Patent Description:
The present disclosure relates to microscopy systems, and specifically to holographic optical microscopy.

Microscopy is a useful tool for imaging of a wide variety of samples, structural features, and objects that cannot be resolved by the unaided human eye. Different microscopy techniques are often employed for imaging of different targets. For example, fluorescent microscopy is often used to image biological samples and tissues, while scanning electron microscopy is often used to image nanoscale features and particles. Many microscopy techniques provide intense excitation radiation to the target to be imaged, which may compromise the function of biological tissues, cause photobleaching of samples, or otherwise degrade the structural integrity or electrical properties of the target. In some instances low intensity microscopy may be used to image sensitive samples and materials, but low intensity techniques require long image capture times, which are not feasible for imaging biological processes, fabrication processes (e.g., atomic layer deposition, or another fabrication to be monitored), or any changes of a sample in real time.

Light sheet microscopy is a high speed, low excitation intensity, 3D imaging technique. During light sheet microscopy, a plane of a material is illuminated using a sheet of light, and a microscope objective is configured to view the plane from an orthogonal direction as compared to the illumination. While light sheet microscopy provides advantages over other known microscopy techniques, excitation light sheets disperse rapidly in refractive tissue, which limits the penetration depth of a target or sample material. A reduced penetration depth is undesirable for many imaging applications including in live cell imaging in which the low excitation intensities of light sheets provide a major advantage, but in which the substrate can be strongly refractive. Further, a reduced penetration depth results in a loss of three-dimensional imaging information, which results in low contrast images of three-dimensional objects and the inability to resolve fine three-dimensional features. Further, if three-dimensional imaging is needed, additional mechanical components and optics may be required to obtain multiple images to generate an adequate three-dimensional image.

One light sheet microscopy approach, known as Lattice Light-Sheet Microscopy (LLSM), uses a convergent lattice of Bessel beams to generate the light sheet. In LLSM the excitation is confined to a plane defined by a lattice of intersecting Bessel beams that self-reinforce as they project through a target. A reflected beam is then viewed orthogonally as compared to the propagation of the excitation, allowing low excitation intensity illumination of planes to be viewed rapidly in sequence with little photobleaching or interacting with the target. The self-reinforcing nature of the Bessel beams increases the penetration depth as compared to other light sheet microscopy techniques. Nevertheless, LLSM is still limited to an imaging depth of approximately <NUM> in live tissue due to optical distortions. Attempts at improving the penetration depth have employed additional optics that has increased the penetration depth to around <NUM>. However, the required additions are technically challenging and require very expensive equipment. Further, imaging capabilities using LLS are limited when moving bulky detection objectives and not practical for many applications (e.g., biological and tissue imaging) because motors and translation stages have limited ranges of motion, and the target may not withstand the motion of the moving objective and optics. Additionally, the deeper penetration depths allow for dispersion and distortion of the lattice to compound with greater distortion due to the penetration of the target. While advances in microscopy techniques have allowed for the imaging of a wide-variety of targets, current methods still suffer from multiple drawbacks such as requiring complex optical setups, high intensity excitation radiation, long image acquisition times, and expensive equipment, among others.

In one aspect, there is a method for performing holographic microscopy. The method includes providing, from a light sheet microscope to a modulator, radiation having a (i) phase, (ii) amplitude, and (iii) Poynting vector, modulating, by the modulator, the phase of the radiation to generate a plurality of beams, and detecting, at a detection plane of a detector module, the plurality of beams. The method further includes generating, by the detector module, a signal indicative of an interference pattern of the plurality of beams, and generating, by a processor, a holographic image from the signal indicative of the interference pattern.

In embodiments, the modulator is a spatial light modulator. In some embodiments, the radiation includes incoherent radiation. Further, in embodiments, the plurality of beams includes two beams have a phase offset of <NUM>°, <NUM>°, <NUM>°, or <NUM>° from each other. In embodiments, the method further includes collecting, by a microscope objective positioned at a first distance from the sample, the radiation from a sample and altering, by an actuator, the distance between the microscope objective and the sample from the first distance to a second distance, wherein the second distance is a different distance than the first distance; and collecting, by the microscope objective positioned at the second distance, the radiation from the sample.

In another aspect, there is a microscopy system including a light sheet microscope configured to provide radiation, the radiation having a (i) phase, (ii) amplitude, and (iii) Poynting vector, the Poynting vector having a direction indicative of a direction of propagation of the radiation. A modulator, disposed along the direction of the Poynting vector, is configured to modulate a phase of the radiation to generate a plurality of beams. A detector module, disposed along the direction of the Poynting vector, is configured to detect, at a detector plane of the detector module, the plurality of beams, and is further configured to generate a signal indicative of an interference pattern of the plurality of beams. A processor is communicatively coupled to the detector module, and is further configured to receive the signal indicative of the interference pattern, and to generate a holographic image from the signal.

Advantages will become apparent to those skilled in the art from the following description of the preferred embodiments, which have been shown and described by way of illustration. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive. The invention is solely defined by the appended claims.

