Abstract:
Devices and methods for lightsheet microscopy using rotational-shear interferometry are provided. Advantages include improved lateral spatial resolution and easier alignment.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates generally to lightsheet microscopy, and more particularly but not exclusively to rotational-shear-interferometer lightsheet microscopes. 
       BACKGROUND OF THE INVENTION 
     Lightsheet Microscopy 
       [0002]    Lightsheet microscopy is a technique for imaging a sample in all three spatial dimensions (“3-D”), in which a “sheet” of light illuminates one slice at a time of the sample under study. This is illustrated in  FIG. 1 . (Lightsheet microscopy is sometimes also referred to as “selective plane illumination microscopy.”) A “detection microscope” records a two-dimensional (“2-D”) image of the region illuminated by the lightsheet. The lightsheet may be scanned step-by-step through the sample, or the sample may be scanned step-by-step through the lightsheet. A 2-D image is recorded at each step. Eventually the entire object may be illuminated and imaged, and the 2-D images fused together with software to make a 3-D image of the sample. 
         [0003]    The 3-D imaging of fluorescent labels in a biological specimen is an example of a common application of lightsheet microscopy. The illumination light may serve to activate the fluorophores which fluoresce in response. The detection microscope may then capture the light emitted by the fluorophores and form a 2-D image of the locations of the fluorescent labels in response to the illumination by the lightsheet. As the lightsheet and/or sample is scanned, a 2-D image is captured at each scan step. A 3-D image may be assembled from the 2-D images, showing the distribution of fluorescent labels within the specimen. 
         [0004]    Many current lightsheet microscopes suffer from at least one of the following difficulties. 
       Exemplary Difficulty #1—Exacting Alignment  
       [0005]    A lightsheet microscope requires alignment between (1) the location of the lightsheet and (2) the region-of-focus of the detection microscope. This alignment requirement is illustrated in  FIGS. 2 and 3 . The alignment is required largely for system focus. Without the alignment the 2-D images recorded by the detection microscope will be blurred, and blurring is undesirable. 
         [0006]    With many current lightsheet microscopes this alignment is exacting. Significant effort and cost are needed to meet the alignment requirement. The “z-offset” and the “tilt-offset” both need to be minimized to a value that is exacting to achieve. As used herein the terms “z-alignment” and the “tilt-alignment” refer to the alignment of such offsets, as defined below under “Definitions.” The exacting nature of these alignments has negative impacts at each stage in the life of the lightsheet microscope. The need for exacting z-alignment and tilt-alignment can complicate the design, manufacture, and/or use of many current lightsheet microscopes. 
         [0007]    For example, the choice of construction materials is constrained to materials able to maintain shape and dimension against even small thermal and mechanical disturbances. These constraints impede the system design. Further, the system designer may be pushed harder to make tradeoffs against other system parameters and thus reduce overall system performance. For example, these constraints may push the system designer to decrease the numerical aperture of the detection microscope in order to increase the depth-of-field of the detection microscope and thus allow for easier system alignment. A decrease in the numerical aperture of a microscope degrades the lateral spatial resolution of the microscope, which is undesirable. Further, exacting alignment requirements may force the system designer to incorporate alignment mechanisms (tip/tilt controls, etc.) with a finer adjustment capability. This complexity may increase system cost and may impede the system designer. 
         [0008]    Furthermore, the exacting alignment requirements make system manufacture more difficult and more expensive. More effort may be required to achieve the alignment requirements. And greater cost may be required since the construction material choices are constrained and since more system complexity may be required. 
         [0009]    Even after the system is manufactured, the exacting alignment requirements may negatively impact the use of the system in the field. The user generally may need to perform a final alignment of the system in the moments before each measurement is performed. More effort may be required if the alignment is more exacting. Related to this is a difficulty with many current lightsheet microscopes described by Huisken in U.S. patent application publication 2011/0115895, the entire contents of which application are incorporated herein by reference: typically, “the [lightsheet] is aligned before the experiment to illuminate the focal plane of the detection lens. This alignment is not changed for individual samples. However, samples differ tremendously in their optical properties. Refraction at the medium-sample interface will divert the [lightsheet] away from the focal plane and result in a blurry image.” This reduces the variety of samples that can be imaged without re-alignment. Furthermore, alignment generally must be maintained throughout the duration of the measurement. The exacting nature of the alignment requirements may make this more difficult. The difficulty is more acute for longer measurements since the alignment must be maintained for a longer time. 
