Optical configurations for high resolution microscopy

Abstract

To provide optical configurations for confocal structured illumination Raman (C-SIM Raman) microscopy systems.SOLUTION: One example includes: a light source configured to project a light; a first set of optical components fixedly aligned along a first optical path, where the first set of optical components includes a vortex phase plate and a quarter wave plate, where the first set of optical components is configured to receive the light; and a second set of optical components configured to be adjusted between a first operating position and a second operating position. When in the first operating position, the second set of optical components is aligned on the first optical path and is configured to expand, in conjunction with the first set of optical components, the light into an expanded light. When in the second operating position, the second set of optical components is not aligned on the first optical path.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] FIELD OF THE DISCLOSURE The present disclosure relates generally to systems and methods for performing high-resolution microscopy, such as Raman spectroscopy. [Background technology]

[0002] Super-resolution imaging is a set of techniques that can be used to increase the resolution of microscopic imaging beyond the diffraction limit of standard confocal microscopy systems. An exemplary super-resolution imaging technique is confocal structured illumination (C-SIM) Raman microscopy, which improves the lateral resolution of Raman microscopy beyond the diffraction limit. C-SIM microscopy involves imaging a sample with a standard confocal scanning spot and separately with a donut-shaped scanning spot. Super-resolution is achieved by subtraction of these two images. Summary of the Invention

[0003] In one aspect, an optical system for structured illumination includes a first set of optical components fixedly aligned with a light beam incident on the optical system and a second set of optical components adjustable between a first operating position and a second operating position. When the second set of optical components is in the first operating position, the light beam is expanded and output as a first output beam. When the second set of optical components is in the second operating position, the light beam passes through the first set of optical components and forms a vortex beam that is output as a second output beam.

[0004] In some embodiments, the optical system is included in a structured illumination microscopy system. The microscopy system includes a light source configured to generate a light beam received by the optical system. The microscopy system includes at least one actuator for adjusting a second set of optical components between a first operating position and a second operating position, an objective lens for directing either the first output beam or the second output beam to the sample, a detector for receiving light from the sample, and a controller, the controller including a processor and non-transitory memory for storing computer-readable instructions. When the computer-readable instructions are executed by the processor, the microscopy system is configured to: adjust the second set of optical components via the actuator to the first operating position, direct the first output beam to the sample, and generate a first image based on the light received from the detector; adjust the second set of optical components via the actuator to the second operating position, direct the second output beam to the sample, and generate a second image based on the light received from the detector; and generate a sample image based on the first image and the second image.

[0005] There is no specific requirement that a system, method, or technique related to C-SIM microscopy include all of the details characterized herein to obtain some benefit from the present disclosure. Thus, the specific examples characterized herein are meant to be exemplary applications of the described techniques, and alternatives are possible. [Brief explanation of the drawings]

[0006] The features and advantages of the present technology will become more apparent from the following detailed description of exemplary embodiments thereof, taken in conjunction with the accompanying drawings. [Figure 1A-C] 1 is a simulated image depicting the subtraction microscopy process. [Figure 1D] 1 is a simulated image depicting the subtraction microscopy process. [Figure 2A] 10A-10C are further images depicting the subtraction microscopy process. [Figure 2B] 2B is a graph providing a cross section of the image of FIG. 2A. [Figure 3] 1 schematically illustrates an exemplary microscopy system. [Figure 4A] 4A and 4B schematically illustrate exemplary C-SIM optics included in the microscopy system of FIG. 3 according to one embodiment. [Figure 4B] 4A and 4B schematically illustrate exemplary C-SIM optics included in the microscopy system of FIG. 3 according to one embodiment. [Figure 5] 4B is an exemplary beam expander implemented with the C-SIM optics of FIG. 4A. [Figure 6] 1 illustrates another exemplary microscopy system. [Figure 7A] Figure 3 includes confocal images captured by the microscopy system. [Figure 7B] 7B is a graph providing a cross section of the image of FIG. 7A. [Figure 8A] 4A and 4B schematically illustrate exemplary C-SIM optics included in the microscopy system of FIG. 3, according to another embodiment. [Figure 8B] 4A and 4B schematically illustrate exemplary C-SIM optics included in the microscopy system of FIG. 3, according to another embodiment. [Figure 9] 1 illustrates exemplary C-SIM optics included in yet another microscopy system. [Figure 10] 1 illustrates exemplary C-SIM optics included in yet another microscopy system. [Figure 11] 1 is a flowchart illustrating an exemplary method for generating a C-SIM image. [Figure 12A] 12 is an image of a toroid shape captured as part of the method of FIG. 11. [Figure 12B] 12 is an image of a toroid shape captured as part of the method of FIG. 11. [Figure 12C] 12 is an image of a toroid shape captured as part of the method of FIG. 11. [Figure 12D]12 is an image of a toroid shape captured as part of the method of FIG. 11. [Figure 13A] 4 is an image depicting the subtraction microscopy process captured by the microscopy system of FIG. 3. [Figure 13B] 4 is an image depicting the subtraction microscopy process captured by the microscopy system of FIG. 3. [Figure 13C] 4 is an image depicting the subtraction microscopy process captured by the microscopy system of FIG. 3. [Figure 14A] FIG. 4 is a confocal image of a matrix of beads captured by the microscopy system of FIG. 3. [Figure 14B] 14B is a graph providing a cross section of the beads in the image of FIG. 14A.

