Abstract:
A volume holographic imaging system, apparatus and/or method enables the projection of a two-dimensional (2D) slice of a four-dimensional (4D) probing object A 4D probing source object is illuminated to emit or scatter an optical field. A holographic element having one or more recorded holograms receives and diffracts the optical field into a diffracted plane beam having spectral information. A 4-f telecentric relay system includes a pupil filter on the relayed conjugate plane of the volume hologram and images the pupil of the volume hologram onto the front focal plane of the collector lens. A collector lens focuses the diffracted plane beam to a 2D slice of the 4D probing source object. The focused 2D slice is projected onto a 2D imaging plane. The holographic element may have multiple multiplexed holograms that are arranged to diffract light from the corresponding slice of the 4D probing source object.

Description:
RELATED APPLICATIONS 
       [0001]    This application claims priority to U.S. Provisional Application Ser. No. 61/250,306, entitled “Phase Contrast Multi-Focal Microscope” filed Oct. 9, 2009, U.S. Provisional Application Ser. No. 61/264,432, entitled “Wavelength-Coded Multi-Focal Microscope” filed Nov. 25, 2009 and U.S. Provisional Application Ser. No. 61/381,369, entitled “System, Method and Apparatus for Contrast Enhanced Multiplexing of Images” filed Sep. 9, 2010, each application in its entirety is incorporated herein by reference. This application is related to International Application PCT, Attorney Docket Number 118648-00120, entitled “System, Method and Apparatus for Phase Contrast Enhanced Multiplexing of Images” filed Oct. 8, 2010 and International Application PCT, Attorney Docket Number 118648-00420, entitled “System, Method and Apparatus for Wavelength-Coded Multi-Focal Microscopy” filed Oct. 8, 2010, each application in its entirety is incorporated herein by reference. 
     
    
     STATEMENT OF GOVERNMENT INTEREST 
       [0002]    The United States government has rights in this application as a result of financial support provided by governmental agencies in the development of aspects of the disclosure. Parts of this work were supported by a grant from the National Institutes of Health, Grant No.: R21CA118167 and the National Science Council Contract No.: NSC-97-2917-1-564-115. 
     
    
     BACKGROUND 
       [0003]    This disclosure relates generally to imaging systems, methods and apparatus, and more particularly to volume holographic imaging systems, methods and apparatus that obtain enhanced images from multiple depths within an object. 
         [0004]    Microscopic imaging systems are beneficial for biomedical and clinical applications. Volume holographic microscopy (VHM) has been developed as a microscopic instrument for biological samples. Volume imaging systems have many useful applications such as spectral and three spatial dimensional biological imaging (known as four-dimensional (4D) imaging), endoscope imaging systems, spectrometers, and the like. 
         [0005]    VHM incorporates multiplexed holographic gratings within a volume hologram to visualize structures at different focal planes in an object. Each focal plane within the object is projected to a different lateral location on the camera. Thus, the entire object volume is imaged slice-wise onto the camera without the need for electrical or mechanical scanning. However, many objects of interest are composed of weak phase features with poor contrast and are barely observable with VHM. 
       SUMMARY 
       [0006]    Embodiments taught herein relate generally to imaging systems, methods and apparatus, and more particularly to volume holographic imaging systems, methods and apparatus that obtain enhanced images from multiple depths within an object. 
         [0007]    An exemplary contrast enhanced multiplexing image system taught herein obtains contrast enhanced information from multiple depths within an object without scanning. A pupil filter is introduced into the Fourier plane of a 4-f telecentric relay system to enhance weak phase information from a volume holographic imaging system. The exemplary system can be expanded to include additional multiplexed holographic gratings within a single volume hologram and, hence, simultaneously image more object slices onto non-overlapping locations on an imaging plane without scanning. 
         [0008]    An exemplary microscope as taught herein includes focusing lenses, a holographic element, relay lenses, a pupil filter and an imaging plane. The lenses, holographic element and pupil filter together project an image onto the imaging plane. The pupil filter is advantageously located at the conjugate plane of the holographic element&#39;s pupil. The holographic element is a volume hologram with one or more multiplexed hologram gratings therein. The multiplexed holographic gratings are located at the Fourier plane of the microscope and are Bragg matched to a different focal plane within an object and simultaneously projected to a different lateral location on the imaging plane. In the exemplary embodiments, the holographic element is recorded in phenanthrenquinone doped poly methyl methacrylate. 
         [0009]    An exemplary volume imaging system for imaging a source object as taught herein includes a holographic element, collector optics and a pupil filter. The holographic element is capable of recording one or more holograms of the source object and is configured to receive and diffract an optical field emitted or scattered from the source object onto one or more diffracted plane beams. The collector optics are configured to focus each of the one or more diffracted plane beams to a two-dimensional slice of the source object, and simultaneously project the focused two-dimensional slice along an optical path onto an imaging plane. The pupil filter is disposed along the optical path to reduce the DC component in the spatial frequency domain of the focused two-dimensional slice of the source object. 
