Abstract:
An illumination system and method for operating the same is disclosed. The illumination system includes a spatial light modulator (SLM), first and second optical systems, a controller and a mask. The SLM is positioned to receive an incident light beam. The first optical system images light leaving the SLM onto the mask that blocks part of the light. The second optical system images light leaving the mask onto a sample to be illuminated. The controller causes the SLM to display an SLM pattern that generates an illumination beam and a spurious light beam from the incident light beam, the illumination beam passing through the mask, wherein the mask includes a fixed part having a plurality of openings and a moveable part that moves in relation to the fixed part and that includes an opening.

Description:
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS 
       [0001]    This application is a conversion of, and claims priority from, U.S. Provisional Patent Application 62/140,369 filed on Mar. 30, 2015, said patent application being incorporated by reference herein. 
     
    
     BACKGROUND 
       [0002]    In fluorescence microscopy, a sample is labeled with a fluorescent dye, and then placed on the microscope using a sample holder. The sample holder positions the sample such that the front focal plane of the microscope&#39;s objective is coinciding with a region of the sample. The sample is then illuminated by one or more beams of light shining through the objective. In response, the fluorescent dye in the sample emits light that is usually at a wavelength that is substantially different from the wavelength of the illuminating light, and hence, the object stained by the dye can be distinguished from objects that did not absorb the dye. The microscope is configured such that light emitted by the sample is, by means of several lenses and mirrors, collected on a detector, typically a camera, which coincides with a plane that is conjugate to the objective focal plane mentioned above. This results in an image of the sample being formed on the detector. 
         [0003]    There are a number of different fluorescence microscopy modes that are distinguished by the type of illumination. Different modes provide different advantages depending on the specific goals of the experiment in which the microscope is being used. Each type of illumination requires a different illumination pattern on the specimen. Typically, each illumination pattern corresponds to a different arrangement of optical elements for forming the desired illumination pattern on the specimen from a light source. The optical elements sometimes include a mask that selectively blocks some light from the source. Different patterns are characterized by different masks, and hence, in switching illumination patterns, the masks must be changed which necessitates keeping a collection of different masks, removing the existing mask and inserting a new mask. 
       SUMMARY 
       [0004]    The present invention includes an illumination system and method for operating the same. The illumination system includes a spatial light modulator (SLM), first and second optical systems, a controller and a mask. The SLM is positioned to receive an incident light beam. The first optical system images light leaving the SLM onto the mask that blocks part of the light. The second optical system images light leaving the mask onto a sample to be illuminated. The controller causes the SLM to display an SLM pattern that generates an illumination light beam and a spurious light beam from the incident light beam, the illumination beam passing through the mask, wherein the mask includes a fixed part having a plurality of openings and a moveable part that moves in relation to the fixed part and that includes an opening. In one aspect of the invention, the incident light beam is a collimated linearly polarized light beam. 
         [0005]    In another aspect of the invention the SLM pattern includes a pattern that diffracts part of the incident light beam to create the illumination beam. The SLM can be a transmissive SLM or a reflective SLM. 
         [0006]    In yet another aspect of the invention, the opening in the moveable part is characterized by a distance from a reference point on the fixed part and wherein the reference point and the SLM pattern are determined by the controller in response to user input specifying one of a plurality of illumination modes, each mode corresponding to a different illumination pattern. 
         [0007]    In another aspect of the invention, the fixed part of the mask includes a transparent slot and a plurality of openings, the moveable part moving in relation to the fixed part such that the moveable part covers the slot. The opening in the moveable part provides an opening in the mask characterized by a position that can be changed by changing the position of the moveable part in the slot. In one aspect, the openings in the fixed part are arranged in opposing pairs. Each pair is centered on a reference point on the fixed part. The slot is characterized by an axis that runs radially through the reference point, the moving part moving in a direction parallel to the axis. 
         [0008]    In another aspect of the invention, the light leaving the SLM includes multiple light beams, and the mask blocks one of the multiple light beams. 