[0007] While the present technology is susceptible to various modifications and alternative forms, specific embodiments have been shown by way of example in the drawings and are herein described in detail. It should be understood, however, that the invention is not intended to be limited to the particular forms disclosed. Rather, the invention is intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims. DETAILED DESCRIPTION OF THE INVENTION

[0008] Confocal structured illumination (C-SIM) Raman microscopy is a technique that can be used to improve the lateral resolution of a Raman microscope beyond the diffraction limit. Using C-SIM (also referred to as subtraction or fluorescence subtraction microscopy), the lateral resolution of the microscope can be improved by up to approximately 40% through a subtraction process. An image acquired by scanning a toroidally shaped focused spot across a sample can be subtracted from an image acquired by scanning with a non-toroidally shaped focused spot (also referred to herein as a confocal microscopy spot). To generate a toroidally shaped focused spot, a vortex generator can be added along the path of a Gaussian beam to generate a vortex beam. The vortex beam can be focused to generate a toroidally shaped spot on the sample plane that includes an internal dark region that can be smaller than the resolution allowed by the objective lens. Typically, a toroidal shape is circular or donut-shaped, but it is understood that other shapes, such as an ellipse, oval, or square, can be toroidal.

[0009] In some examples, the vortex generator may include a vortex phase plate (VPP). A quarter-wave plate may be placed after the VPP to add circular polarization to the vortex beam, thereby improving the contrast between the center and edge of the donut-shaped scanning spot. In other examples, the vortex generator may include an azimuthal polarizer that forms azimuthal polarized light. In some examples, the vortex generator may include a spatial light modulator. A spatial light modulator may be used in conjunction with an azimuthal polarizer to generate a toroid-shaped scanning spot. For example, a beam may be directed through a polarizer to generate both radially and azimuthal polarized light, and the radial component may be removed using a spatial light modulator (spatial filter), leaving the azimuthal polarized light, which is focused using a lens system to form the toroid-shaped scanning spot. A spatial light modulator (SLM) typically has a matrix of elements (e.g., 1000 × 1000), each of which can be selectively turned on and off. In some instances, linearly polarized light is directed at the SLM and a specific mask (a series of matrix elements programmed as on / off) is input. The light can be reflected or transmitted through the SLM. After interacting with the SLM, the light becomes circularly polarized and then enters the sample, forming a toroid-shaped scanning spot. When the images are subtracted, the diameter of this dark region can define the improved lateral resolution. Thus, in C-SIM microscopy and related difference microscopy techniques, two images are acquired by scanning two different tightly focused scanning spots, and the images are subtracted from each other to improve the lateral resolution of the system.

[0010] To generate a C-SIM image, the microscopy system must acquire perfectly aligned non-toroidal and toroidal images of the same imaging area. Applicant recognizes that because the illumination beam must be switched between a Gaussian beam and a vortex beam, care must be taken to ensure that switching the illumination beam does not affect the position of the corresponding beam spot on the sample surface. Because super-resolution images captured using C-SIM microscopy are on the micrometer to nanometer scale, it is important that the optical system involved in capturing the super-resolution image experiences minimal movement. Any changes in the optical system can introduce errors in the super-resolution image, including undesired blur and noise. In particular, movement of the vortex generators can result in inaccurate imaging. Regular movement of the optical system combined with temperature changes can also induce drift in the optical system, compromising the accuracy and reliability of the measurements.

[0011] To address the above problems, an optical system is used to generate structured illumination. The optical system includes a first set of optical components fixedly aligned with a light beam entering the optical system and a second set of optical components adjustable between a first operating position and a second operating position. The second set of optical components can be moved to a first operating position to generate a first output beam and can be moved to a second operating position to generate a second output beam. In the first operating position, an input light beam is expanded, and the expanded beam (i.e., the first output beam) is used to acquire a first image (e.g., a non-toroidal image or a confocal image). In the second operating position, the input light beam passes through the second set of optical components to form a vortex beam. The vortex beam can be output as a second output beam. The vortex beam is used to acquire a second image (e.g., a toroidal image). By fixedly aligning the first set of optical components with respect to the input light beam, the first set of optical components, i.e., the optical components for generating the vortex beam, are not moved when the output beam is switched between the expanded beam and the vortex beam. In this way, by keeping the high-sensitivity vortex-generating optics stationary with respect to the input light beam, the stability of the output beam from the optical system for structured illumination is maintained, ensuring high-quality super-resolution image generation.

[0012] The first set of optical components includes a vortex generator that forms the input light beam into a vortex beam. The vortex generator may include a VPP, an azimuthal polarizer, or a spatial light modulator (SLM). In one example, the vortex generator includes a VPP. The vortex generator may optionally include a quarter-wave plate. In another example, the vortex generator includes an SLM. In yet another example, the vortex generator includes an SLM and an azimuthal polarizer. All of the optical components in the first set of optical components may be aligned along a first optical axis.

[0013] The second set of optical components includes at least one beam expander, which may include multiple lenses positioned adjacent to each other or may have other optical components therebetween.

[0014] In some embodiments, an input light beam passes through the first set of optical components when the second set of optical components is in either the first or second operating position. The beam expander of the second set of optical components may be formed by two lenses. When the second set of optical components is in the first operating position, the input light beam passes sequentially through a first lens of the two lenses, the first set of optical components, and a second lens of the two lenses before being output as a first output beam. The vortex generator of the first set of optical components may be positioned near an intersection between the first lens and the second lens. The focal points of the first and second lenses may substantially overlap at the intersection. In one example, the first lens and the second lens may be separated by a distance of 10 inches to 14 inches, such as about 12 inches. The beam expander is configured such that the beam diameter of the light passing through the vortex generator is relatively small and such that the input light beam can be expanded without being affected by the vortex generator. The beam diameter of the light passing through the vortex generator may depend on the numerical aperture (NA) of the objective lens and the wavelength of the light. For example, with a high NA value of the objective lens (e.g., NA>1) and a wavelength of 532 nm, the beam diameter of the light passing through the vortex generator may be less than about 20 mm. In some examples, the beam diameter of the light passing through the vortex generator is less than 200 mm. When the second set of optical components is in the second operating position, the first and second lenses are moved away from the first optical axis of the first set of optical components so that the input light beam does not pass through the first lens or the second lens. A vortex beam is generated by passing the input light beam through the vortex generator of the first set of optical components. The vortex beam is output as a second output beam.