An incoherent holographic microscopy detection method and associated system are disclosed. The microscopy technique employs incoherent holographic microscopy lattice light-sheet (IHLLS) imaging to generate three-dimensional images of a target. The method employs Fresnel incoherent correlation holography to generate the three-dimensional images. The incoherent imaging disclosed utilizes a scanning geometry with two imaging planes, reduces optical design and hardware complexity, and overall system cost as compared to other microscopy techniques. Further, the disclosed system and method allows for the generation of amplitude and phase three-dimensional images using numerical processing by digital holography.

Digital holography is a powerful three-dimensional (3D) imaging tool. In digital holography the 3D image of a sample volume is formed from determining and analyzing a complex-amplitude distribution of radiation. Most common digital holography techniques use coherent light, such as lasers, to illuminate a sample or target to image the target. The use of coherent light introduces noise from speckle and spurious interference, which limits the imaging resolution and contrast. Further, coherent light sources often provide high intensity excitation radiation which may damage, or otherwise be unsuitable, for imaging of certain materials (e.g., biological samples, tissues, electrically active materials, etc.). The incoherent holographic imaging described herein employs spatially incoherent light (e.g., fluorescent light, black body radiation, etc.) to form holograms and produce images with improved spatial resolution than conventional microscopy techniques.

The incoherent holography method and system disclosed utilizes a modified dual-lens Fresnel incoherent correlation holography technique to produce a complex hologram and to provide a reconstruction distance needed for the reconstruction of the hologram. As described herein, the IHLLS system exhibits a significant contrast improvement over other imaging techniques for imageing of beads and neuronal structures within a biological test sample, as well as quantitative phase imaging. The IHLLS also demonstrates improved transverse imaging resolutions as compared to traditional lattice light-sheet technique.

The disclosed system and method enable the generation of 3D holographic images without requiring the movement of any of a sample stage or detection microscope objective, which simplifies the system setup and reduces the need for mechanical components and controls. Further, by moving only galvanometric mirrors, the described system provides increased resolution and image accuracy as compared to known glass-optics LLS schemes. While not requiring the movement of system components, it may be desirable to perform a deeper volumetric reconstruction for a holographic image, and as such, the described IHLLS may include mechanical components. IHLLS allows for holographic volume reconstruction from fewer position displacements than other holography 3D imaging techniques reducing volume image acquisition time. For example, the described system may reconstruct a holographic image using <NUM> spatial positions of an objective, whereas traditional methods would require nearly <NUM> positions for the same objective. Capturing and reconstructing images at multiple objective positions may also improve axial resolution to achieve better localization of ample points and improve axial resolution of images. The optical and mechanical design of the described IHLLS system expands the applicability of lattice light-sheet systems and could open entirely new imaging modalities in all light sheet imaging instruments. The incoherent holography configuration described could be added as an accessory, or as an add-on feature, to any imaging system that uses Bessel or Gaussian beams for scanning and imaging.

<FIG> is a schematic diagram of an embodiment of a holographic microscopy detection apparatus <NUM> for performing IHLLS as described herein. The holographic microscopy apparatus <NUM> includes a modulator <NUM> and a detector module <NUM>. Radiation <NUM> is provided to the modulator <NUM> by a light sheet microscope, and the modulator <NUM> modulates the radiation <NUM> to generate a plurality of beams <NUM> from the radiation <NUM>. The radiation <NUM> may be incoherent radiation that has a phase, amplitude, wavelength, and a Poynting vector indicative of the propagation direction of the radiation <NUM>. In embodiments, the radiation <NUM> may be emitted continuous wave radiation or pulsed radiation before reaching the modulator, and the modulated plurality of beams <NUM> may be continuous or pulsed beams. The plurality of beams <NUM> is illustrated in <FIG> as a single beam for visual clarity, generated by two diffractive lenses superimposed on the modulator, referred to and further described herein as IHLLS <NUM>. In implementations, each beam of the plurality of beams may co-propagate along a direction of a same Poynting vector, or beams of the plurality of beams <NUM> may propagate along different Poynting vectors. The plurality of beams <NUM> may include two beams, three beams, four or more beams with at least two of the beams of the plurality of beams <NUM> being spatiotemporally overlapped. At least two of the beams of the plurality of beams <NUM> must be partially, or entirely, spatiotemporally overlapped to generate an interference pattern between the overlapped beams.

The detector <NUM> is disposed along a propagation direction of the plurality of beams <NUM> (e.g., along one or more Poynting vectors of a beam of the plurality of beams), with the detector <NUM> configured to detect the interference pattern of the overlapped beams. The detector <NUM> may include one or more of a CMOS detector, a charge-coupled device (CCD), one or more photodiodes or photodiode arrays, a photovoltaic detector, a photomultiplier tube, a metal-oxide-semiconductor (MOS) capacitor, or an infrared sensor. The detector <NUM> is configured to generate a signal indicative of the detected interference pattern, and to provide the generated signal to a processor <NUM> for performing hologram generation, hologram reconstruction in amplitude and phase, and image processing. Further, the processor <NUM> may be configured to perform any required processing, calculations, or functions described herein to generate a holographic image.