       Exemplary Difficulty #2—Limited Lateral Spatial Resolution 
       [0010]    Another difficulty with many current lightsheet microscopes is the limitation on the lateral spatial resolution that can be achieved by the detection microscope. Indeed, many current lightsheet microscopes use one or more conventional microscopes for the detection microscope. The lateral spatial resolution of a conventional microscope is limited in a known way. This is a limit to image quality. 
         [0011]    Thus, there is a need in the art for improved lightsheet microscopes that may address, for example, one or more of the above-noted difficulties or additional difficulties. 
       SUMMARY OF THE INVENTION 
       [0012]    In one of its aspects the present invention provides a RSI lightsheet microscope that combines lightsheet illumination and rotational-shear interferometry. 
         [0013]    As used herein, “2-D” stands for two-dimensional; “3-D” stands for three-dimensional; “MTF” Stands for modulation transfer function; and, “RSI” stands for rotational-shear interferometer. 
       Definitions 
       [0014]    As used herein the following terms have the following meanings. 
         [0015]    The term “conventional imager” refers to an imager that forms a direct image on a detector. The term “conventional microscope” refers to a microscope that forms a direct image on a detector. An RSI imager is not a conventional imager since the data recorded by the detector needs to be processed to infer an image. Likewise, an RSI microscope is not a conventional microscope. 
         [0016]    The term “lightsheet microscope” refers to a system that includes both lightsheet illumination and a detection microscope. The terms “conventional microscope” and “RSI microscope” each refer only to the detection microscope, not the lightsheet illumination system. 
         [0017]    The “region-of-focus” of an imaging system refers to the 3-D region over which an object point will appear “in focus” in the 2-D image. In  FIGS. 1 ,  2 ,  3 ,  5 ,  6 ,  9 A,  9 B,  9 C, and  9 D, a pair of dashed lines marks the boundaries of the “region-of-focus” of the detection microscope. For example, in  FIG. 1  the region-of-focus is labeled  103 . An object point located between the pair of dashed lines will appear in focus in the 2-D image. An object point located outside the region-of-focus will appear blurred in the 2-D image. 
         [0018]    The “depth-of-field” of an imaging system refers to the distance over which the “region-of-focus” extends in the “z” direction. In  FIGS. 1 ,  2 ,  3 ,  5 ,  6 ,  9 A,  9 B,  9 C, and  9 D the “depth-of-field” is the distance between the two dashed lines in the “z” direction. 
         [0019]    The “plane-of-mid-focus” refers to the plane within the region-of-focus halfway between the two ends of the region-of-focus in the “z” direction. For example, in  FIG. 1  the “plane-of-mid-focus” is halfway between the two dashed lines that mark region-of-focus  103 . 
         [0020]    In a system with “field curvature” aberration, the mid-focus surface may be curved rather than planar. For simplicity of discussion, systems discussed here are approximated to have no “field curvature.” The same concepts apply when “field curvature” is present. 
         [0021]    “Tilt-offset” refers to the angle between (1) the plane of the lightsheet and (2) the plane-of-mid-focus of the detection microscope. This is illustrated in  FIG. 2 . For example, in  FIG. 2  the “tilt-offset” is labeled  204  and has a value of 22 degrees. This value was chosen for clarity and ease of illustration. In  FIGS. 1 ,  3 ,  5 ,  9 A,  9 B,  9 C, and  9 D the “tilt-offset” is zero. 
         [0022]    “Tilt-alignment” refers to the alignment that minimizes the “tilt-offset.” The “tilt-alignment” can involve adjustment of (1) the tilt angle of the lightsheet, (2) the tilt angle of the region-of-focus of the detection microscope, or (3) a combination of (1) and (2). 
         [0023]    “z-offset” refers to the distance in the “z” direction between (1) the plane of the lightsheet (the plane through the center of the lightsheet) and (2) the plane-of-mid-focus of the detection microscope after correcting for “tilt-offset.” This is illustrated in  FIG. 3 . In  FIG. 1  and  FIG. 2  the “z-offset” is zero. In  FIG. 3  the “z-offset” is non-zero. 