[0015] In some embodiments, the input light beam bypasses the first set of optical components when the second set of optical components is in the first operating position. The second set of optical components may include a mirror that deflects the input light beam away from the first optical axis of the second set of optical components in the first operating position. The deflected beam then passes through a beam expander before outputting as the first output beam. In one example, the beam expander may be part of the second set of optical components that is moved between two operating positions. In another example, the beam expander may be fixedly attached to the first and second optical components and not moved between operating positions.

[0016] The first set of optical components may include a beam splitter for directing a portion of the light beam passing through the first set of optical components to a second detector (such as a polarimeter) for monitoring the polarization state of the vortex beam. The beam splitter may be used during the calibration process and removed during sample measurement.

[0017] In some embodiments, the input light beam has a wavelength between about 200 nanometers and 900 nanometers. For example, the light may have a wavelength of about 532 nanometers.

[0018] In some embodiments, the second set of optical components is located on a mechanical slider configured to move the second set of optical components between a first operating position and a second operating position. In some embodiments, the system includes a controller configured to control the mechanical slider to adjust the second set of optical components between the first operating position and the second operating position. In some examples, the optical system for structured illumination is used in a microscopy system. The microscopy system includes a light source configured to generate a light beam; an optical system for receiving the light beam and generating a first output beam or a second output beam; at least one actuator for adjusting a second set of optical components between a first operating position and a second operating position; an objective lens for directing either the first output beam or the second output beam to a sample; a detector for receiving light from the sample; and a controller, wherein the controller includes a processor and a non-transitory memory for storing computer-readable instructions, such that when the computer-readable instructions are executed by the processor, the microscopy system is configured to: adjust, via the actuator, the second set of optical components to the first operating position, direct the first output beam to the sample, and generate a first image based on the light received from the detector; adjust, via the actuator, the second set of optical components to the second operating position, direct the second output beam to the sample, and generate a second image based on the light received from the detector; and generate a C-SIM image based on the first image and the second image.

[0019] The first and second output beams may be directed to the sample surface via a microscope including a scanner and at least an objective lens. The scanner scans the beam over the sample area. The objective lens focuses the beam at the sample surface. The first and second output beams are aligned when they enter the microscope.

[0020] The first and second images may be acquired by scanning the first and second output beams, respectively, over the sample area. The first image may be a non-toroidal image, and the second image may be a toroidal image. The microscopy system may include a pinhole for confocal imaging. Thus, the non-toroidal image may be a confocal image. The pinhole may be effectively formed by an optical fiber. In one example, the first image is acquired before acquiring the second image. In another example, the image is acquired after acquiring the second image.

[0021] 1A-1D illustrate an exemplary C-SIM imaging process 100 using simulated data. FIG. 1A shows the ground truth geometry of a series of point-source objects 102a, 102b arranged to be imaged with a structured illumination microscopy system. As shown, the objects 102a, 102b are simulated spots of fluorescein-stained nanoscale beads. FIG. 1B shows a simulated confocal image (or non-toroidal image) of the objects 102a, 102b convolved with a focused Gaussian beam. Due to the diffraction limit, the image appears as a distorted blob 104 that omits the spaced-apart dual nature of the objects 102a, 102b. FIG. 1B shows an image acquired with a typical confocal microscope. FIG. 1C shows a simulated vortex image acquired by convolving the objects 102a, 102b with a focused toroidal beam. The diameter of a focused toroidal beam (including both bright and dark regions) is typically larger than the diameter of a focused Gaussian (or non-toroidal) beam. As can be seen in the image of Figure 1C, holes 106a, 106b become visible, forming a somewhat similar distorted blob 108.

[0022] Figure 1D shows the pixel intensities of the toroidal image weighted and subtracted from the pixel intensities of the confocal image to form a subtracted image, or difference image (i.e., a C-SIM image). In the subtracted image, isolated blobs 110a, 110b are seen that have more characteristics in common with the real objects 102a, 102b, demonstrating the improved lateral resolution available to the microscope used to collect the image. By using the subtraction technique, the improved lateral resolution allows for differentiation between simulated spots 102a, 102b that would otherwise be indistinguishable.

[0023] As shown, the intensity of the toroidal image is weighted by a gamma factor. Thus, one image (I 非トロイダル ) is acquired using a confocal scanning spot, and the other images (I トロイダル ) is acquired using a toroidal scanning spot, and the image acquired with the toroidal scanning spot is usually scaled by an arbitrary parameter before being subtracted from the Gaussian image to produce a C-SIM image (I C-SIM ) and determining the intensity of the super-resolution image may generally be of the form: I C-SIM =I 非トロイダル -(Gamma)*I トロイダル where gamma is an arbitrary parameter that can be expressed as: As disclosed in U.S. patent application Ser. No. 18 / 296,859, entitled "Adaptive subtraction for C-SIM Microscopy," which is incorporated herein by reference in its entirety, this can be determined based on the peak of the non-toroidal beam imaging pixel intensity and the peak of the toroidal beam imaging intensity over the imaging area.

[0024] Figures 2A-2B illustrate another exemplary C-SIM process using experimental data. A set of sub-resolution fluorescent beads dried on a coverslip was imaged. The beam width was approximately 0.5 μm. Figure 2A shows a non-toroidal image 200, a toroidal image 202, and a C-SIM image 204 acquired from the same bead. The resulting C-SIM image 204 was generated by subtracting the toroidal image 202 from the non-toroidal image 200. Figure 2B compares the signal intensity at the same cross section of the bead in the non-toroidal (confocal) image 200 and the C-SIM image. The width of the bead captured in the confocal image is 0.73 μm, and the width of the bead captured in the C-SIM image 204 is 0.45 μm. The beam width is estimated based on the full-width at half maximum of the Gaussian-fitted intensity profile.