In embodiments, the holographic microscopy apparatus <NUM> may include additional optical elements for performing wavelength filtering, polarization filtering, reflection, magnification, lensing, beam splitting, amplitude modulation, phase modulation, or for performing another optical process. In the embodiment illustrated in <FIG>, the holographic microscopy apparatus <NUM> includes a first lens 112a and a second lens 112b configured to operate together as a telescope to magnify, or demagnify, the radiation <NUM>. It may be desirable to increase or decrease the size of a wavefront of the radiation <NUM> according to the size of the wavefront <NUM> as it enters the holographic microscopy apparatus <NUM>, and/or according to a size of an active area of the modulator <NUM>. The active area of the modulator <NUM> is an area of the modulator <NUM> that actively modulates the phase and/or amplitude of the radiation <NUM>. To prevent loss of radiation <NUM> it may be desirable for the first and second lenses 112a and 112b to magnify the wavefront of the radiation <NUM> to a size that is equal to, or smaller than, the active area of the modulator <NUM>. While the first and second lenses 112a and 112b are described herein as magnifying the radiation <NUM>, other elements may be employed to magnify the radiation <NUM>. For example, a mirror, spatial light modulator, telescope, objective, or other magnifying element may be used to magnify the radiation <NUM>.

The holographic microscopy apparatus <NUM> may further include a third lens 114a and a fourth lens 114b configured to operate together as a telescope to magnify the plurality of beams <NUM>. In embodiments, it may be desirable to increase or decrease the size of one or more beams of the plurality of beams <NUM> according to the size of a detection area of the detector module <NUM>, and/or the size of the active area of the modulator <NUM>. The detector module <NUM> may include one or more sensors that have defined detection areas for detecting one or more of the beams of the plurality of beams <NUM>, and/or for detecting interference between beams of the plurality of beams <NUM>. To prevent loss of radiation <NUM>, it may be desirable to for the third and fourth lenses 114a and 114b to magnify one or more beams of the plurality of beams <NUM> to a transverse size that is equal to, or smaller than an overall detection area of the detector module <NUM> (i.e., the combined detection area of the one or more sensors of the detection module <NUM>). While the third and fourth lenses 114a and 114b are described herein as magnifying the plurality of beams <NUM>, other elements may be employed to magnify the plurality of beams <NUM>. For example, a mirror, spatial light modulator, telescope, objective, or other magnifying element may be used to magnify the plurality of beams <NUM>.

The holographic microscopy apparatus <NUM> may include a wavelength filter <NUM> disposed along a direction of the Poynting vector of the radiation <NUM> configured to filter out wavelengths of the radiation <NUM>. In embodiments, the wavelength filter <NUM> may include one or more of a low-pass filter, high-pass filter, notch filter, bandpass filter, or another type of wavelength filter. The holographic microscopy apparatus <NUM> may further include a polarizer <NUM> disposed along a direction of the Poynting vector of the radiation <NUM> configured to filter out polarizations of the radiation <NUM>. In embodiments, the polarizer <NUM> may include one or more polarizers configured to transmit, or filter out, horizontally polarized radiation, vertically polarized radiation, diagonally polarized radiation, circularly polarized radiation, elliptically polarized radiation, or a superposition of polarizations. The polarizer <NUM> may include a polarizer configured to transmit radiation having a polarization parallel to an active axis of the modulator <NUM>. The wavelength filter <NUM> and the polarizer <NUM>, may provide filtering and polarizing of the radiation to increase a signal-to-noise (SNR) ratio of the radiation <NUM>, to further improve an SNR of the signal generated by the detector module <NUM>. The apparatus <NUM> may further include mirrors <NUM> for directing the radiation <NUM> and the plurality of beams <NUM> throughout the apparatus <NUM> as required. Further, in embodiments, the holographic microscopy apparatus may include one or more spatial filters, amplitude modulators, phase modulators, mirrors, wavelength filters, beam splitters, diffractive elements, prisms, lenses, refractive elements, or other optical elements. In addition to modulating the radiation <NUM> to form the plurality of beams <NUM>, the modulator <NUM> may additionally be configured to modulate the radiation <NUM> to correct for aberrations and distortions due to other optical elements of the apparatus <NUM>. For example, the modulator <NUM> may provide phase corrections due to imperfections of lens surfaces, imperfections of mirror surfaces, index of refraction inconsistencies, debris, or another optical aberration. Additionally, the modulator <NUM> may be configured to provide a phase to the radiation <NUM> to correct for phase errors due to the modulator <NUM> itself.