         [0024]    “z-alignment” refers to the alignment that minimizes the “z-offset.” The “z-alignment” can involve adjustment of (1) the location of the lightsheet, (2) the location of the region-of-focus of the detection microscope, or (3) a combination of (1) and (2). 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0025]    The foregoing summary and the following detailed description of exemplary embodiments of the present invention may be further understood when read in conjunction with the appended drawings, in which: 
           [0026]      FIG. 1  schematically illustrates the concept of lightsheet microscopy; 
           [0027]      FIG. 2  schematically illustrates the meaning of the terms tilt-offset and tilt-alignment; 
           [0028]      FIG. 3  schematically illustrates the meaning of the terms z-offset and z-alignment; 
           [0029]      FIG. 4  schematically illustrates a lightsheet microscope with a rotational-shear interferometer in accordance with the present invention; 
           [0030]      FIG. 5  schematically illustrates why the z-alignment is easier to perform when the depth-of-field of the detection microscope is large; 
           [0031]      FIG. 6  schematically illustrates why the tilt-alignment is easier to perform when the depth-of-field of the detection microscope is large; 
           [0032]      FIG. 7  schematically illustrates the light path in one embodiment of a rotational-shear interferometer; 
           [0033]      FIG. 8  schematically illustrates one method of operation; 
           [0034]      FIGS. 9A ,  9 B,  9 C, and  9 D further schematically illustrate a method of operation; and 
           [0035]      FIG. 10  schematically illustrates an alternate embodiment involving a lightsheet microscope with two rotational-shear interferometers in accordance with the present invention. 
       
    
    
     DETAILED DESCRIPTION 
       [0036]    Turning now to the figures,  FIG. 1  schematically illustrates the concept of lightsheet microscopy.  FIG. 1  illustrates part of a lightsheet microscope representing a snapshot at one scan step of a sample  100  under study. Sample  100  could be a biological specimen, for example. Sample  100  is three-dimensional and extends in the “+/−x”, “+/−y”, and “+/−z” directions. (Mutually orthogonal coordinate axes indicate the “x”, “y”, and “z” directions, with each arrow on each coordinate axis pointing in the positive direction along the axis. For example, the “+y” direction is to the right on the page.) For simplicity of illustration, only a two-dimensional slice of sample  100  is drawn. 
         [0037]    Lightsheet  101  enters sample  100  in the direction indicated by arrow  102  and represented by a pair of thick lines. The lightsheet  101  is parallel to the x-y plane and has a slight curvature, customary of the behavior of a Gaussian beam. A Gaussian beam is one form of illumination used to generate a lightsheet. The most-narrow part of lightsheet  101  is located near the center of sample  100 . Lightsheet  101  expands slightly in the “+/−z” direction as there is an increase in the distance from the most-narrow part of lightsheet  101 . This comports with the behavior of a Gaussian beam. The detection microscope is not shown in  FIG. 1 . Light from sample  100  travels in the “+z” direction to a detection microscope. A region-of-focus  103  is centered in the “z” direction on lightsheet  101 .  FIG. 1  is meant to be compared directly to  FIGS. 2 ,  3 ,  5 , and  6 . These Figures are all drawn on the same scale, each of which includes a region-of-focus. The depth-of-field is the same in  FIGS. 1-3 . 
         [0038]      FIG. 2  schematically illustrates the meaning of the terms tilt-offset and tilt-alignment. Like  FIG. 1 ,  FIG. 2  illustrates part of a lightsheet microscope representing a snapshot at one scan step of a sample  200  under study. Light from sample  200  travels in the “+z” direction to the detection microscope. The detection microscope is not shown. The region-of-focus is labeled  203 . A difference between  FIG. 2  and  FIG. 1  is that in  FIG. 2  the lightsheet  201  is tilted. The lightsheet  201  is tilted about the “x” axis, and arrow  202  indicates the direction the lightsheet  201  enters sample  200 . 
         [0039]    In  FIG. 2  there is a sufficiently-large tilt-offset  204  between lightsheet  201  and region-of-focus  203  as to produce blur in the recorded image. Parts of the illuminated region of sample  200  are outside region-of-focus  203 . Light from points outside the region-of-focus of the detection microscope contributes to blur in the recorded image. To avoid blur in the recorded image, only a small tilt-offset  204  is allowed. The region of the sample illuminated by the lightsheet  201  (within the field-of-view of interest) should fit completely within the region-of-focus of the detection microscope. This alignment requirement is the tilt-alignment. The system illustrated in  FIG. 2  does not have proper tilt-alignment, because the tilt-offset  204  is too large. 