[0025] 3 illustrates a block diagram of an exemplary microscopy system 300. The microscopy system 300 includes a light source 302 that emits a light beam 304 and C-SIM optics 306 that receive the light beam 304 from the light source 302. The C-SIM optics 306 receives the light beam 304 and outputs an output beam 308. The output beam 308 is received by a dichroic filter 314 (or, in some implementations, a dichroic mirror).

[0026] The output beam 308 passes through the dichroic filter 314 and enters the microscope 322. The microscope 322 includes at least a scanner and an objective lens. The microscope 322 directs and focuses the output beam 308 toward the sample 326. The scanner can scan the output beam 308 over a sample region of the sample 326. The microscope 322 receives sample light 328 from the sample 326 and transmits the sample light 328 through the dichroic filter 314 to a wavelength-selective element 315. The wavelength-selective element 315 can receive the sample light 328 through an input port 317, such as a pinhole. The wavelength-selective element 315 selectively transmits a wavelength range of the sample light 328 to a detector 316. The wavelength-selective element can include one or more of a filter, a monochromator, and a spectrometer. The detector 316 can be, for example, a camera integrated within or connected to a spectrometer. The electrical signals are transmitted from the detector to a controller 330, which includes an electronic processor 313 and a memory 332. The controller 330 (i.e., by executing software stored in memory 332 in the electronic processor 313) performs an analysis of the light detected from the sample (e.g., sample light 328) based on the electrical signals from the detector 316.

[0027] The microscopy system 300 may be a structured illumination microscopy system, and the output beam 308 from the C-SIM optics 306 may be either a non-toroidal beam or a toroidal beam. The non-toroidal and toroidal beams are aligned. The input port 317 may be a confocal pinhole. The signal detected from the detector may include both fluorescence and Raman signals. An optical fiber may be used in any of the optical paths shown in FIG. 3 as an optical waveguide. For example, the sample light 328 reflected from the dichroic filter 314 may be coupled to one end of an optical fiber, and the other end of the optical fiber may be coupled to the wavelength-selective element 315. The end of the fiber that receives light from the dichroic filter 314 may serve as the input port 317.

[0028] Controller 330 may have more or fewer components than those illustrated. Controller 330 is suitable for the application and configuration and may include, for example, multiple electronic processors, multiple data storage devices (memory modules), or a combination thereof.

[0029] The memory 332 is a random-access memory. memory (RAM) devices (e.g., static RAM (SRAM) devices, magnetic RAM (MRAM) devices, dynamic RAM (DRAM) devices, resistive RAM (RRAM) devices, or conductive-bridging RAM The memory may include one or more local or remote memory devices, such as RAM, CBRAM (devices), hard drive-based memory devices, solid state memory devices, network drives, cloud drives, or any combination of memory devices.

[0030] In some implementations, the microscopy system 300 includes more or fewer optical components than illustrated in Figure 3. For example, additional mirrors may be provided to direct the light beam.

[0031] 4A-4B illustrate an example of the C-SIM optics 306 in different operating positions. The C-SIM optics 306 includes a first slider 400 and a second slider 402. A VPP 404 and a quarter-wave plate 406 are located between the first slider 400 and the second slider 402. The VPP 404 and the quarter-wave plate 406 are located along an optical path 408 and form, for example, a first set of optical components. A light beam 304 from the light source 302 travels along the first optical path 408.

[0032] The first slider 400 includes, for example, a first lens 410 and a linear polarizer 412, which form a second set of optical components. The second slider 402 includes a second lens 414. The first slider 400 and the second slider 402 move to position the first lens 410 and the second lens 414 or the linear polarizer 412 in the light path 408 so that the light beam 304 passes through either the first lens 410 and the second lens 414 or the linear polarizer 412. The linear polarizer 412 includes an optical system that converts the incident light into a fully linearly polarized state.

[0033] In Figure 4A, the second set of optical components of the C-SIM optics 306 is in a first operating position. In particular, in the first operating position, the light beam 304 input to the C-SIM optics 306 is expanded and output as a first output beam 411 (which may be, for example, output beam 308 of Figure 3) to the microscope 322 as a non-toroidal shaped beam. In the first operating position, the first slider 400 and the second slider 402 are positioned such that the first lens 410 and the second lens 414 are aligned along the optical path 408. The light beam 304 from the light source 302 (shown in Figure 3) travels along the first optical path 408, sequentially passing through the first lens 410, the VPP 404, the quarter-wave plate 406, and the second lens 414.

[0034] Considered together, the first lens 410 and the second lens 414 form a beam expander 500, as shown in FIG. 5. The dashed lines are ray traces of light passing through the beam expander. The beam expander 500 may be, for example, a Keplerian beam expander that provides approximately 1x, 2x, or 3x beam expansion to the light beam 304. In the example of FIG. 5, the first lens 410 and the second lens 414 have relatively long focal lengths such that the distance between the first lens 410 and the second lens 414 is between approximately 10 inches and 14 inches. In other implementations, the distance between the first lens 410 and the second lens 414 may be greater than 10 inches. Due to this long focal length, light passing through the space between the first lens 410 and the second lens 414 occupies a narrow space in the central region between the first lens 410 and the second lens 414. The vortex generators, in this example VPP 404 and quarter wave plate 406, are located in a narrow space on either side of intersection point 502 so that the beam diameter passing through the vortex generators is small, so that light beam 304 experiences minimal perturbation from the vortex generators. Intersection point 502 is the intersection of the focal points of first lens 410 and second lens 414. The expanded beam is provided as output beam 308, which fills the back lens of the objective of microscope 322.