The holographic microscopy apparatus <NUM> is configured to be an addition to, or add-on to, another imaging system. As illustrated in <FIG>, the holographic microscopy apparatus <NUM> is, according to the invention, added onto an LLSM imaging system <NUM>, with the LLSM imaging system <NUM> configured to provide the radiation to the holographic microscopy apparatus <NUM>. The LLSM imaging system <NUM> includes a radiation source <NUM> that provides illumination radiation <NUM> to an illumination focusing element <NUM>. The illumination focusing element <NUM> focuses the illumination radiation <NUM> onto a target <NUM>, also refered to as a sample, to image the target <NUM>. The target <NUM> is disposed on a lattice stage <NUM>. The target <NUM> and the lattice stage <NUM> reflect the illumination radiation <NUM>, and an objective <NUM> is physically configured to collect the reflected illumination radiation. The objective <NUM> magnifies the reflected radiation and directs the reflected radiation toward the holographic microscopy apparatus <NUM> as the radiation <NUM> provided to the holographic microscopy apparatus <NUM>. In embodiments, additional, coupling optics may be employed to direct the radiation <NUM> from the objective <NUM> to the modulator <NUM> of the holographic microscopy apparatus <NUM>, thereby optically coupling the imaging system <NUM> to the holographic microscopy apparatus <NUM>. For example, as illustrated in <FIG>, a mirror <NUM> may direct the radiation <NUM> from the objective <NUM> of the LLSM imaging system <NUM> to the holographic microscopy apparatus <NUM>. Therefore, the LLSM system <NUM> is a source of the radiation <NUM> for the holographic microscopy apparatus <NUM>. In embodiments, an actuator may be physically coupled to the objective <NUM> to control a position of the objective <NUM>. The actuator may be configured to alter a distance between the objective <NUM> and the target <NUM>. In embodiments, the actuator may be a galvanometer, translation stage, piezoelectric device, or another actuator. Further, in embodiments, an actuator may be physically coupled to the lattice stage <NUM> to alter the position of the lattice stage <NUM>.

<FIG> is a flow diagram of a method <NUM> for performing IHLLS imaging as described herein. The method <NUM> of <FIG> is performed by the holographic microscopy apparatus <NUM> of <FIG>. Referring now simultaneously to <FIG> and <FIG>, the method <NUM> includes providing the radiation <NUM> to the modulator <NUM> (block <NUM>). The modulator <NUM> is configured to modulate the radiation <NUM> according to modulation phase and amplitude profiles. The modulation phase and amplitude profiles are configured such that the modulator <NUM> modulates the radiation <NUM> to generate a plurality of beams <NUM> (block <NUM>). In embodiments, the modulator <NUM> may include a spatial light modulator (SLM) configured to reflect and modulate the radiation <NUM> as the plurality of beams <NUM>. In other embodiments, the modulator <NUM> may be a modulator that transmits the radiation <NUM> to form the plurality of beams <NUM>.

The method <NUM> further includes detecting the plurality of beams <NUM> (block <NUM>). The detector module <NUM> is configured to detect one or more of the plurality of beams, or to detect an interference pattern of two or more beams of the plurality of beams <NUM>. The detector module <NUM> generates a signal indicative of the detected plurality of beams, and/or the detected interference pattern (block <NUM>). The detector module <NUM> provides the signal to the processor <NUM> and the processor <NUM> generates a holographic image from the signal (block <NUM>). The processor <NUM> may perform one or more machine executable instructions that cause the processor to perform one or more signal processing techniques, image processing techniques, optical analyses, transformations, or other processes for generating the holographic image. The generated holographic image may be presented to a user via a user interface, stored in a memory, provided to another storage device, provided to another system, and/. or further processed.

<FIG>, <FIG> are schematic diagrams of optical layouts for an IHLLS system <NUM>. <FIG> is a schematic diagram of an IHLLS system having single diffractive lens configuration, while <FIG> are schematic diagrams of an IHLLS system having two diffractive lenses. <FIG>, <FIG> include a source of radiation <NUM> providing radiation <NUM> along an optical axis A to a first lens 312a. In <FIG>, <FIG>, the radiation <NUM> has a Poynting vector with a direction parallel to the optical axis A. The first lens 312a focuses the radiation and a second lens 312b collimates the radiation. Together, the first and second lenses 312a and 312b perform as a telescope to magnify the radiation <NUM>. A modulator <NUM> modulates the radiation <NUM> to generate a first beam 310a, illustrated in <FIG>, and a second beam 310b, illustrated in <FIG>, that co-propagate along the optical axis A. For the single diffractive lens configuration of <FIG>, the modulator <NUM> modulates the radiation <NUM> to form a single output beam <NUM>. A third and fourth lens 314a and 314b perform together as a second telescope to further magnify and focus the first and second beams 310a and 310b. The first beam 310a is focused to a first focal plane 330a, and the second beam 310b is focused to a second focal plane 330b that is at a different location along the optical axis A than the first focal plane 330a. A detector <NUM> is disposed along the optical axis A to detect the first and second beams <NUM> a and 310b, and any interference thereof.

To provide maximum interference of the first and second beams 310a and 310b at the detector <NUM>, it may be desirable for the first and second beams 310a and 310b to have as much transverse overlap as possible at the detector <NUM>. For example, as shown in <FIG> and <FIG>, the modulator <NUM> may modulate the radiation <NUM> such that the transverse size of the first beam 310a and second beam 310b are equal at the detector. Although, the first focal plane 330a of the first beam 310a is along the optical axis A a distance before the detector <NUM>, and the second focal plane 330b of the second beam 310b is along the optical axis A a distance behind the detector <NUM>. Therefore, while the first and second beams 310a and 310b may have a maximum amount of transverse overlap at the detector <NUM>, there may be a phase difference between the first beam 310a and second beam 310b at the detector <NUM>. The modulator <NUM> may be configured to focus the first and second beams 310a and 310b anywhere along the optical axis, and further, may modulate the first and second beams 310a and 310b to provide a desired phase offset between the first and second beams 310a and 310b.