         [0040]      FIG. 3  schematically illustrates the meaning of the terms z-offset and z-alignment. Like  FIGS. 1 and 2 ,  FIG. 3  illustrates part of a lightsheet microscope representing a snapshot at one scan step of a sample  300  under study. Light from sample  300  travels in the “+z” direction to the detection microscope. The detection microscope is not shown. The region-of-focus is labeled  303 . A difference between  FIG. 3  and  FIG. 1  is that in  FIG. 3  the region-of-focus is offset in the “z” direction from lightsheet  301 . Arrow  302  indicates the direction the lightsheet  301  enters sample  300 . In  FIG. 3  there is a sufficiently-large z-offset  304  between lightsheet  301  and region-of-focus  303  as to produce blur in the recorded image. The illuminated region of sample  300  is outside region-of-focus  303 . Light from points outside the region-of-focus  303  of the detection microscope produces blur in the recorded image. 
         [0041]    To avoid blur in the recorded image, only a small z-offset  304  is allowed. The region of the sample  300  illuminated by the lightsheet  301  (within the field-of-view of interest) should fit completely within the region-of-focus  303  of the detection microscope. This alignment requirement is the z-alignment. The system illustrated in  FIG. 3  does not have proper z-alignment, because the z-offset  304  is too large. 
         [0042]      FIG. 4  schematically illustrates a RSI (rotational shear interferometer) lightsheet microscope  450  in accordance with the present invention. The RSI lightsheet microscope  450  includes an optical source/optics  401  that, in cooperation with an objective  402 , generates a lightsheet used to illuminate a sample  400 . Specifically, the objective  402 , which may be a conventional microscope objective, is disposed between the optical source/optics  401  and sample  400  to create and deliver the lightsheet to the sample  400 . Light from sample  400  is collected by a collection objective  403 , which may also be a conventional microscope objective, and is delivered to an RSI  404 . The objectives  402 ,  403  do not need be conventional microscope objectives; other suitable optical elements for creating the lightsheet and collecting light from the sample  400 , respectively, may be used. As seen in  FIGS. 5 and 6 , the RSI lightsheet microscope  450  provides enhanced performance with regard to z-alignment and tilt-alignment, due, in part to the increased depth-of-field of the RSI  404 . In particular,  FIG. 5  schematically illustrates why the z-alignment is easier to perform when the depth-of-field of the detection microscope is large. 
       Operation 
       [0043]    There are several manners in which the embodiment  450  can be operated. Some examples are as follows. This list is not meant to be limiting. 
       Exemplary Manner of Operation #1 
       [0044]    The lightsheet and the RSI microscope are both held fixed with no adjustment. The sample is translated (and possibly rotated) in steps through the lightsheet. At each step the RSI records a snapshot. This process is repeated until the entire sample has been imaged. 
       Exemplary Manner of Operation #2 
       [0045]    In this scenario the depth-of-field of the RSI microscope is large enough to encompass the entire sample. The system is set up so the sample is completely contained within the depth-of-field. The lightsheet is scanned through the entire sample. At each scan step the RSI microscope records a snapshot. No intermediate refocusing of the RSI microscope is required. 
       Exemplary Manner of Operation #3 
       [0046]    In this scenario, the depth-of-field of the RSI microscope is not large enough to encompass the entire sample. The sample is held fixed in location and orientation throughout the measurement. The lightsheet is scanned through the sample in steps. The RSI microscope must be refocused one or more times as the lightsheet is scanned. 
         [0047]    A flowchart is drawn in  FIG. 8 . The steps outlined in the flowchart are further illustrated in  FIGS. 9A ,  9 B,  9 C, and  9 D. 
         [0048]    The first step is step  800 . The RSI focus is adjusted so the region-of-focus is near one end of the sample under study. This is further illustrated in  FIG. 9A . Sample  900  is the sample under study. Region-of-focus  901  has been located near one end of sample  900 . 