[0035] In FIG. 4B , the second set of optical components of the C-SIM optics 306 is in a second operating position, forming a vortex (toroidal-shaped) beam. In the second operating position, the first slider 400 and the second slider 402 are positioned such that the first lens 410 and the second lens 414 are removed from the optical path 408 and the linear polarizer 412 is aligned along the optical path 408. The optical beam 304 is not expanded, but instead converted by the VPP 404 and the quarter-wave plate 406 into a vortex beam (second output beam) 418. The second output beam 418 is provided as the output beam 308 and transmitted to the microscope 322 (as shown in FIG. 3 ).

[0036] The first slider 400 and the second slider 402 are mechanical sliding components that transition the second set of optical components between a first operating position and a second operating position. The first set of optical components, including the VPP 404 and the quarter-wave plate 406, are fixedly aligned with the input light beam 304. The first set of optical components may be fixed to a base plate (shown in FIG. 6 ) and do not move out of the optical path 408. Movement of the first slider 400 and the second slider 402 may be initiated by an operator of the microscopy system 300 or may be controlled by a controller associated with the microscopy system 300 (e.g., controller 330).

[0037] In some implementations, the C-SIM optics 306 includes a beam splitter 416 located between the quarter-wave plate 406 and the second slider 402. The beam splitter 416 can split off a small portion of the light beam 304 (e.g., 10% of the light beam 304) to a separate measurement device, such as a polarimeter 604 (shown in FIG. 6 ).

[0038] FIG. 6 illustrates an exemplary microscopy system 600 having C-SIM optics 306 configured similarly to FIGS. 4A and 4B . Microscopy system 600 may be substantially similar to microscopy system 300 shown in more detail. In the example of FIG. 6 , VPP 404 and quarter-wave plate 406, along with other optical components, are rigidly secured to a base plate 602. In addition, microscopy system 600 includes a polarimeter 604 that receives a portion of light from beamsplitter 416. In microscopy system 600, focusing optics 608 are located optically after dichroic filter 314. Focusing optics 608 focuses sample light 328 onto a fiber optic cable 606 that is connected to a spectrometer (such as spectrometer 312). The exemplary microscopy system 600 may also include multiple mirrors 610 for directing light traveling through microscopy system 600. For example, a first mirror 610A and a second mirror 610B direct the output beam 308 from the second slider 402 to the dichroic filter 314. A third mirror 610 directs the third light 318 from the dichroic filter 314 to the microscope 322 and directs the scattered light 328 from the microscope 322 to the dichroic filter 314.

[0039] Figure 7A illustrates a first C-SIM image 702, a second C-SIM image 704, and a third C-SIM image 706 of an imaged 0.5 μm fluorescent bead. C-SIM images 702-706 were captured using the microscopy system 300. The size of the imaged beads was measured using a full-width half-maximum technique extracted from a line scan taken through the center of the bead. Figure 7B illustrates a plot showing the experimental (solid line) and theoretical (dashed line) signal intensity along the cross-section of the fluorescent bead in the C-SIM image plotted against the theoretical signal intensity. The measured signal is Gaussian, indicating no obvious aberrations. Furthermore, the FWHM of the measured data is close to the theoretical signal intensity, demonstrating the system's capability for super-resolution imaging.

[0040] 8A-8B show another exemplary embodiment of C-SIM optics 306. In this example, the first set of optical components includes a vortex generator. The vortex generator includes a vortex phase plate 404 and a quarter-wave plate 406. The first optical components may optionally include a beam splitter 416. The second set of optical components includes a beam expander 806. The beam expander 806 may be formed by two lenses, such as lenses 410 and 414 shown in FIGS. 4A and 4B. The second set of optical components may optionally include one or more mirrors (such as mirrors 802, 804, 808, and 810) for directing the input beam 304 through the beam expander when the second set of optical components is in the first operating position. Some of the second set of optical components (such as mirrors 802 and 810) may be mounted on sliders 400 and 402. Similar to the configurations of FIGS. 4A and 4B, sliders 400 and 402 may be individually or collectively actuated by one or more actuators.

[0041] In Figure 8A, the second set of optical components of the C-SIM optics 306 is in a first operating position. In the first operating position of Figure 8A, the light beam 304 is redirected away from the optical path 408, thus bypassing the vortex generator. The light beam 304 is redirected away from the VPP 404, the quarter-wave plate 406, and the beam splitter 416 by the first mirror 802. The first slider 400 and the second slider 402 are aligned such that the first mirror 802 and the second mirror 810 are aligned along the optical path 408. The light beam 304 from the light source 302 (shown in Figure 3) is directed by the first mirror 802 to the beam expander 806, which expands the beam to produce the expanded output beam 411, which is a non-toroidal shaped beam. 8A , in some cases, the C-SIM optics 306 also include a third mirror 804 for directing light from the first mirror 802 to a beam expander 806, and a fourth mirror 808 for directing an expanded output beam 411 from the beam expander 806 to a second mirror 810. The expanded output beam 411 is provided as the output beam 308 described above with respect to FIG. 3 and fills the back lens of the objective of the microscope 322.

[0042] In FIG. 8B , the second set of optical components of the C-SIM optics 306 is in a second operating position. In particular, in the second operating position, the C-SIM optics 306 is configured to generate a vortex beam, such as a toroidal-shaped beam, such as a donut-shaped focused spot at the sample plane. In the second operating position, the first slider 400 and the second slider 402 are positioned such that the first mirror 802 and the second mirror 810 are excluded from the optical path 408. Therefore, in this position, the optical beam 304 is not expanded but instead converted into a narrow vortex beam 418 by the VPP 404 and the quarter-wave plate 406. The narrow vortex beam 418 is provided as the output beam 308, described above with respect to FIG. 3 , and transmitted to the microscope 322. The optical beam can pass through the first and second sliders through apertures on the sliders.