<FIG>, <FIG> include distance parameters between the optical elements of the IHLLS system <NUM>. <FIG> illustrates the schematics of an IHLLS with only one diffractive lens, referred to as IHLLS <NUM>, and <FIG> illustrate the schematics of an IHLLS with two diffractive lenses, referred to as IHLLS <NUM>. Distance d<NUM> is the distance between the source of radiation <NUM> and a microscope objective MO. The distance d<NUM> is the distance between the microscope objective and the first lens 312a. The distance d<NUM> is the distance between the first lens 312a and the second lens 312b, distance d<NUM> is the distance between the second lens 312b and the modulator <NUM>, distance d<NUM> is the distance between the modulator <NUM> and the third lens 314a, distance d<NUM> is the distance between the third lens 314a and the fourth lens 314b, distance d<NUM> is the distance between the fourth lens 314b and the either the first focal plane 330a or the second focal plane 330b as illustrated by <FIG> respectively, and distance d<NUM> is the distance between the detector <NUM> and the first and second focal planes 330a and 330b as illustrated in <FIG> respectively.

To generate a holographic image as described in the IHLLS method and system described herein, a hologram must be obtained from the interference pattern of multiple beams at the detector <NUM>. For example, using the illustrations of <FIG>, the radiation <NUM> may be one or more Bessel beams having positive spherical wavefronts. As such, the interference between the first and second beams 310a and 310b, which are focused to the first and second focal planes 330a and 330b, may be determined by the expression <MAT> where Q is the quadratic phase function <MAT> θ is the phase shift of the SLM, fd<NUM> is the focal length of a first diffractive lens, and fd<NUM> is the focal length of a second diffractive lens. The first and second diffractive lenses are superimposed on the modulator <NUM>, and the first lens generates the first beam 310a and having a focus at the first focal plane 330a, and the second lens and C<NUM> = C<NUM> = <NUM> are constants. Therefore, employing the optical system and method of <FIG> and <FIG>, with EQS. <NUM> and <NUM>, the interference of a plurality of beams at a detector may be determined, and a holographic image may be generated. It should be noted, that by setting either one of the superimposed lens focal lengths to infinity, either fd<NUM> = ∞ or fd<NUM> = ∞, EQ. <NUM> reduces to a single lens term weighted by either C<NUM> or C<NUM>, effectively creating a single lens superimposed on the SLM. The single lens superimposed SLM will be described further herein in reference to single lens IHLLS imaging measurements.

To demonstrate the described IHLLS, a holographic apparatus according to the apparatus <NUM> of <FIG> was constructed. Similar to the illustration of <FIG>, an LLS system, such as the LLS system <NUM> of <FIG>, provided the radiation <NUM> to the constructed apparatus. For clarity, references to <FIG>, <FIG>, <FIG> will be used for the following examples of performing IHLLS imaging.

To demonstrate IHLLS imaging, an SLM was employed as the modulator <NUM> and the radiation <NUM> had a wavelength of <NUM>. The SLM was a phase SLM (Meadowlark; <NUM> × <NUM> pixels), that was recalibrated to phase shift the radiation <NUM> to form two beams as the plurality of beams <NUM>. The SLM was configured to apply a full range of <NUM> to 2π phase shift over its working range of <NUM> gray levels.

The detector module <NUM> was a CMOS ORCA camera and various components of the apparatus <NUM> and the LLS <NUM> were controlled by a Labview platform (National Instruments). A custom diffraction method was developed using MATLAB (Mathworks, Inc. ), and the complex hologram was propagated and reconstructed at a focal plane using the custom diffraction method.

Performance of the holographic microscopy apparatus <NUM> was compared to conventional LLS imaging by superimposing two different lens configurations onto the active surface of the SLM, (i) a single lens was superimposed on the SLM was to generate one beam from the radiation <NUM>, and (ii) two diffractive lenses were superimposed on the SLM to generate two beams as the plurality of beams <NUM>. The single lens configuration is referred to herein as IHLLS <NUM>, and the two diffractive lens configuration is referred to herein as IHLLS <NUM>.