         [0049]    The next step is step  801 . The lightsheet is placed at its initial location within the sample. This corresponds to lightsheet  902 . Arrow  903  illustrates the direction lightsheet  902  enters sample  900 . Lightsheet  902  is not right at the edge of region-of-focus  901 . Instead lightsheet  902  is separated from the edge of region-of-focus  901  by a distance labeled  908 . There is a tradeoff a user makes in choosing a value for distance  908 . A small value for distance  908  means more of the sample can be scanned before the RSI microscope must be refocused. A large value for distance  908  eases the z-alignment and tilt-alignment of the system. 
         [0050]    The next step is step  802 . The RSI microscope records a snapshot of the light from the sample. 
         [0051]    The next step is step  803 . Step  803  is a yes/no branch. If the lightsheet has been scanned through the intended area of the region-of-focus, the “yes” branch is followed and step  805  is next. Otherwise the “no” branch is followed and step  804  is next. 
         [0052]    Lightsheet  902  is not at the end of the intended area of region-of-focus  901 , so the “no” branch is followed to step  804 . In step  804  the lightsheet location is stepped. Then as indicated with the arrow, the next step is  802 , where a snapshot is again recorded with the RSI. This loop is repeated until the lightsheet location is at the end of the intended area of illumination of region-of-focus  901 . Lightsheet  906  indicates the last intended location for the lightsheet. Lightsheet  906  is a distance  909  from the edge of region-of-focus  901 . This is a buffer. The tradeoff is the same as was discussed in connection with buffer  908 . 
         [0053]    The lightsheet is stepped through a number of locations from location  902  to location  906 . Location  904  is in the middle. Ellipses indicate that additional steps are taken between the lightsheet locations that are drawn. Location  904  is an intermediate location for the lightsheet during the scan. Arrow  903  shows the direction lightsheet  902  enters sample  900 . Arrow  905  shows the direction lightsheet  904  enters sample  900 . Arrow  907  shows the direction lightsheet  906  enters sample  900 . 
         [0054]    When the lightsheet is at location  906  and step  803  is reached, the “yes” branch is followed to step  805 . 
         [0055]    Step  805  is another yes/no branch. If the entire sample has been imaged then the “yes” branch is followed to step  807 , which is the close of the flowchart. Otherwise the “no” branch is followed to step  806 . 
         [0056]    In step  806  the location of the RSI region-of-focus is stepped. Compare  FIG. 9A  to  FIGS. 9B ,  9 C, and  9 D. In going from one of these Figures to the next, the lightsheet region-of-focus is stepped through the sample. This involves moving from step  806  to step  802  as indicated in  FIG. 8 , and repeating the loop until the “no” branch is followed from step  805  to step  807 . 
         [0057]    At each location of the lightsheet region of focus, the lightsheet is scanned through as before in connection with  FIG. 9A . 
         [0058]    In  FIG. 9B  lightsheets  911 ,  913 , and  915  travel in the directions indicated by arrows  912 ,  914 , and  916  respectively. Markers  917  and  918  indicate the length of each buffer region. The region-of-focus is labeled  910 . The sample is again labeled  900 . 
         [0059]    In  FIG. 9C  lightsheets  920 ,  922 , and  924  travel in the directions indicated by arrows  921 ,  923 , and  925  respectively. Markers  926  and  927  indicate the length of each buffer region. The region-of-focus is labeled  919 . The sample is again labeled  900 . 
         [0060]    In  FIG. 9D  lightsheets  929 ,  931 , and  933  travel in the directions indicated by arrows  930 ,  932 , and  934  respectively. Markers  935  and  936  indicate the length of each buffer region. The region-of-focus is labeled  928 . The sample is again labeled  900 . 
         [0061]    As indicated by the flowchart in  FIG. 8 , the last location of the lightsheet within one region-of-focus is the same as the first location of the lightsheet within the next region-of-focus. This provides data that assists with co-registration of the 2-D images recorded from different regions-of-focus. 
         [0062]      FIG. 5  schematically illustrates the z-alignment performance of the RSI lightsheet microscope  450  by showing a snapshot at one scan step of a sample  500  under study. Here a lightsheet  501  illuminates sample  500 , and arrow  502  indicates the direction lightsheet  501  enters sample  500 , with light from sample  500  traveling in the +z direction to the detection microscope, i.e., the RSI  404  and collection objective  403 . The magnitude of the z-offset in  FIG. 5  is the same as magnitude of z-offset  304  in  FIG. 3 . A difference between  FIG. 5  and  FIG. 3  is that in  FIG. 5  the detection microscope has a larger depth-of-field. The depth-of-field for region-of-focus  503  in  FIG. 5  is larger (by a factor of approximately four) than the depth-of-field for region-of-focus  303  in  FIG. 3 .  FIG. 5  and  FIG. 3  are drawn on the same scale as each other. The factor-of-four difference is only an example used for illustrative purposes. Other values are possible. 