         [0009]    In yet another aspect of the invention, the SLM pattern and the moveable part of the mask are configured such that light leaving the mask provides one of a plurality of predetermined illumination patterns, the one of the plurality of predetermined illumination patterns is determined by user input. 
         [0010]    In still a further aspect of the invention, the one of the plurality of predetermined illumination patterns is adapted for total internal reflection microscopy, inclined illumination microscopy, two dimensional structured illumination microscopy (SIM), three dimensional SIM, or epifluorescence bright field microscopy. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0011]      FIG. 1  illustrates the basic optical elements of a light source according to the present invention. 
           [0012]      FIG. 2  is one embodiment of a mask that can be utilized in the arrangement shown in  FIG. 1 . 
           [0013]      FIGS. 3A-3C  illustrate various mask configurations that are obtained by positioning moveable part  32  relative to disk  31 . 
           [0014]      FIG. 4  illustrates a cross-sectional view of a microscope beam path for total internal reflection microscopy and inclined illumination microscopy. 
           [0015]      FIG. 5  illustrates a cross-sectional view of a microscope beam path for two dimensional SIM. 
           [0016]      FIG. 6  illustrates a cross-sectional view of a microscope beam path for bright field epifluorescence microscopy. 
           [0017]      FIG. 7  illustrates a cross-sectional view of a microscope beam path for three dimensional SIM. 
           [0018]      FIGS. 8A and 8B  illustrate an exemplary light source according to one embodiment of the present invention. 
           [0019]      FIGS. 9A and 9B  illustrate an SLM pattern for creating an inclined beam with a smaller, adjustable diameter that can be used in embodiments of the present invention to create inclined illumination. 
           [0020]      FIGS. 10A and 10B  illustrate another exemplary light source according to one embodiment of the present invention. 
           [0021]      FIGS. 11A and 11B  illustrate another SLM pattern for creating an inclined beam with a smaller, adjustable diameter that can be used in embodiments of the present invention to create inclined illumination. 
       
    
    
     DETAILED DESCRIPTION 
       [0022]    The present invention includes a light source for illuminating specimens for viewing in a microscope or other instrument. The light source can provide a number of different illumination patterns depending on the settings of a mask and the programming of an SLM. The manner in which the present invention provides its advantages can be more easily understood with reference to  FIG. 1 , which illustrates the basic optical elements of a light source according to the present invention. Illumination system  10  illuminates an object at location  17 . Light from a collimated linearly polarized light source  11 , which is typically a laser, is split into one or more beams by SLM  12 . The details of the beam or beams depend on the particular illumination pattern that is to be generated. SLM  12  is controlled from a controller  13  which is under the control, either directly or indirectly, of the user of the system. The light beams generated by SLM  12  are focused onto a mask  15  by a first lens assembly  14 . Mask  15  has one or more openings through which the desired light beam or beams pass. The beam or beams leaving mask  15  are imaged by a second lens assembly  16  onto the specimen in the desired pattern. 
         [0023]    Refer now to  FIG. 2 , which is one embodiment of a mask that can be utilized in the arrangement shown in  FIG. 1 . Mask  30  has two parts that move with respect to one another. Disk  31  is a disk having a number of circular openings  34  disposed around the outer edge of disk  31  at a constant radius from the center of disk  31 . The openings are arranged in three pairs of opposing apertures. The diameter of disk  31  is several millimeters in one embodiment, although different sizes could be utilized depending on the size of the optical elements shown in  FIG. 1 . Disk  31  also includes a slot  33  that extends beyond the center of disk  31  so that light can pass along a line through the center of disk  31 . 
         [0024]    Mask  30  also includes a moveable part  32  that has a width that is slightly larger than the width of slot  33 . Moveable part  32  has one opening  35  that is positioned such that the combination of moveable part  32  and disk  31  can provide an opening along the axis  37  of slot  33  that can be positioned along a radius of disk  31  at any position between the center of disk  31  and the radius at which the circular openings  34  are placed. The mask also includes a linear actuator that moves moveable part  32  along axis  36  so that slot  33  is covered except for the location at which opening  35  is positioned. 