         [0010]    An exemplary method for imaging an object in four-dimensions and real time in which an emitted or scattered optical field of an object is received by a holographic element which diffracts the received optical field into one or more diffracted plane beams. The diffracted plane beams are focused into a two-dimensional slice of the object and filtered. The filtered two-dimensional slice is projected onto an imaging plane. When two or more slices of the object are projected, the slices are simultaneously projected to non-overlapping regions of the imaging plane. The filtering step is performed using a pupil filter. The diffraction is based on one or more Bragg degeneracy properties. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0011]    The foregoing and other objects, aspects, features, and advantages of exemplary embodiments will become more apparent and may be better understood by referring to the following description taken in conjunction with the accompanying drawings, in which: 
           [0012]      FIG. 1  depicts an illustrative diagrammatic view of a recording arrangement for multiplexing holographic gratings within a volume hologram as taught herein. 
           [0013]      FIG. 2  depicts an illustrative diagrammatic view of an exemplary volume holographic microscope as taught herein. 
           [0014]      FIG. 3  depicts an illustrative diagrammatic view of the 4-f telecentric relay system of the volume holographic microscope of  FIG. 2 . 
           [0015]      FIG. 4  is a flow diagram depicting an illustrative method for practicing an embodiment of an exemplary volume holographic imaging system as taught herein. 
           [0016]      FIG. 5  illustrates an image of a mouse colon sample obtained by a conventional volume holographic microscope. 
           [0017]      FIG. 6  illustrates an image of the mouse colon sample used in  FIG. 5  obtained by an exemplary volume holographic microscope as taught herein. 
       
    
    
     DESCRIPTION OF EXEMPLARY EMBODIMENTS 
       [0018]    In accordance with various embodiments taught herein are volume holographic imaging systems that employ a pupil filter to obtain contrast enhanced images from multiple depths within an object. An exemplary volume holographic imaging system can obtain contrast enhanced information from multiple depths within biological samples without scanning. An exemplary volume holographic imaging system enhances weak phase information of the displayed images which are from different depths within biological samples by introducing a pupil filter at the plane conjugate to the volume holographic pupil during imaging. This enhances weak phase features from multiple depths. An exemplary volume holographic imaging system images the entire object volume in real time without electrical or mechanical scanning, and provides enhanced edge and contrast information at all slices simultaneously. The volume hologram imaging system may be a microscope, spectroscope, endoscope, and the like and may be known as an enhanced volume holographic microscope (E-VHM). 
         [0019]    A mouse colon placed in the exemplary volume holographic imaging system as taught herein results in two-depth resolved images separated by approximately 50 μm simultaneously displayed on an imaging plane. With the enhanced volume holographic method for contrast enhancement, the exemplary imaging system improves contrast of objects over the conventional VHM methods. 
         [0020]      FIG. 1  illustrates an exemplary recording arrangement  100  for multiplexing holographic gratings, or recording multiple holographic gratings, within a volume hologram  124  using a source of electromagnetic radiation such as a collimated laser beam. A holographic grating may be created in a transmissive volume hologram by recording the interference pattern of two mutually coherent light beams. In an exemplary embodiment, a collimated laser beam, not shown, is split into a reference arm  115  and a signal arm  117 . A point source  120  along the reference arm  115  is formed by lens  116 . The point source  120  provides the source of electromagnetic radiation along the reference arm  115  which interferes with the signal arm  117  to record a grating in the multiplexed volume hologram  124 . More than one grating is formed in multiplexed volume hologram  124  by varying the position of the point source  120  in the reference arm, by moving lens  116  while lens  118  stays fixed, between different exposures of electromagnetic radiation from the collimated laser beam. The nominal inter-beam angle θ is the angle between signal arm  117  and reference arm  115  at the volume hologram  124  surface and is changed by Δθ between exposures. 
         [0021]    In some embodiments, the nominal inter-beam angle in air is 68°, Δθ is 1°, and Δz is 50 μm. In the same embodiment, the recording medium of volume hologram  124  is phenanthrenquinone doped poly methyl methacrylate (PQ-doped PMMA) and the collimated laser beam is an argon-ion (Ar + ) laser operating at a wavelength of approximately 488 nm. 