         [0063]    In  FIG. 5  the system is in focus even though the z-offset is not zero. The system has proper z-alignment. By comparison, the system in  FIG. 3  is not in focus even though the z-offset is the same in  FIGS. 3 and 5 . The large depth-of-field in  FIG. 5  is what allows the system to be in z-alignment despite the non-zero z-offset. In  FIG. 5  a larger z-offset would be needed to place the system out of alignment. This illustrates why the z-alignment is easier to perform when the depth-of-field of the detection microscope is large. 
         [0064]      FIG. 6  schematically illustrates the tilt-alignment performance of the RSI lightsheet microscope  450  by showing a snapshot at one scan step of the sample  600  under study. In particular,  FIG. 6  illustrates why the tilt-alignment is easier to perform when the depth-of-field of the detection microscope is large. A lightsheet  601  illuminates sample  600 , and arrow  602  indicates the direction lightsheet  601  enters sample  600 , with light from sample  600  traveling in the +z direction to the detection microscope. The magnitude of the tilt-offset in  FIG. 6  is the same as the magnitude of the tilt-offset  204  in  FIG. 2 . A difference between  FIG. 6  and  FIG. 2  is that in  FIG. 6  the detection microscope has a larger depth-of-field. The depth-of-field for region-of-focus  603  in  FIG. 6  is larger (by a factor of approximately four) than the depth-of-field for region-of-focus  603  in  FIG. 2 .  FIG. 6  and  FIG. 2  are drawn on the same scale as each other. The factor-of-four difference is only an example used for illustrative purposes. Other values are possible. 
         [0065]    In  FIG. 6  the system is in focus even though the tilt-offset is not zero. The system has proper tilt-alignment. By comparison the system in  FIG. 2  is not in focus even though the tilt-offset is the same in  FIGS. 2 and 6 . The large depth-of-field in  FIG. 6  is what allows the system to be in tilt-alignment despite the non-zero tilt-offset. In  FIG. 6  a larger tilt-offset would be needed to place the system out of alignment. This illustrates why the tilt-alignment is easier to perform when the depth-of-field of the detection microscope detection microscope is large. Thus,  FIGS. 5 and 6  illustrate the effects of having a relatively larger depth-of-field provided by the RSI  404  as contrasted with the relatively smaller depth-of-field illustrated in  FIGS. 1-3  associated with a conventional microscope, such as exists on many current lightsheet microscopes. That is, the depth-of-field illustrated in  FIG. 1-3  is the depth-of-field of a conventional microscope, whereas the depth-of-field illustrated in  FIGS. 5 and 6  is the depth-of-field of an RSI microscope  450  in accordance with the present invention. 
       Rotational-Shear Interferometer 
       [0066]    Turning now to the rotational-shear interferometer  404  more specifically, the RSI  404  is an instrument in which light entering through an aperture is split into two beams. The two beams are recombined so as to produce interference fringes. The fringes can be analyzed to infer an image of the scene in front of the RSI  404 . (As used herein, when an RSI is used in this manner, it is referred to as an RSI imager, and when an RSI is used more-specifically in a microscope configuration, it is referred to as an RSI microscope.) The angle of rotational-shear can be set to different values, depending on the application. When the angle of rotational-shear is 180 degrees, there term “180-degree RSI imager” is used herein. 
         [0067]    The imaging performance of an RSI microscope may compared to the imaging performance of a conventional microscope in the cases where the following two conditions are met. (1) The entrance pupil of each microscope is the same distance from the object being imaged. (2) The sizes of the entrance pupils of the two microscopes are the same as each other. Under these two conditions, the following two comparisons can be made. 
       (a) Long Depth-of-Field  
       [0068]    The depth-of-field of the RSI microscope is long compared to the depth-of-field of the conventional microscope. 