Optical simulations of IHLLS were performed in two steps. First, the distances between each sequential optical component and the focal length of fSLM of a diffractive lens superimposed on the SLM, were calculated to match the transversal pixel magnification of <NUM> for both conventional LLS and IHLLS <NUM> (<FIG>) configurations, using OpticsStudio (Zemax, LLC) optical design. It was determined that with the radiation <NUM> having an emission wavelength of <NUM>, and with an overall transversal magnification set to <NUM>, the distances should be about , d<NUM> = <NUM> mm, d<NUM> = <NUM> mm, d<NUM> = <NUM> mm, d<NUM> = <NUM> mm, d<NUM> = <NUM> mm, d<NUM> = <NUM> mm, the distance between the fourth lens 314b to the detector <NUM> should be about <NUM> (i.e., d<NUM> = <NUM> mm), and the focal length of the single diffractive lens superimposed on the SLM was determined to be fSLM = <NUM> mm. For the first step, the transversal magnification was checked by imaging a USAF <NUM> resolution target, group <NUM>, element <NUM>, with both conventional LLS and the IHLLS <NUM> setup using a white light source. <FIG> is a y-x LLS image of the resolution target, and <FIG> is an x-y IHLLS <NUM> image of the resolution target. As evidenced by comparing the cross-sections, along the dashed lines, of group <NUM> of <FIG>, and element <NUM> of <FIG>, the IHLLS <NUM> provides the same magnification of <NUM> as the conventional LLS system. Additionally, an optimization of a multi-configuration optical system, as shown in <FIG>, was performed to calculate the focal lengths of two diffractive lenses superimposed on a spatial light modulator to provide maximum overlap of the two beams at the plane of the detector <NUM> and keeping all of the distances d<NUM> ÷ d<NUM> fixed as determined in the above for the previous step. After performing the optimization, the values of the two focal lengths were found to be fd<NUM> = <NUM> and fd2 = <NUM> mm. , which were used for the design of two diffractive lenses. The two lenses focus at a distance d<NUM> = <NUM> in the front of the camera, <FIG>, and at d<NUM> = <NUM> behind the camera, <FIG>, respectively. In implementation, the distance d<NUM> + d<NUM> may need to be tuned by ±<NUM> depending on tolerances and imperfections of optical parameters of other elements of the system (e.g., tolerances of lenses, tolerances of phase resolution of the SLM, etc.). (i.e., d7_1 + d<NUM> = <NUM> mm, when calculating fd<NUM>, and d<NUM> + d<NUM> = <NUM> mm, when calculating fd<NUM>. <FIG> are plots of the normalized image intensity cross-sections for the group <NUM>, and element <NUM> respectively, of the resolution target according to the images of <FIG>. In both of the plots of <FIG> there is a complete overlap of the intensities, which means a complete matching of the transversal magnification of the two modules, the LLS detection path and the IHLLS <NUM> detection path.

As previously discussed, to achieve high diffraction efficiency, and therefore high a contrast interference pattern at the detector <NUM>, the first and second beams 310a and 310b formed by the SLM must overlap at a detection plane of the detector module <NUM>. To determine the various distances between, and optical parameters of, the elements of the ILSSM system <NUM>, the OpticsSetup optical design software was used. Keeping all the distances d<NUM> to d<NUM> constant, and superimposing two diffractive lenses onto the SLM, a multi-configuration optical system was determined having the transverse height of the two beams equal in size at the camera plane (e.g., at the detector <NUM>). It was determined that, to provide a maximal overlap of the two beams at the detector <NUM>, the first focal plane 330a should be at a focal distance of fd<NUM> = <NUM> from the SLM, and the second focal plane 330b should be at a focal distance of fd<NUM> = <NUM> from the SLM. As previously mentioned, the various distances of the first and second focal planes 330a and 330b are approximate values, and in practice, they may be tuned by ±<NUM> to ±<NUM>, or more, depending on tolerances and imperfections of optical parameters of other elements of the system.

An IHLLS <NUM> imaging system was built according to the setups of <FIG>, <FIG> using the optical element distances and focal distances determined by the simulations, and by superimposing two lenses on the SLM. The constructed IHLLS imaging system was used to image fluorescent latex beads that fluoresce at <NUM> and <NUM> (λesc=<NUM>, λem = <NUM>, L-<NUM>, Molecular Probes, USA). The beads were placed in a solution having a concentration of <NUM>% of the solid beads, and the solution was diluted to a ratio of <NUM>:<NUM> with distilled water. The solution was centrifuged in a desktop centrifuge for <NUM> minute and clean coverslips were prepared by applying <NUM>µL of the solution as a thin layer that was left to dry. After drying, a cover slip was mounted in a sample holder of a conventional LLS system under distilled water. Three tests were performed to evaluate the performance of the IHLLS <NUM> imaging system. The first test was used a conventional LLS pathway having a z-galvonometer control the position of a microscope objective to collect images of multiple z-planes of the <NUM> flourescent beads. The z-galvonometer was stepped in increments of δzLLS = <NUM> through the focal plane of a 25x Nikon objective which was simultaneously moved the same distance with a z-piezo controller for a displacement range of Δzgalvo = <NUM>. The transverse scanning area of each image was <NUM>×<NUM><NUM> for the conventional LLS system. A second set of images of the <NUM> fluorescent beads was obtained using the IHLLS <NUM> with an SLM superimposed lens focal length of fSLM = <NUM>. Both the z-galvonometer and z-piezo were stepped by the same increment of δzLLS = <NUM> through the focal plane of the objective for the same displacement Δzgalvo = <NUM> as in the conventional LLS measurements. The two measurements described above, using the conventional LLS and IHLLS <NUM> imaging systems, were repeated to image the <NUM> fluorescent beads.

The two measurements described above, using the conventional LLS and IHLLS <NUM> imaging systems, were repeated to image the <NUM> fluorescent beads. For both the conventional LLS and the IHLLS <NUM> systems, the <NUM> beads were imaged with a transverse scanning area of <NUM>×<NUM><NUM>, a galvanometer displacement of Δzgalvo = <NUM>, by increments of δzLLS = <NUM>. For conventional LLS, the scanning area is typically limited to about <NUM>×<NUM><NUM>, with some operating up to <NUM>×<NUM><NUM>. Therefore, multiple 54x54 µm<NUM> images were obtained in a mosaic-fashion by moving the sample, to form a single image at a given z-galvonometer position. This multiple image mosaicing requires substantially longer acquisition time and images registration for an LLS system than the disclosed IHLLS <NUM>, and the IHLLS <NUM>, systems.