         [0025]    Refer now to  FIGS. 3A-3C , which illustrate various mask configurations that are obtained by positioning moveable part  32  relative to disk  31 . Referring to  FIG. 3A , opening  35  of moveable part  32  is positioned at varying distances to the optical axis. In one aspect of the invention, a linear actuator  36  moves part  32  as shown by the arrows. Actuator  36  can be attached to a surrounding structure or to disk  31 . This configuration is intended to be used with a pattern on the programmable light source that creates diffraction orders at positions such that one position coincides with the aperture of the movable part. This results in a single off-axis beam to pass through the mask. This configuration is utilized in total internal reflection fluorescence (TIRF) microscopy and inclined illumination configurations. 
         [0026]    In TIRF microscopy, a single beam of illuminating light exits the objective at a substantial angle to the optical axis. The beam also exits the objective at a position that is shifted sideways such that the beam crosses the front focal plane of the objective at the intersection of this focal plane and the optical axis. This means that the illuminated region on the sample at this focal plane is centered on the optical axis. The advantage of TIRF microscopy is that it can reduce background signals. 
         [0027]    In inclined illumination, a single beam is used in a mode similar to TIRF. However, the angle used is shallower. The beam may be smaller in diameter than the field of view of the microscope; however, other beam diameters could be utilized. Using a smaller diameter avoids illuminating light passing through the sample above and below the objective&#39;s focal plane. Such out-of-focus illuminating light rays can reduce the image contrast, as they can cause the background to fluorescence. In addition, the illuminating beam can be positioned sequentially at different lateral regions of the sample to achieve a complete coverage in illumination. Images are taken sequentially at each beam step. The advantage of this mode is that it reduces background signals. 
         [0028]    Refer now to  FIG. 4 , which illustrates a cross-sectional view of a microscope beam path for TIRF and inclined illumination. A single beam of light  302  coming from a collimated light source  304  propagates at an angle to the optical axis  306  and is focused by several lenses  301 . The beam passes through a mask  30  with moveable part  32  positioned to create an off-center aperture. Mask  30  also blocks stray light. Mask  30  also blocks unwanted light beams if the light source emits other unwanted beams at the same time at different angles. The beam exits the last (i.e. objective) lens as a collimated beam of a smaller diameter and intersects front focal plane  305  of the objective at an angle. It should be noted that it is the specific lens arrangement and not the mask that leads to the smaller beam diameter on the sample. 
         [0029]    Refer now to  FIG. 3B . Here, moveable part  32  is positioned such that opening  35  is covered by the underlying portion of disk  31  leaving only the three pairs of opposing apertures on the mask open. This configuration can be used in two dimensional SIM. Here, two beams exit the objective at two angles to the optical axis of opposing signs. Both beams are shifted sideways by individual distances such that the two beams intersect the front focal planes at a region centered on the optical axis. Again, this is to ensure that the illuminated region on the front focal plane is centered on the optical axis. The advantage of two dimensional SIM is that the pattern generated by the interference of the two beams can be used to increase the lateral resolution of the microscope. 
         [0030]    Refer now to  FIG. 5 , which illustrates a cross-sectional view of a microscope beam path for two dimensional SIM illumination. Two beams of light  602 A and  602 B coming from a collimated light source  604  propagate at an angle to the optical axis  606  and are focused by several lenses  601 . The beams pass through mask  30  utilizing two off-center apertures. Mask  30  also blocks unwanted light beams if the light source emits other unwanted beams at the same time at different angles. The beams exit the last (i.e. objective) lens as two collimated beams of a smaller diameter and intersect the front focal plane  605  of the objective at an angle. 
         [0031]    Refer now to  FIG. 3C . In the third configuration moveable part  32  is positioned to provide a central aperture in the mask. This configuration can be used, depending on the pattern displayed on the programmable light source, either for bright field illumination or for three dimensional SIM. In epifluorescence bright field microscopy, the illuminating beam exits the objective along the optical axis, i.e. orthogonal to the front focal plane of the objective. The advantage of bright field microscopy is that it is simple and robust. 