         [0069]    The reason the RSI microscope has a long depth-of-field is as follows. Consider an object point located front of the RSI microscope. The object is on the axis of the RSI microscope. Light from the object point generates two wavefronts incident on the RSI detector. Now move the object point along the axis of the RSI microscope to a new location a short distance away. There is a change in the curvature of the two wavefronts incident on the RSI detector. The magnitude of the change-in-curvature is the same for both wavefronts. The change-in-curvature is common-mode. Interferometers are generally insensitive to common-mode changes. The fringe pattern recorded by the RSI detector does not change much with the movement of the object point along the imager&#39;s axis. For this reason, the RSI microscope has a long depth-of-field. 
       (b) Superior Lateral Spatial Resolution 
       [0070]    Under the scenario where spatially-incoherent light is used to image the scene, a 180-degree RSI imager is characterized by a modulation transfer function (MTF) superior to that of a conventional imager. The MTF is superior by up to a factor of two, as measured by the area under the MTF curve. The MTF is a measure of the lateral spatial resolution of the imaging system. 
         [0071]    The image generated by an RSI imager is a conical projection of the 3-D scene in front of the RSI. The vertex of the cone is the center of the RSI imager&#39;s entrance pupil. 
       RSI Layout 
       [0072]      FIG. 7  illustrates a layout for a simple version of an RSI microscope, including an object point from the sample under study, and including a model for the objective lens. The system illustrated in  FIG. 7  is drawn as 2-dimensional. Most current RSIs are 3-dimensional.  FIG. 7  is limited to 2 dimensions for simplicity of illustration. The figure provides the information needed without the complication of 3-dimensional drawings. 
         [0073]    The lenses illustrated in  FIG. 7  are illustrated as “thin lenses,” as is common in the field of optics. 
         [0074]    Object point  700  emits light towards the objective, such as objective  403 . In the thin-lens model illustrated in  FIG. 7 , the objective (which typically has many optical surfaces internally) is represented by a single thin lens  701 . Lens  701  collimates the light. The light travels to aperture  702 , commonly referred to in the field of optics as the “system stop.” Aperture  702  truncates the beam. The beam propagates to thin-lenses  703  and  704 . Optics  703  and  704  work together to image aperture  702  to detectors  709  and  710 . After leaving lens  704  the light propagates to beamsplitter  705 . One beam travels to fold mirrors  706  and  707 , then to beamsplitter  708 . The other beam travels to fold mirrors  711 ,  712 , and  713 , then to beamsplitter  708 . Two beams are incident on each detector  709  and  710 . The two beams respond in a counter-tilt fashion to movement of object point  700  within the “x-y” plane. The counter-tilt is due to the fact that one beam experiences an odd number of reflections while the other beam experiences an even number of reflections. 
         [0075]    As illustrated in  FIG. 7 , an object point at location  700  generates wavefronts on detectors  709  and  710  that are flat. When the object point is moved to a different location along the z-axis, the wavefronts incident on detectors  709  and  710  are no longer flat. I refer to the RSI as being at “best focus” when the wavefronts incident on detectors  709  and  710  are flat. There are various ways to adjust the RSI focus. One way is to adjust the locations of lenses  703  and  704 , possibly including the separation between the two lenses, in the “+/−z” direction. 
       Advantages 
       [0076]    As demonstrated above, the RSI lightsheet microscope  450  provides multiple advantages. For example, compared to many current lightsheet microscopes, the tilt-alignment and the z-alignment are less exacting. This mitigates one or more of the negative impacts listed above under Difficulty #1. The reason the tilt-alignment and the z-alignment are less exacting is as follows. The depth-of-field of the detection microscope of the exemplary RSI lightsheet microscope  450  is larger than the depth-of-field of the detection microscope in many current lightsheet microscopes. A larger depth-of-field makes it easier to perform the tilt-alignment and z-alignment. In addition, compared to the detection microscope in many current lightsheet microscopes (a conventional microscope), the lateral spatial resolution of the detection microscope of the exemplary RSI lightsheet microscope  450  is superior (as measured by the area under the MTF curve) when using spatially-incoherent light, which mitigates Difficulty #2 noted above. 