<FIG> are tomographic images of the <NUM> fluorescent beads obtained by the conventional LLS system and the IHLLS <NUM> system, respectively. The images of <FIG> were obtained using <NUM> steps of the z-galvonometer and have transverse scanning areas of <NUM> x <NUM><NUM>. <FIG> are tomographic images of the <NUM> fluorescent beads obtained by the conventional LLS system and the IHLLS <NUM> system respectively. The images of <FIG> were obtained using <NUM> steps of the z-galvonometer and have transverse scanning areas of 208x208 µm<NUM>. The Bessel beam profiles used to obtain each of the images of <FIG> though 5D are displayed in the upper left corners of <FIG> respectively.

In the measurements of the fluorescent beads, the IHLLS <NUM> system has an increased transverse scanning area and shorter imaging times than conventional LLS. One reason for these improvements is the larger potential scanning area of the IHLLS <NUM> system as compared to the conventional LLS system. It was also determined from <FIG> that the IHLLS <NUM> system exhibited a decrease in image resolution, in both lateral and axial directions, due to a blurring effect of the SLM due to the superimposed lens that has a focal distance of infinity, as previously described.

The IHLLS <NUM> system was also used to image the <NUM> and <NUM> fluorescent beads. The measurements performed by the IHLLS <NUM> system also translated the z-gavlonometer according to the total galvanometer translation, and displacement intervals as was used for the conventional LLS and IHLLS <NUM> measurements. The two beam wavefronts interfered at the detector <NUM> and created Fresnel holograms captured by the detector <NUM>. The modulator <NUM> was configured to provide multiple sets of beams with each set having two beams with a different phase offset. Each set of two beams was generated by the modulator <NUM> at a different time than other sets of beams to generate a time-series of four different sets of beam pairs. Each set of beam pairs had a phase offset of either <NUM>°, <NUM>°, <NUM>°, or <NUM>° (i.e., phases of <NUM>, π/<NUM>, π, and 3π2). An image was captured at each of the four phase offsets at each position of the z-galvonometer. Therefore, each image plane in the z direction of the image target included four images, with each image capturing the interference pattern generated by a corresponding phase offset of the generated beams. The four images, for a given galvanometer position, were used to determine a complex amplitude of the wavefronts at the detector <NUM> according to the equation <MAT> where A(u, v) is an amplitude profile of the image, each term IH is an hologram image intensity for one of the four phase images, and ϕ(u, v) is the phase profile of the image according to <MAT>.

<FIG> are arrays of holographic images obtained by the IHLLS <NUM> system of the <NUM> and <NUM> fluorescent beads, respectively. Each column of the image arrays of <FIG> includes a set of images obtained at a given z-galvonometer position. A z-galvonometer position of zgalvo = <NUM> provides an image of a top layer of beads, while a position of zgalvo = -<NUM> provides images of a bottom layer of beads, and positions of ±<NUM>,±<NUM>, and <NUM>, provide images of beads that are equidistant between the top and bottom layers. Images of other z-galvonometer positions were obtained and are not presented in <FIG>. The rows of images include a top row of IHLLS holograms detected at the camera or detector <NUM>, a middle row of IHLLS phase images of ø as determined by EQ. <NUM>, and a bottom row of IHLLS reconstructed images of the complex amplitude of EQ. The phase images of the middle row contain depth dependent phase information derived from the IHLLS holograms of the top row of images. The complex holograms were propagated and reconstructed at a desired focal plane for increasing resolution and contrast using a custom diffraction Angular Spectrum method (ASM) routine in MATLAB (Mathworks, Inc. ) to generate the bottom row of IHLLS reconstructed images. The ASM was implemented instead of a Fresnel reconstruction since the ASM can reconstruct a wave field at any distance from the hologram plane with no restriction on the propagation distance. In embodiments, the reference plane for depth scanning is taken as the plane where the position of the z-galvanometric mirror coincides with the z-piezo stage objective position, which is the middle plane of the camera FOV. The sample is in focus at this position, referred to as 'Z-galvo <NUM>'. In embodiments, other reconstruction method schemes may be implemented as a combination of ASM and Fresnel methods. The ASM may be implemented for the in-focus samples and the Fresnel implemented for reconstruction of the out-of-focus samples.

<FIG> are images of the maximum composite projections of all of the reconstructed complex amplitudes at the z-galvonometer mirror positions z-galvo of <NUM>, ± <NUM>, ± <NUM>, and ± <NUM> for the <NUM> and <NUM> fluorescent beads, respectively, with high resolution and contrast. The images of <FIG> include an extended scanning range of ± <NUM>.