         [0022]    Other materials may be used as a recording medium. By way of example, Aprilis ULSH-500, LiNbO 3  including Zn-doped LiNbO 3  and DuPont photopolymers may be used as recording material. (See Atsushi Sato et al, Applied Optics vol. 42, pp. 778-784, (2003), Yasuo Tomita et al, Optics Express vol. 14, pp. 5773-5778 (2006), and Raymond K. Kostuk et al, Applied Optics vol. 38, pp. 1357-1363 (1999)). Those skilled in the art will appreciate that each material has a range of sensitivity for recording and that another source of electromagnetic radiation with appropriate wavelength in the corresponding range of sensitivity may be used for recording. With proper fabrication, the multiplexed holographic gratings within a volume hologram can operate at wavelengths longer than the recording wavelength of signal arm  117  and reference arm  115 . (See Y. Luo, P. J. Gelsinger, J. K. Barton, G. Barbastathis, and R. K. Kostuk, Opt. Lett. Vol. 33, 566-568 (2008) which is incorporated by reference herein in its entirety). In the same embodiment, the diffraction efficiencies of the two multiplexed gratings are approximately 40% and approximately 35%, the thickness of the PQ-doped PMMA recording material is approximately 1.5 mm, and the numerical apertures of lens  116  and lens  118  are 0.65 and 0.55, respectively. 
         [0023]      FIG. 2  illustrates an exemplary imaging system  200  which may be an E-VHM system as taught herein. The system  200  includes an optional source of electromagnetic radiation  201 , an objective lens  222 , the multiplexed volume hologram  124 , relay lenses  232  and  234 , a pupil filter  236 , a collector lens  226 , and an imaging plane  240 . Source  201  emits an electromagnetic field along signal arm  203  to object  210 . An objective lens  222  acts to collimate the optical field emitted or scattered from the object  210 . The collimated field passes through the multiplexed volume hologram  124  towards relay lenses  232  and  234 . The emitted holographic representation from the multiplexed volume hologram  124  is relayed by lenses  232  and  234  towards the pupil filter  236 . The filtered representation from the pupil filter  236  is collected by the collector lens  226  which projects images to the imaging plane  240 . In an exemplary embodiment, the multiplexed volume hologram  124  has two multiplexed gratings. Each grating is Bragg matched to a different two-dimensional (2D) slices of the object  210  taken along the y-axis at first focal plane  212  and second focal plane  214 . Thus, in the same embodiment, images of focal planes  212  and  214  are simultaneously projected by the system  200  to non-overlapping lateral locations,  242  and  244 , respectively, on the image plane  240 . The gratings are diffractive elements consisting of a periodic phase or absorption perturbation throughout the entire volume of the holographic element. When a beam of incident light satisfies the Bragg phase matching condition it is diffracted by the periodic perturbation. Those skilled in the art would appreciate that Bragg matched refers to satisfying the Bragg matching condition which occurs when the diffraction efficiency of a transmissive volume hologram is maximized. 
         [0024]    In an exemplary embodiment, the multiplexed volume hologram  124  is located at the Fourier plane of the objective lens  222 . Similarly, the imaging plane  240  is located at the Fourier plane of the collector lens  226 . In the same embodiment, the distance f o  is the distance between the second focal plane  214  and the objective lens  222 . Those skilled in the art would appreciate that the grating within multiplexed volume hologram  124  that is Bragged matched to the second focal plane  214  is located a distance of f o  from the objective lens  222 . Relatively positioned between the multiplexed volume hologram  124  and the collector lens  226  is a relay system composed of relay lenses  232  and  234 . Pupil filter  236  is located such that it images the pupil of the multiplexed volume hologram onto the front focal plane of the collector lens  226 . The distance f c  is the distance between the pupil filter  236  and the collector lens  226 , which is the same distance between the collector lens  226  and the imaging plane  240 . 
         [0025]    In exemplary embodiments, the source of electromagnetic radiation may be a plurality of coherent light sources, a broadband light source such as a dispersed white-light source with chromatic foci, a plurality of light emitting diodes or the like. The imaging plane  240  may be part of a charge couple device or camera which may be connected to or part of a computer, projector, or other such device. In some embodiments, the pupil filter may be any amplitude filter resulting in enhanced contrast information such as an opaque mask, gray scale mask, or the like. In some embodiments, the use of an opaque mask may result in a significant loss in intensity of the resulting image but this may be corrected by using a cooled charge-coupled device with high dynamic range as part of the imaging plane  240 . 
         [0026]      FIG. 3  depicts an exemplary placement relationship of the relay system located between lenses  232  and  234  of the imaging system of  FIG. 2 . The relay system located between lenses  232  and  234  is a 4-f telecentric system. The distance f R  is the distance between the multiplexed volume hologram  124  and the relay lens  232 . The distance between the relay lenses  132  and  134  is two times the length of distance f R . The distance f R  is also the distance between the relay lens  234  and the pupil filter  236 . Pupil filter  236  is therefore located on the conjugate plane of the multiplexed volume hologram  124  relayed through the 4-f telecentric relay system, i.e. on the 4-f telecentric relay system&#39;s Fourier plane. The pupil filter  236  eliminates the DC component in the spatial frequency domain to achieve the enhanced volume holographic method, as taught herein. This enhancement is observed in parallel at all the multiplexed focal planes (slice-wise images from multiple depths within object  210 ) of the exemplary imaging system  200 . 