[0043] Microscopy system 300 and microscopy system 600 are merely examples, and additional microscopy systems including fixed or stationary vortex phase plates are possible. For example, Figures 9-10 illustrate microscopy system 900. In Figure 9, microscopy system 900 is in a first operating position. In Figure 10, microscopy system 900 is in a second operating position. The optical components found in microscopy system 900 may operate similarly to their counterparts in microscopy system 300, but are arranged in a different manner.

[0044] The microscopy system 900 includes a light source 902 configured to emit light 904, such as a laser beam. The light 904 is emitted toward a first slider 906, which includes a transmissive portion, such as a hole, and a reflective portion, such as a mirror. In a first operating position, the reflective portion of the first slider 906 directs the light through a beam expander 926 (see FIG. 9 ). After expansion, the light 904 is reflected by a steering mirror 928 to a second slider 908, which also has a transmissive portion and a reflective portion. After expansion, the reflective portion of the second slider 908 directs the light 904 to a dichroic filter 918 and a detector 920. The light 904 is reflected by the dichroic filter 918 to a third mirror 922 and toward a microscope 924.

[0045] When the first slider 906 and the second slider 908 move from the first operating position to the second operating position, the optical path of the light 904 is changed. Specifically, as illustrated in FIG. 10 , the open portion of the first slider 906 is aligned along the optical path of the light 904, and the light 904 travels through the first slider 906 (bypassing the beam expander 926). The light 904 travels through the first slider 906, is reflected by the first mirror 910, and is directed toward the VPP 912 and the quarter-wave plate 914. The light 904 travels through the VPP 912 and the quarter-wave plate 914 to the second slider 908. In the second operating position, the open portion of the second slider 908 is aligned along the optical path of the light 904, and the light 904 travels through the second slider 908. Light 904 traveling through second slider 908 is reflected by second mirror 916 to dichroic filter 918 and detector 920. Light 904 is reflected by dichroic filter 918 to third mirror 922 and toward microscope 924.

[0046] Thus, in microscopy system 900, both beam expander 926 and VPP 912 are rigidly fixed and do not need to be adjusted during measurements. Rather, additional mirrors are moved in or out of the path of light 904 to direct the light along the appropriate path.

[0047] 11 is a flowchart illustrating an exemplary method 1100 for generating a C-SIM image. Method 1100 may be executed by controller 330 and may be performed using any of the exemplary microscopy systems described herein. The steps of method 1100 are described iteratively for explanatory purposes. The various steps described herein with respect to method 1100 may be performed simultaneously, in parallel, or in an order different from the illustrated sequential and iterative execution method. In some cases, various steps may be omitted from method 1100.

[0048] At block 1102, the method 1100 includes calibrating the microscopy system 300. Calibrating the microscopy system may include aligning the first and second output beams generated in the first and second operating positions of the second set of optical components, respectively. The microcopy system may be calibrated using a reference sample. For example, prior to measurement, a toroidal image of a 0.5 μm diameter fluorescent bead may be collected using the vortex phase plate 404 in the optical path. In other words, the toroidal image may be acquired with the C-SIM component 306 of the microscopy system in the second operating position. Examples of such images are shown in FIGS. 12A, 12B, 12C, and 12D. In each image, a donut shape is visible, and the uneven intensity in the outer donut region is the result of photobleaching. These images can be used to ensure that the C-SIM optical component 306 is aligned before detailed imaging. In some cases, the operation of block 1102 is optional, and calibration may not be performed for every sample or before every scanning operation.

[0049] At block 1104, the method 1100 includes loading a sample into the microscope 322. As one non-limiting example, following the alignment verification (block 1102), a sample of sulfur is loaded into the microscope 322.

[0050] At block 1106, the method 1100 includes scanning the first output beam to acquire a non-toroidal image of the sample. For example, to acquire a non-toroidal image of the sample, the C-SIM optics 306 is positioned in a first operating position such as that shown in FIG. 4A, FIG. 8A, or FIG. 9. An exemplary image of a sample (e.g., sulfur) acquired by scanning a non-toroidal shaped scanning spot having a pixel size of 0.2 μm is shown in FIG. 13A.

[0051] At block 1108, the method 1100 includes scanning the second output beam to acquire a toroidal image of the sample. For example, to acquire a toroidal image of the sample, the C-SIM optics 306 is positioned in a second operating position as shown in FIG. 4B, FIG. 8B, or FIG. 10. An exemplary image of a sulfur sample acquired by scanning a toroidal-shaped scanning spot having a pixel size of 0.2 μm is shown in FIG. 13B.

[0052] In block 1110, method 1100 includes generating a C-SIM image based on the non-toroidal image (acquired in block 1106) and the toroidal image (acquired in block 1108). For example, the image of FIG. 13C represents a weighted subtraction of the images of FIGS. 13A and 13B, representing a super-resolution image. In the example of FIGS. 13A-13C, the toroidal image of FIG. 13B is subtracted from the non-toroidal image of FIG. 13A. The super-resolution image of FIG. 13C contains significantly more surface and edge detail compared to the images of FIGS. 13A and 13B. A complex set of intensity inhomogeneities is visible in FIG. 13C. In some examples, block 1108 is performed before block 1106. That is, the toroidal image is acquired before the non-toroidal image.

