computer  1005  performs calculations for controlling a light steering device within optical system  1020  to steer the collimated light beam through plane  1025  at a designated incidence angle. Similarly, in a case where system  1000  is being employed to illuminate plane  1025  with a convergent light beam, computer  1005  performs calculations for controlling the light steering device within optical system  1020  to steer the convergent light beam to a target position in plane  1025 . Computer  1005  also controls positioning of lenses within optical system  1020  to focus the light beams. For examples of some of the calculations performed by computer  1005 , see the discussions above concerning (a) calibration of galvanometer positions, (b) automated calibration of FRAP galvanometer positions, and (c) automated calibration of TIRF galvanometer positions. 
     Computer  1005  includes a processor  1008  and a memory  1009  that contains instructions that are executable by processor  1008 . Upon execution of the instructions, processor  1008  performs methods that include the evaluation of the characteristic of the image, the calculations of settings for controlling optical system  1020 , and thus the ultimate control of optical system  1020  to perform methods that include the various operations described herein. 
     Although system  1000  is described herein as having the instructions for processor  1008  installed into memory  1009 , the instructions can be tangibly embodied on an external computer-readable storage medium, e.g., a storage medium  1007 , for subsequent loading into memory  1009 . Storage medium  1007  can be any conventional storage medium, including, but not limited to, a floppy disk, a compact disk, a magnetic tape, a read only memory, or an optical storage medium. The instructions could also be embodied in a random access memory, or other type of electronic storage, located on a remote storage system and coupled to memory  1007 . 
     Moreover, although computer  1005  is described herein as having the instructions installed in memory  1009 , and therefore being implemented in software, the operation of computer  1005  could be implemented in any of hardware, firmware, software, or a combination thereof. 
     System  1000  creates optimal illumination conditions for total internal reflection fluorescent (TIRF) microscopy. The illumination is uniform across a large field of view, does not have the interference fringes seen in other illumination systems, and the penetration depth of the evanescent wave can be very rapidly varied. System  1000  creates illumination conditions for fluorescence recovery after photobleaching (FRAP) or photoactivation experiments. 
     The quality of TIRF illumination that system  1000  produces is superior to systems that use stationary beams. The systems that use stationary beams suffer from interference fringes, flaring, shadowing and other types of non-uniformity. System  1000 , by scanning the beam in a trajectory of beams with equal inclination angles, averages out such artifacts. 
     System  1000  allows the illumination angle of the incident beam to be varied in milliseconds or less, and provides an extremely rapid method for achieving multi-angle TIRF microscopy over a large range of incident angles. Computer  1005  will use an automated search algorithm, with feedback control, to automatically optimize TIRF conditions for many types of objectives and dichroic filter cubes. 
     FRAP/Photoactivation TIRF and the Objective Lens 
     FRAP experiments require selected regions of a specimen being observed to be photobleached. Conclusions can be made about the dynamics of molecules by monitoring the kinetics and extent of fluorescence recovery after FRAP experiments and by examining the spatial pattern of fluorescence recovery. A correlate approach is to photoactivate dyes or molecules either so as either to manipulate the cellular milieu (e.g. releasing caged calcium, etc.) or to track the cellular fate of photoactivated molecules, which may through photoactivation be brighter or have an altered spectral characteristics. 
     Since the bleaching/photoactivation must be localized to a region of interest, it is commonly accomplished by scanning a focused beam of light across a region of interest using a raster scan or other area-covering beam trajectory. Alternatively, FRAP may be achieved using selective illumination with epifluorescent light that is passed through an adjustable mask (e.g. a rectangle of adjustable width and height) at a conjugate image plane. While less expensive, the latter is slow to adjust as it&#39;s typically done manually and bleaching/photoactivating small objects, multiple objects, or other shapes (e.g. a circle) is not possible. Thus, for speed and flexibility a raster scanned approach is generally preferred. 
     Generally the area bleached is a sub-region of area observed so that both are positioned at the focal plane of the microscope objective. This specimen focal plane has corresponding image planes at the image plane in the observation beam path of the microscope and also on the excitation beam path (in most microscopes). 
     Different Requirements for FRAP/Photoactivation Versus TIRF Illumination. 
     For FRAP/photoactivation experiments a scanned beam (or mask) must be focused on an image plane of the microscope. The size of the smallest FRAP/photoactivated point is determined by the wavelength of the illuminating light, the numerical aperture (NA) of the objective and the extent to which the back focal plane of the objective is filled. The microscope optics (including the objective lens) relays this focus to the specimen image plane. Assuming the scanning optics are telecentric and the objective is infinity corrected the light will pass through the center of the microscope objective lens pupil as a collimated beam. 
     For TIRF illumination a collimated beam is required at the specimen image plane. Unlike FRAP, for TIRF illumination the laser beam must be focused on the outer rim of the back focal plane of the objective lens. For TIR to occur two criteria must be met: (i) light must go from medium of high refractive index (typically glass) to lower refractive index (e.g. aqueous medium, cell cytosol, etc.) and (ii) the incidence angle of the light with respect to the optical axis (normal) must be greater than the critical incidence angle for the specimen—coverslip interface being observed. For the latter these high incidence angle foci occur near the outer perimeter of the pupil aperture. If a collimated beam is incident at the image plane of the microscope it is relayed to the specimen image plane. At the pupil of the objective lens this light will come to a sharp focus. 
     System  1000  can be used in applications such as (a) multi-angle TIRF microscopy, (b) FRAP in combination with TIRF, (c) photoactivation in combination with TIRF, (d) optical trapping in combination with TIRF, (e) in vitro or in vivo imaging, and (f) material science and local activation of a surface, e.g., for lithography. 
     The techniques described herein are exemplary, and should not be construed as implying any particular limitation on the present invention. It should be understood that various alternatives, combinations and modifications could be devised by those skilled in the art. The present invention is intended to embrace all such alternatives, modifications and variances that fall within the scope of the appended claims.