<FIG> present the transverse full-width at half-maximum (FWHM) values for images obtained by the conventional LLS, IHLLS <NUM>, and IHLLS <NUM> systems for the <NUM> beads and the <NUM> beads, respectively. A custom peak-finding routine was used to identify the locations for each bead. The peak-finding routine defined a region of interest (ROI) around each potential bead in each tomographic and holographic reconstructed image (i.e., the images of <FIG>, &a, and 7B). A least square fit to a 2D Gaussian function was performed in each region of interest and the center-of-mass position and FWHM in x and y for each ROI was determined. For the images of the <NUM> beads, conventional LLS had a FWHM of <NUM>, IHLLS <NUM> had a FWHM value of <NUM>, and the IHLLS <NUM> system had a FWHM value of <NUM>. The calculations for the <NUM> beads show an LLS FWHM of <NUM>, IHLLS <NUM> FWHM of <NUM>, and a IHLLS <NUM> FWHM of <NUM>. The FWHM values of the IHLLS <NUM> and IHLLS <NUM> systems are a factor of <NUM> to <NUM> times smaller than in LLS. With a * p-value ≤ <NUM>, ** p-value ≤ <NUM>, and *** p-value ≤ <NUM>, where p-value ≤ <NUM> is a statistically significant difference and p-value > <NUM> is not statistically significant difference.

The disclosed system and method for performing IHLLS may be useful for imaging of sensitive materials such as biological samples and tissues. For example, imaging of neuronal cells may provide the foundation for understanding many diseases in the human brain. Understanding and analyzing the physiological behavior of the neuronal cells requires the ability to observe cell structure and dynamics quantitatively to cellular and subcellular levels. The described IHLLS imaging system and method provides a noninvasive contrast imaging technique that uses low intensity radiation, which preserves the integrity of the cell. Further, neuronal cells are 3D structures that extend throughout tissue in all directions and many conventional microscopy methods are unable to resolve the cellular structures into 3D images. Conventional approaches are unable to image in the millisecond temporal range at multiple depths simultaneously. The disclosed IHLLS system allows for the 3D holographic imaging of neurons and other cells at imaging speeds capable of capturing physiological responses and cellular dynamics.

As an example, neuronal cells could respond electrically to chemical and electrical inputs. The electrical response could rapidly spread throughout the neuronal cell structure. The electrical response rapidly spreads throughout the neuronal cell structure. The electrical activity in the neuronal cell causes a change in the refractive index of the cell, which, can be imaged over time by the disclosed IHLLS imaging techniques. To demonstrate the observation of cellular behavior, an IHLLS system was constructed to image biological samples.

Quantitative phase cell imaging was performed using an IHLLS <NUM> system according to the description of the IHLLS <NUM> system above. The target to be imaged was a lamprey spinal cord ventral horn neuron with dendrites that were sufficiently large to cover the whole detector FOV of 208x208 µm<NUM>. Images of the target neuron were obtained using the conventional LLS system, the IHLLS <NUM> system, and the IHLLS <NUM> system. <FIG> is an array of images of the target neuronal cell obtained by each of the conventional LLS, IHLLS <NUM>, and IHLLS <NUM> systems.

<FIG>and <FIG> are tomographic images obtained by using conventional LLS and IHLLS <NUM>, respectively. The conventional LLS system had maximum transverse imaging area of <NUM>×<NUM><NUM>, and therefore, features of the target cannot be resolved outside of that area. Each of the images of <FIG>and <FIG> was obtained using a z-galvonometer and z-piezo stage objective that were positioned across a range of <NUM> over <NUM> steps. Deconvolution sharpening of the raw data was performed to reduce blur and enhance fine details of the images of <FIG>and <FIG> are images obtained by the IHLLS <NUM> system for the max projection of three reconstructed amplitude images from IHLLS holograms recorded at zgalvo = <NUM>, <NUM>, and -<NUM>. The reconstructed holographic images of <FIG>provide similar image quality as the LLS image of <FIG>, with the IHLLS <NUM> images resolving a larger imaging area of the target or sample (i.e., the neuronal cell). <FIG>provide quantitative phase information that is unable to be obtained by the conventional LLS system. The phase images of <FIG>are important for understanding the physiology and pathophysiology of various cells, tissues, and other types of biological samples. <FIG>are images of the phase information after a bandpass filter has been applied to the phase information. The bandpass filter applied in <FIG> allows for the attenuation of certain spatial frequencies in the images to accentuate specific features of the cell to be observed and further studied.

Claim 1:
A microscopy system comprising:
a light sheet microscope (<NUM>, <NUM>) configured to provide radiation (<NUM>), the radiation (<NUM>) having a (i) phase, (ii) amplitude), and (iii) Poynting vector, the Poynting vector having a direction indicative of a direction of propagation of the radiation;
a modulator (<NUM>, <NUM>) disposed along the direction of the Poynting vector, the modulator (<NUM>, <NUM>) configured to modulate a phase of the radiation to generate a plurality of beams (<NUM>);
a detector module (<NUM>) disposed along the direction of the Poynting vector, the detector module (<NUM>) configured to detect, at a detector plane of the detector module, the plurality of beams, the detector module (<NUM>) further configured to generate a signal indicative of an interference pattern of the plurality of beams; and
a processor (<NUM>) communicative coupled to the detector module (<NUM>), the processor (<NUM>) configured to receive the signal indicative of the interference pattern, and further configured to generate a holographic image from the signal.