[0053] FIG. 14A illustrates another exemplary C-SIM image 1400 of a matrix of 1.0 μm diameter polystyrene beads acquired by the methods and systems described herein. The polystyrene beads are dried on a coverslip, and the image is collected with a 100× 0.95 NA objective. As shown in the confocal image 1400, the beads are densely packed together in a matrix-like formation and are symmetrical and regularly sized. A line scan 1405 is taken across the center of one bead to capture its cross-section, as shown in FIG. 14B. The full width at half maximum of the bead is slightly over 1.0 μm. This width reflects not only the width of the bead itself, but also the convolution of the bead with the confocal scan spot, which increases the width of the bead captured in the image.

[0054] Although the examples described herein primarily refer to both the VPP and the quarter-wave plate being adjacently positioned, in some cases the quarter-wave plate may be omitted so that light passes through the VPP rather than the quarter-wave plate during image capture. Additionally, in some implementations, the VPP may be replaced by other optical systems (e.g., spatial light modulators) capable of generating vortex beams.

[0055] Furthermore, although the sliders described herein with respect to microscopy system 300, microscopy system 600, and microscopy system 900 are illustrated and described as linear mechanical sliders that move laterally, other optical repositioning devices such as rotating mechanical wheels or other manual or motorized positioning devices may also be implemented.

[0056] Thus, the implementations described herein provide systems, methods, computing and storage devices, and computer-readable media for capturing C-SIM microscope images using fixed optics. As discussed above, the implementations described herein provide for adjusting between confocal and toroidal imaging modes without adjusting a vortex phase plate. The implementations described herein improve the reliability and reproducibility of super-resolution imaging. Thus, the implementations disclosed herein provide improvements to super-resolution imaging.

[0057] As set forth above in the detailed description of the invention, reference is made to the accompanying drawings which form a part hereof, in which like numerals designate like parts throughout and which show, by way of illustration, implementations which may be practiced. It is to be understood that other implementations may be utilized and structural or logical changes may be made without departing from the scope of the present disclosure. Accordingly, the detailed description of the invention set forth above should not be construed in a limiting sense.

[0058] Various operations may be described sequentially as multiple separate actions or operations as is most helpful in understanding the subject matter disclosed herein. However, the order of description should be construed as implying that these operations are necessarily order-dependent. In particular, these operations may not be performed in the order presented. The operations described may occur in a different order than in the described implementation. Various additional operations may be performed and / or described operations may be omitted in additional implementations.

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In the event of a conflict, the present document, including definitions, shall control. Exemplary methods and systems are described below; however, methods and systems similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. The systems, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0060] As used herein, the terms "comprises," "includes," "having," "having," "can," "includes," and variations thereof are intended to be open-ended transitional phrases, terms, or clauses that do not exclude the possibility of additional acts or structures. The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0061] The modifier "about" used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (e.g., includes at least the degree of error associated with measurement of the particular quantity). The modifier "about" should also be considered to disclose a range defined by the absolute values ​​of the two endpoints. For example, the phrase "about 2 to about 4" also discloses the range "2 to 4." The term "about" can refer to plus or minus 10% of the indicated number. For example, "about 10%" can indicate a range of 9% to 11%, and "about 1" can mean 0.9 to 1.1. Other meanings of "about" may be apparent from the context, such as rounding; for example, "about 1" can also mean 0.5 to 1.4.

[0062] As used herein, the term "or" is intended to mean an inclusive "or" rather than an exclusive "or." That is, unless otherwise specified or clear from context, "X employs A or B" is intended to mean all natural inclusive permutations. That is, if X employs A, X employs B, or X employs both A and B, then "X employs A or B" satisfies all of the foregoing examples. Furthermore, the articles "a" and "an" as used in this specification and the accompanying drawings should generally be construed to mean "one or more" unless otherwise specified or clear from context to refer to the singular form.

[0063] With respect to the recitation of numerical ranges herein, each intervening number is expressly contemplated to the same degree of precision. For example, for the range 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0064] The present disclosure will now be described with reference to the drawings, wherein like reference numerals are used to refer to like elements throughout. In the following description, for purposes of explanation, numerous specific details are set forth in order to provide an improved understanding of the present disclosure. It may be apparent, however, that the systems and methods of the present disclosure can be practiced without one or more of these specific details. In other instances, well-known structures and devices are shown in block diagram form to facilitate description of the present systems and methods.

[0065] term Implementations of the present disclosure are disclosed in the following sections.

[0066] Item 1: An optical system for structured illumination, comprising: a first set of optical components fixedly aligned with a light beam entering the optical system; and a second set of optical components adjustable between a first operating position and a second operating position, wherein when the second set of optical components is in the first operating position, the light beam is expanded and output as a first output beam, and when the second set of optical components is in the second operating position, the light beam passes through the first set of optical components to form a vortex beam that is output as the second output beam.

[0067] Item 2: The optical system of item 1, wherein each optical component of the first set of optical components is aligned along a first optical axis.

[0068] Item 3: The optical system of item 1 or 2, wherein the first set of optical components includes a vortex generator.

[0069] Item 4: The optical system of item 3, wherein the second set of optical components includes at least one beam expander.

[0070] Item 5: The optical system of item 4, wherein the light beam passes through the first set of optical components when the second set of optical components is in the first operating position.

[0071] Item 6: An optical system as described in Item 5, wherein the beam expander includes a first lens and a second lens, and when the second set of optical components is in the first operating position, the light beam passes sequentially through the first lens, the vortex generator, and the second lens.

[0072] Item 7: The optical system described in Item 6, wherein in the first operating position, the light beam entering the optical system is focused at an intersection between the first lens and the second lens, and the cross section of the beam passing through the vortex generator is 20 mm to 200 mm or less.

[0073] Item 8: The optical system of item 7, wherein the vortex generator includes a vortex phase plate and a quarter-wave plate, and the intersection point is between the vortex phase plate and the quarter-wave plate.

[0074] Item 9: The optical system of item 8, wherein the first lens and the second lens are separated by 10 to 14 inches.