         [0032]    Refer now to  FIG. 6 , which illustrates a cross-sectional view of a microscope beam path for bright field microscopy. A single beam of light  402  coming from a collimated light source  404  propagates along the optical axis  406  and is focused by several lenses  401 . The beam passes through mask  30  which is configured to provide a central aperture. Mask  30  blocks stray light. The beam exits the last (i.e. objective) lens as a collimated beam of a smaller diameter and intersects the front focal plane  405  of the objective vertically. 
         [0033]    In three dimensional SIM, two beams are configured in a manner similar to that used for two dimensional SIM, while a third beam is configured in the same manner as for bright field microscopy. The three beams interfere at the sample. The advantage of three dimensional SIM is that the resulting interference pattern can be used to increase the resolution of the microscope in all three dimensions. 
         [0034]    Refer now to  FIG. 7 , which illustrates a cross-sectional view of a microscope beam path for three dimensional SIM illumination. Here, the light source generates three beams of light  702 A- 702 C. Beams  702 A and  702 B propagate at an angle with respect to optical axis  706 . Beam  702 C propagates parallel to optical axis  706 . The beams are focused by lenses  701 . The beams pass through mask  30  that is using two off-center apertures and one centered aperture formed by positioning moveable part  32  so that the aperture is centered on disk  31 . Mask  30  blocks stray light and is also useful if the light source emits other unwanted beams at the same time at different angles. The beams exit the last (i.e. objective) lens  701  as three collimated beams of a smaller diameter and intersect the front focal plane  705  of the objective at an angle. 
         [0035]    The above-described embodiments of the present invention utilize a mask having a fixed portion that is in the shape of a disk. However, other mask shapes having similarly placed openings could be utilized. It is the positions of the openings that provide the advantage, not the shape of the fixed and moveable parts. 
         [0036]    Refer now to  FIGS. 8A and 8B , which illustrate an exemplary light source according to one embodiment of the present invention that can be used in the configurations described above.  FIG. 8A  is a cross-sectional view of a light beam processed by an SLM  202  and  FIG. 8B  is a front view of the pattern created on SLM  202 . For the purposes of the present discussion a transmissive SLM is a device that imposes, on a beam of light that passes through the device, a set of localized shifts in phase, amplitude, or both. In contrast, a reflective SLM is a device that imposes, on a beam of light reflected off the device, a set of localized shifts in phase, amplitude or both. The SLMs, both reflective and transmissive, are usually segmented into a rectangular or square lattice of pixels. The phase and amplitude shifts are uniform over the region covered by an individual pixel, but can vary between pixels. The pixels&#39; values for phase and amplitude shifts are addressable by software, and can vary at a frequency of at least several different values per second. Between the pixels may lie a “dead zone” that is not controllable by software and that may block or reflect or attenuate light at a constant rate. 
         [0037]    Retelling to  FIG. 8A , a collimated beam of coherent light  201  impacts SLM  202  positioned at a substantially vertical angle to the optical axis  205 . The SLM can be programmed to provide a pattern of pixels in which each pixel introduces a particular phase shift into the light passing through that pixel. An example of a pattern that provides a diffraction grating is illustrated in  FIG. 8B . In this pattern, horizontal stripes that introduce large phase shifts are shown as bright bands, alternating periodically with equally wide regions that impart small phase shifts, shown as dark bands. This pattern results in the SLM acting as an interference grating. Part of the collimated light impacting the SLM is diffracted as shown at  204 . To simplify the drawing only the first diffraction orders are shown. Part of the collimated light gets transmitted to form beam  203 , as the grating is finitely effective, and hence, not all of the light is diffracted. This light source can flexibly produce inclined beams at an angle dependent on the pattern displayed. By rotating the pattern on the SLM, the pairs of diffracted beams are also rotated around the optical axis. This is useful to create the rotated interference patterns on the sample as described for the SIM above. However, this source will always emit beams in pairs of opposite angles, and will always produce a beam parallel to the optical axis, necessitating the mask as described above. 
