Optical configuration for high resolution microscopy

Abstract

The invention relates to an optical configuration for high resolution microscopy. An optical configuration for a confocal structured illumination Raman (C-SIM Raman) microscopy system is provided. One example includes: a light source configured to project 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 operational position, the second set of optical components is aligned on the first optical path and is configured to expand the light into expanded light in conjunction with the first set of optical components. When in the second operational position, the second set of optical components are not aligned on the first optical path.

Description

TECHNICAL FIELD

[0001] The present disclosure relates generally to systems and methods for performing high resolution microscopy, such as Raman spectroscopy. BACKGROUND

[0002] Super-resolution imaging is a set of techniques that can be used to increase the resolution of microscopy beyond the diffraction limit of standard confocal microscope systems. One example super-resolution imaging technique is confocal structured illumination (C-SIM) Raman microscopy, which increases the lateral resolution of a Raman microscope beyond the diffraction limit. C-SIM microscopy includes imaging a sample with a standard confocal scanning spot and separately with a circular ring scanning spot. Super-resolution is achieved through subtraction of these two images. SUMMARY

[0003] In one aspect, an optical system for structured illumination includes a first set of optical components fixedly aligned with respect to 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. 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, which is output as a second output beam.

[0004] In some aspects, the optical system is included in a structured illumination microscopy system. The microscopy system includes a light source configured to generate a light beam, which is received by the optical system. The microscopy system includes at least one actuator to adjust the second set of optical components between the first operating position and the second operating position, an objective lens to direct the first output beam or the second output beam to a sample, a detector to receive light from the sample, and a controller including a processor and a non-transitory memory to store 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 in the first operating position via the actuator, direct the first output beam toward the sample and generate a first image based on light received from the detector, adjust the second set of optical components in the second operating position via the actuator, direct the second output beam toward the sample and generate a second image based on light received from the detector, and generate a sample image based on the first image and the second image.

[0005] Systems, methods, or techniques related to C-SIM microscopy are not specifically required to include all of the details characterized herein to obtain some of the benefits according to the present disclosure. Thus, the specific examples characterized herein are intended as example applications of the described techniques and alternatives can also be employed. BRIEF DESCRIPTION OF DRAWINGS

[0006] The features and advantages of the present technology will become more apparent from a detailed consideration of the following specific implementations of the present technology in conjunction with the accompanying drawings, in which:

[0007] FIG. 1A , FIG. 1B , FIG. 1C and FIG. 1D are simulated images depicting a subtraction microscopy process.

[0008] FIG. 2A are further images depicting a subtraction microscopy process.

[0009] FIG. 2B is a graph providing a cross-section of the images of FIG. 2A .

[0010] FIG. 3 An example microscopy system is schematically illustrated.

[0011] FIG. 4A and FIG. 4B schematically illustrate example C-SIM optical components included in a microscopy system according to one embodiment. FIG. 3

[0012] FIG. 5 is an example beam expander implemented with C-SIM optical components of FIG. 4A .

[0013] FIG. 6 Another example microscopy system is illustrated.

[0014] FIG. 7A includes confocal images captured by a microscopy system of FIG. 3 .

[0015] FIG. 7B is a graph providing a cross-section of the images of FIG. 7A .

[0016] FIG. 8A and FIG. 8B schematically illustrate example C-SIM optical components included in a microscopy system according to another embodiment. FIG. 3

[0017] FIG. 9 and FIG. 10 ​​Example C-SIM optical components included in yet another microscopy system are shown.

[0018] FIG. 11 is a flowchart showing an example method for generating a C-SIM image.

[0019] FIG. 12A FIG. 12B FIG. 12C and FIG. 12D are annular images captured as part of the method of FIG. 11

[0020] FIG. 13A FIG. 13B and FIG. 13C are images depicting a subtraction microscopy process captured by the microscopy system of FIG. 3

[0021] FIG. 14A is a confocal image of a matrix of beads captured by the microscopy system of FIG. 3

[0022] FIG. 14B is a graph providing a cross-section of a bead within an image of FIG. 14A

[0023] 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 will herein be described in detail. It should be understood, however, that the application is not to be limited to the particular DETAILED DESCRIPTION

[0024] 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 known as differential microscopy or fluorescence differential interference microscopy, the lateral resolution of a microscope can be improved by up to about 40% through a subtraction process. An image acquired by scanning with a non-annular focal spot (also referred to herein as a confocal microscopy spot) can be subtracted from an image acquired by scanning an annular focal spot across a sample. To produce an annular focal 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 produce an annular spot at the sample plane that contains an inner dark region that can be smaller than the resolution allowed by the objective lens. Typically, the annular shape is circular or circular ring-shaped, but it should be understood that other shapes can also be annular, such as elliptical, oval, square, etc.