[0075] Item 10: An optical system described in any of items 3 to 9, wherein the second set of optical components further includes a linear polarizer, and the light beam passes through the linear polarizer before passing through the vortex generator when the second set of optical components is in the second operating position.

[0076] Item 11: The optical system of item 3, wherein the light beam bypasses the first set of optical components when the second set of optical components is in the first operating position.

[0077] Item 12: The optical system described in Item 11, wherein the second set of optical components includes at least one mirror, and the light beam is deflected by the mirror so as to bypass the first set of optical components when the second set of optical components is in the first operating position.

[0078] Item 13: The optical system according to any one of Items 1 to 12, wherein the first output beam and the second output beam are aligned.

[0079] Item 14: The optical system according to any one of Items 1 to 13, wherein the optical components of the second set of optical components are fixedly attached to each other.

[0080] Item 15: The optical system described in Item 14, wherein at least one optical component of the second set of optical components is fixedly mounted on a movable structure, and the second set of optical components is adjusted between a first operating position and a second operating position by operating the movable structure.

[0081] Clause 16: A structured illumination microscopy system, comprising: a light source configured to generate a light beam; an optical system for structured illumination according to any of clauses 1 to 15 for receiving the light beam and generating a first output beam or a second output beam; at least one actuator for adjusting a second set of optical components between a first operating position and a second operating position; an objective lens for directing either the first output beam or the second output beam to the sample; a detector for receiving light from the sample; and a controller, the controller comprising: a processor; and a computer readable instruction memory for storing computer readable instructions. and a non-transitory memory for adjusting, when the computer-readable instructions are executed by the processor, the microscopy system is configured to: adjust, via the actuator, the second set of optical components to a first operating position, direct the first output beam to the sample, generate a first image based on the light received from the detector; adjust, via the actuator, the second set of optical components to a second operating position, direct the second output beam to the sample, generate a second image based on the light received from the detector; and generate a sample image based on the first image and the second image.

[0082] Item 17: The microscopy system of item 16, wherein the light from the sample includes a Raman shift.

[0083] Item 18: A microscopy system as described in Item 16 or 17, wherein at least one optical component of the second set of optical components is fixedly mounted on a movable structure, the movable structure including a slider, and the actuator is configured to slide the slider to move the second set of optical components between a first operating position and a second operating position.

[0084] Item 19: A microscopy system described in any of items 16 to 18, further comprising a scanner for scanning the first output beam or the second output beam over the sample area to obtain a first sample image and a second sample image.

[0085] Item 20: A microscopy system as described in Item 19, wherein the microscopy system is configured to scan the second output beam over the sample area before scanning the first output beam over the sample area.

Claims

1. 1. An optical system for structured illumination, comprising: a first set of optical components fixedly aligned with respect to a light beam incident on the optical system; a second set of optical components adjustable between a first operating position and a second operating position; when the second set of optical components is in the first operating position, the light beam is expanded and output as a first output beam; an optical system, wherein when the second set of optical components is in the second operating position, the light beam passes through the first set of optical components to form a vortex beam that is output as a second output beam.

2. The optical system of claim 1 , wherein each optical component of the first set of optical components is aligned along a first optical axis, and the first set of optical components includes a vortex generator.

3. The optical system of claim 2 , wherein the second set of optical components includes at least one beam expander.

4. 4. The optical system of claim 3, wherein the light beam passes through the first set of optical components when the second set of optical components is in the first operating position.

5. 5. The optical system of claim 4, wherein the beam expander includes a first lens and a second lens, and when the second set of optical components is in the first operating position, the light beam passes sequentially through the first lens, the vortex generator, and the second lens.

6. 6. The optical system of claim 5, wherein in the first operating position, the light beam entering the optical system is focused at an intersection between the first lens and the second lens, and a cross section of the beam passing through the vortex generator is between 20 mm and 200 mm or less.

7. The optical system of claim 6 , wherein the vortex generator includes a vortex phase plate and a quarter wave plate, and the intersection point is between the vortex phase plate and the quarter wave plate.

8. 8. The optical system of claim 7, wherein the first lens and the second lens are separated by 10 to 14 inches.

9. 9. The optical system of claim 2, wherein the second set of optical components further comprises a linear polarizer, and wherein the light beam passes through the linear polarizer before passing through the vortex generator when the second set of optical components is in the second operating position.

10. The optical system of any one of claims 2 to 8, wherein the light beam bypasses the first set of optical components when the second set of optical components is in the first operating position.

11. 11. The optical system of claim 10, wherein the second set of optical components includes at least one mirror, and the light beam is deflected by the mirror to bypass the first set of optical components when the second set of optical components is in the first operating position.

12. The optical system of claim 1 , wherein the first output beam and the second output beam are aligned.

13. The optical system of claim 1 , wherein the optical components of the second set of optical components are fixedly attached relative to each other.

14. 1. A structured illumination microscopy system comprising: a light source configured to generate a light beam; an optical system according to claim 1 for receiving the light beam and generating the first output beam or the second output beam; at least one actuator for adjusting the second set of optical components between the first operating position and the second operating position; an objective lens for directing either the first output beam or the second output beam to a sample; a detector for receiving light from the sample; a controller, the controller including a processor and a non-transitory memory for storing computer readable instructions, the computer readable instructions, when executed by the processor, causing the structured illumination microscopy system to: adjusting the second set of optical components to the first operating position via the actuator; directing the first output beam at the sample and generating a first image based on light received from the detector; adjusting the second set of optical components to the second operating position via the actuator; directing the second output beam at the sample and generating a second image based on the light received from the detector; a structured illumination microscopy system configured to generate an image of the sample based on the first image and the second image.

15. The structured illumination microscopy system of claim 14 , wherein the light from the sample includes a Raman shift.

Images