         [0038]    Refer now to  FIGS. 9A and 9B , which illustrate an SLM pattern for creating an inclined beam with a smaller, adjustable diameter that can be used in embodiments of the present invention to create inclined illumination. As noted above, an inclined illumination arrangement requires a smaller beam that is positioned at multiple locations and which leaves the light source at an angle. The pattern of phase shifts shown in  FIG. 9B  displayed on the SLM  202  is separated in two regions. In the first region shown at  202 A, the periodicity will be such that the diffraction angles will be suitable to allow light to pass through the off-axis aperture of the mask through the opening in the moveable part  32  as illustrated in  FIG. 3A . This region creates the two beams shown at  1304 A and  1304 B from input beam  1301 . These beams have smaller diameters than input beam  1301 . The offset of the beams relative to the center line of input beam  1301  is determined by the offset of region  202 A relative to the center line of input beam  1301 . In region  202 B, the periodicity of the pattern is smaller than that of region  202 A, resulting in a steeper diffraction angle. This region produces the two beams shown at  1303 . These beams will be stopped by mask  30 . SLM  202  is positioned in a plane in the microscope conjugate to the sample plane; hence, the two regions will correspond to two regions on the sample. Therefore, if this pattern is used in conjunction with a mask as described above, the size of the two regions determines which part of the sample will be illuminated by an inclined beam. By reprogramming the SLM, different illuminating beams of arbitrary size, shape, and position can be obtained. 
         [0039]    In the above-described embodiments the SLM is shown as a conductive structure in which the incident light is directed into the SLM from one side and part of the light that passes through the SLM is utilized. However, embodiments which utilize a reflective SLM can also be constructed. In such embodiments, the incident light is directed to the SLM from one side and the light source is arranged to create a collimated light beam that is diffracted by the SLM to create a plurality of light beams that are reflected from the SLM and that enter lens assembly  14 . Refer now to  FIGS. 10A and 10B , which illustrate another embodiment of a light source according to the present invention.  FIG. 10A  is a cross-section view of light source  1400 , and  FIG. 10B  is an end view of SLM  1402  shown in  FIG. 10A . Light source  1400  utilizes a reflective SLM to provide the light beams shown in  FIG. 8A . Refer now to  FIG. 10A . The incoming light beam  1401  is reflected from a reflective SLM  1402  that has the diffraction grating pattern shown in  FIG. 10B . The incoming light is diffracted into beams  1460  and  1404 . Beam  1460  being a reflected beam that is not diffracted, and analogous to beam  203  shown in  FIG. 8A . 
         [0040]    Refer now to  FIGS. 11A and 11B , which illustrate another embodiment of a light source according to the present invention.  FIG. 11A  is a cross-section view of light source  1500 , and  FIG. 11B  is an end view of SLM  1502  shown in  FIG. 11A . Light source  1500  utilizes a reflective SLM to provide the light beams shown in  FIG. 9A . Refer now to  FIG. 11A . The incoming light beam  1501  is reflected from a reflective SLM  1502  that has the diffraction grating pattern shown in  FIG. 11B . The incoming light is diffracted into beams  1504  by the portion of the pattern shown at  1502 A. The portion of the pattern shown at  1502 B generates light beams  1505  and  1506 . 
         [0041]    While the above embodiments utilize an SLM that is programmed to provide a diffraction grating, other patterns could be utilized. Any pattern that provides the multiple beams discussed above could, in principle, be utilized. In addition, it should be noted that the pattern on the SLM need not be rectangular. It should also be noted that SLMs that introduce changes in intensity as well as changes in phase could also be utilized. 
         [0042]    The above-described embodiments of the present invention have been provided to illustrate various aspects of the invention. However, it is to be understood that different aspects of the present invention that are shown in different specific embodiments can be combined to provide other embodiments of the present invention. In addition, various modifications to the present invention will become apparent from the foregoing description and accompanying drawings. Accordingly, the present invention is to be limited solely by the scope of the following claims.