[0025] ​​​​​​​In some examples, the vortex generator can include a vortex phase plate (VPP). A quarter wave plate can be positioned behind the VPP to add circular polarization to the vortex beam, improving the contrast between the center and the edge of the circular ring-shaped scan spot. In other examples, the vortex generator can include an azimuthal polarizer to form azimuthally polarized light. In some examples, the vortex generator can include a spatial light modulator. The spatial light modulator can be used with the azimuthal polarizer to produce a ring-shaped scan spot. For example, the beam can be directed through a polarizer to produce both radial and azimuthal polarization, and the radial component can be removed with a spatial light modulator (spatial filter) to leave the azimuthally polarized light, which is then focused using a lens system to form a ring-shaped scan spot. A spatial light modulator (SLM) typically has a matrix of elements (e.g., 1000 x 1000), each of which can be selectively turned on and off. In some examples, linearly polarized light is directed onto the SLM and a specific mask (a set of matrix elements programmed to be on / off) is input. The light can be reflected or transmitted through the SLM. After interacting with the SLM, the light is circularly polarized and subsequently incident on the sample, forming a ring-shaped scan 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 differential microscopy techniques, two images are acquired by scanning two different tightly focused scan spots, and these images are subtracted from each other in order to improve the lateral resolution of the system.

[0026] To generate a C-SIM image, the microscopy system needs to acquire a perfectly aligned non-ring-shaped image and a ring-shaped image of the same imaged area. Applicants have recognized that, since the illumination beam needs to be switched between a Gaussian beam and a vortex beam, care needs to be taken to ensure that the switching of the illumination beam does not affect the position of the corresponding beam spot on the sample plane. Since the super-resolution images captured using C-SIM microscopy are on the scale of microns to nanometers, it is important that the optical system involved in the capture of the super-resolution images undergoes minimal movement. Any changes in the optical system can lead to errors within the super-resolution images, including unwanted blurring and noise. In particular, movement of the vortex generator can lead to inaccurate imaging. Regular movement of the optical components combined with temperature variations can also induce drift within the optical system, compromising the accuracy and reliability of the measurement results.

[0027] To address the above issues, an optical system is used to generate structured illumination. The optical system includes a first set of optical components that are fixedly aligned with respect to an input beam of light entering the optical system, and a second set of optical components that are adjustable between a first operating position and a second operating position. The second set of optical components can be moved to the first operating position to generate a first output beam, and to the second operating position to generate a second output beam. At the first operating position, the input beam is expanded, and the expanded beam (i.e., the first output beam) is used to acquire a first image (such as a non-ring image or a confocal image). At the second operating position, the input beam passes through the second set of optical components and forms a vortex beam. The vortex beam can be output as the second output beam. The vortex beam is used to acquire a second image (such as a ring image). By fixedly aligning the first set of optical components with respect to the input beam, the first set of optical components (i.e., the optical components used to generate 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 highly sensitive vortex-generating optical components stationary with respect to the input beam, the stability of the output beam from the optical system for structured illumination is maintained, which ensures high-quality super-resolution image generation.

[0028] The first set of optical components includes a vortex generator for forming the input beam into a vortex beam. The vortex generator can include a VPP, an azimuthal polarizer, or a spatial light modulator (SLM). In one example, the vortex generator includes a VPP. The vortex generator can 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 can be aligned along a first optical axis.

[0029] The second set of optical components includes at least one expander. The expander can include a plurality of lenses positioned adjacent to each other, or have other optical components between the plurality of lenses.

[0030] In some embodiments, the input beam passes through the first set of optical components when the second set of optical components is in the first operational position or the second operational position. The beam expander of the second set of optical components can be formed by two lenses. When the second set of optical components is in the first operational position, the input beam sequentially passes through a first one of the two lenses, the first set of optical components, and a second one of the two lenses before being output as the first output beam. The vortex generator of the first set of optical components can be positioned near an intersection between the first lens and the second lens. The focal points of the first lens and the second lens can substantially overlap at the intersection. In one example, the first lens and the second lens can be separated by a distance of between 10 inches and 14 inches, such as approximately 12 inches. The beam expander is configured such that the beam diameter of light passing through the vortex generator is relatively small, and such that the input beam can be expanded without being affected by the vortex generator. The beam diameter of light passing through the vortex generator can depend on the numerical aperture (NA) of the objective lens and the wavelength of the light. For example, using a high NA value (e.g., NA > 1) for the objective lens and a wavelength of 532 nm, the beam diameter of light passing through the vortex generator can be less than approximately 20 mm. In some examples, the beam diameter of light passing through the vortex generator is less than 200 mm. When the second set of optical components is in the second operational position, the first lens and the second lens are moved away from the first optical axis of the first set of optical components such that the input beam does not pass through the first lens or the second lens. A vortex beam is generated by passing the input beam through the vortex generator of the first set of optical components. The vortex beam is output as the second output beam.