         [0027]      FIG. 4  depicts an exemplary method of imaging an object defined in four-dimensional space and real time using an exemplary volume holographic imaging system as taught herein. In step  400 , multiplexed volume hologram  124  receives an optical field that has been scattered or emitted from the object  210  of interest. In some embodiments, the scattered or emitted optical field may be processed by one or more optical elements, such as the objective lens  222 , to focus the received optical field onto the volume hologram  124 . In step  410 , a grating within the multiplexed volume hologram  124  diffracts the received optical field into one or more plane beams. The plane beam is a holographic representation of a 2-D slice of the object  210  taken at a plane within the object  210  that is Bragg matched to the grating in the volume hologram  124 . In step  420 , the Fourier transform of the plane beam is formed by the relay lenses  232  and  234  at an intermediate plane located at the pupil filter  236 . In step  430 , the Fourier transform of the plane beam is filtered by the pupil filter  236  to a filtered Fourier transform of the plane beam. In step  440 , the Fourier transform of the plane beam diffracted from the pupil filter  236  is projected onto an imaging plane  240 . In some embodiments, the volume hologram  124  has two or more gratings recorded therein. In the same embodiment, the number of 2-D images that are simultaneously projected onto the imaging plane  240  in a non-overlapping manner correspond to the number of gratings. Advantageously, the multiple images are simultaneously projected to non-overlapping portions of the imaging plane. 
         [0028]      FIG. 5  depicts two depth resolved images of a mouse colon obtained using a conventional VHM.  FIG. 6  depicts two depth resolved images of the mouse colon of  FIG. 5  obtained using the exemplary imaging system  200  using an opaque mask as the filter  236 . The images in  FIGS. 5 and 6 , were obtained by the PQ-doped PMMA volume hologram  124  with two multiplexed gratings each grating imaging in parallel a different slice through the object: one slice just below the tissue surface and one approximately 75 μm in to the tissue. The mouse colon was illuminated using a red LED with central wavelength of approximately 630 nm and spectral bandwidth of approximately 25 nm using the exemplary imaging system  200  of  FIG. 2 . An Olympus objective lens (ULWDMSPlan 50X), a Mitutuyo collector lens (MPlanAPO20X), and an Andor iXon CCD array (Andor X-2647) were used to produce the images of  FIGS. 5-6 . The field of view of this embodiment was approximately 1 mm by 0.8 mm. 
         [0029]      FIGS. 5 and 6  further include the contrast ratio of four arbitrarily selected regions along an arbitrarily selected vertical line on each image (right-hand side inset of  FIG. 5  and  FIG. 6 ). Regions  501 ,  503 ,  505  and  507  in  FIG. 5  correspond to regions  601 ,  603 ,  605 , and  607 , respectively, in  FIG. 6 . Each region  601 ,  603 ,  605 , and  607  has a higher contrast ratio than the corresponding region  501 ,  503 ,  505  and  507 . In particular, each region,  601 ,  603 ,  605 , and  607 , has a higher amplitude than its corresponding region,  501 ,  503 ,  505  and  507 , resulting in a higher sustained peak contrast ratio value for each region  601 ,  603 ,  605 , and  607 . Thus at the four arbitrarily selected regions, in the two images, the contrast ratio in  FIG. 5  using the conventional VHM system was lower than at the same location in  FIG. 6  using the exemplary imaging system  200  as taught herein. 
         [0030]    Advantageously the exemplary imaging systems taught herein increase the identification of structures, such as the turbid media depicted in  FIGS. 5 and 6 . The resulting images are contrast-enhanced, two-dimensional and observable in real time. Furthermore, exemplary imaging systems as taught herein can be applied to both fluorescence and non-fluorescence imaging and collects both spectral and spatial information of an object without mechanically scanning in the X-Y-Z direction for a given field of view. 
         [0031]    Although the teachings herein have been described with reference to exemplary embodiments and implementations thereof, the disclosed methods, systems and apparatus are not limited to such exemplary embodiments/implementations. Rather, as will be readily apparent to persons skilled in the art from the description taught herein, the disclosed methods, systems and apparatus are susceptible to modifications, alterations and enhancements without departing from the spirit or scope hereof. Accordingly, all such modifications, alterations and enhancements within the scope hereof are encompassed herein.