         [0077]    In addition to the particular exemplary RSI lightsheet microscope  450  disclosed above, further variations are included within the scope of the present invention. For example, the lightsheet microscope  450  may include more than one lightsheet source, such as counter-propagating, co-planar lightsheet illumination of a sample as disclosed for example in U.S. patent application publication 2011/0115895. In addition, the RSI lightsheet microscope  450  can use more than one detection microscope. For example, a second detection microscope may view the sample from a perspective 180 degrees away from the first detection microscope. This is illustrated in  FIG. 10 . Sample  1000  is illuminated by the lightsheet generated by  1001  and  1002  working in concert. Objective  1003  presents light from the sample to RSI  1004 . Objective  1005  presents light from the sample to RSI  1006 . Reference  1050  refers to the entire system. More than two detection microscopes may also be used. Furthermore, a microscope objective may be used for both transmission of light to the sample and receipt of light from the sample. 
         [0078]    Still further, adaptive optics may be incorporated into RSI lightsheet microscopes in accordance with the present invention. One use of adaptive optics is to compensate for the otherwise-detrimental light-scattering properties of the sample. Additionally, the RSI  404  may be constructed and used in a number of configurations, such as a Michelson or Mach-Zehnder configuration. 
         [0079]    Moreover, the rotational-shear angle of the RSI can be set to different values. There are different ways to set the rotational-shear angle to a given value. For example consider the case of a 180-degree rotational-shear angle. This corresponds to a counter-tilt of the two beams incident on the RSI detector. One way to produce a counter-tilt is to use an odd number of reflections in one arm of the interferometer and an even number of reflections in the other arm. A different way to produce a counter-tilt is to send the light in one arm of the interferometer through an intermediate focus within the arm. 
         [0080]    One can also adjust the angle at which the two beams are incident on the detector. For a point at the center of the field-of-view, the two resulting beams can be incident on the detector at normal incidence or at some different angle (e.g., +/−3 degrees). If the two beams are incident at normal incidence, there will be ambiguity (the twin image problem). If the angle-of-incidence of each beam corresponding to the center of the field-of-view is large enough, the twin image problem is avoided. 
         [0081]    Still further, the RSI may be used in a modified form known as a quadrature-phase interferometer. 
         [0082]    The RSI  404  may also use fringe-scanning to obtain a time series of exposures with different phase differences between the two arms of the interferometer. The RSI  404  may be configured to compensate or correct for differences in the polarization response of the two arms of the interferometer, for example by the addition of phase plates. The RSI  404  may further be configured to achromatize the fringe pattern to increase the spectral bandwidth of the RSI  404 . The RSI  404  may use mirrors that may or may not contain a roofline through the middle of the mirror, and may optionally include a prism to steer light. 
         [0083]    Different types of beamsplitters may also be used within the RSI  404 , such as cube or pellicle beam splitters, or even a glass plate that reflects off one of its external surfaces. There are also different ways to convert the fringe pattern recorded on the RSI detector into an image. One method is to Fourier-transform the fringe pattern, and a second method is to fit the fringe pattern with a set of orthogonal functions. In the case of a sparse image, a procedure exists to convert the fringe pattern recorded on the RSI detector into an image with spectral information for each point in the image. 
         [0084]    Sometimes the lightsheet activates quantum dots rather than fluorophores. Sometimes it is scattered lightsheet-light from small particles like beads that is used for the imaging. 
         [0085]    An RSI lightsheet microscope  450  in accordance with the present invention may be used in conjunction with a technique like Photo-Activated Localization Microscopy (PALM) or Stochastic Optical Reconstruction Microscopy (STORM). PALM and STORM are used for imaging on a spatial scale smaller than the wavelength of light. An RSI lightsheet microscope  450  used in accordance with the present invention may be configured for two-photon lightsheet microscopy. 
         [0086]    Different techniques can be used to generate the lightsheet, such as a cylindrical lens or the rapid scanning of an axial beam. Different beams can be used in the lightsheet, such as Gaussian beams or Bessel beams. 
         [0087]    There are other techniques for stepping the lightsheet through the sample (or the sample through the lightsheet). For example the stepping may skip regions of the sample known to be empty of interesting targets. 
         [0088]    These and other advantages of the present invention will be apparent to those skilled in the art from the foregoing specification. Accordingly, it will be recognized by those skilled in the art that changes or modifications may be made to the above-described embodiments without departing from the broad inventive concepts of the invention. It should therefore be understood that this invention is not limited to the particular embodiments described herein, but is intended to include all changes and modifications that are within the scope and spirit of the invention as set forth in the claims.