[0031] In some embodiments, the input beam bypasses the first set of optical components when the second set of optical components is in the first operational position. The second set of optical components can include a mirror that deflects the input beam away from the first optical axis of the second set of optical components in the first operational position. The deflected beam then passes through a beam expander before being output as the first output beam. In one example, the beam expander can be part of the second set of optical components that moves between the two operational positions. In another example, the beam expander can be fixedly mounted relative to the first set of optical components and does not move between the operational positions.

[0032] The first set of optical components can 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 can be used during a calibration process and removed during sample measurements.

[0033] In some aspects, the input beam has a wavelength of between approximately 200 nanometers and 900 nanometers. For example, the light can have a wavelength of approximately 532 nanometers.

[0034] In some aspects, the second set of optical components is located on a mechanical slide configured to move the second set of optical components between a first operating position and a second operating position. In some aspects, the system includes a controller configured to control the mechanical slide 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 the second set of optical components between a first operating position and a second operating position; an objective lens for directing the first output beam or the second output beam to a sample; a detector for receiving light from the sample; a controller including a processor and a non-transitory memory for storing computer readable instructions that, when executed by the processor, configure the microscopy system to: adjust the second set of optical components in the first operating position via the actuator; direct the first output beam toward the sample and generate a first image based on light received from the detector; adjust the second set of optical components in the second operating position via the actuator; direct the second output beam toward the sample and generate a second image based on light received from the detector; and generate a C-SIM image based on the first image and the second image.

[0035] The first output beam and the second output beam can be directed to the sample surface via a microscope including a scanner and at least one objective lens. The scanner causes the beams to scan over the sample region. The objective lens focuses the beams at the sample plane. The first output beam and the second output beam are aligned as they enter the microscope.

[0036] The first image and the second image can be acquired by scanning the first output beam and the second output beam, respectively, over the sample region. The first image is a non-annular image, and the second image is an annular image. The microscopy system can include a pinhole for confocal imaging. As such, the non-annular image can be a confocal image. The pinhole can be effectively formed by an optical fiber. In one example, the first image is acquired before the second image. In another example, the image is acquired after the second image.

[0037] FIGS. 1A-1D An example C-SIM imaging process 100 using simulated data is shown. FIG. 1A is a ground truth shape of a set of point source objects 102a, 102b arranged to be imaged with a structured illumination microscopy system. As shown, the objects 102a, 102b are simulated light points of fluorescently dyed nanoscale beads. FIG. 1Bis a simulated confocal image (or non-ring image) of the object 102a, 102b convolved with a focused Gaussian beam. Due to the diffraction limit, the image appears as a skewed blob 104, omitting the separated doublet nature of the object 102a, 102b. FIG. 1B represents an image obtained with a typical confocal microscope. FIG. 1C is a simulated vortex image obtained by convolving the object 102a, 102b with a focused ring beam. The diameter of the focused ring beam (including both bright and dark regions) is typically larger than the diameter of the focused Gaussian (or non-ring) beam. The holes 106a, 106b become visible to form a somewhat similar skewed blob 108, as FIG. 1C shown in the image of

[0038] FIG. 1D shows the pixel intensity of the ring image weighted and subtracted from the pixel intensity of the confocal image to form a subtracted or difference image (i.e., a C-SIM image). In the subtracted image, the separated blobs 110a, 110b are seen, which have more in common with the actual object 102a, 102b, demonstrating the improved lateral resolution obtainable for the microscope used to collect the images. By using the subtraction technique, the simulated light points 102a, 102b that would have otherwise been indistinguishable can be distinguished with improved lateral resolution.

[0039] As shown, the intensity of the ring image is weighted by a gamma factor. Thus, with one image (I 非环形 ) taken using a confocal scanning light point and another image (I 环形 ) taken using a ring scanning light point, and with the image taken with the ring scanning light point typically scaled by an arbitrary parameter before being subtracted from the Gaussian image to generate the C-SIM image (I C-SIM ), the equation for determining the intensity of the super-resolution image can generally be in the following form:

[0040] I C-SIM = I 非环形 - γ * I 环形

[0041] where γ is an arbitrary parameter. The arbitrary parameter can be determined based on a peak of the non-ring beam imaging intensity and a peak of the ring beam imaging intensity across the imaged area, as disclosed in U.S. Application No. 18 / 296,859, entitled “Adaptive subtraction for C-SIM Microscopy,” the entirety of which is hereby incorporated by reference.

[0042] FIGS. 2A-2BAnother example C-SIM process is shown using experimental data. A set of sub-resolution fluorescent beads dried on a coverslip were imaged. The width of the beam was approximately 0.5 um. FIG. 2A Non-ring image 200, ring image 202, and C-SIM image 204 taken from the same bead are shown. The resulting C-SIM image 204 was generated by subtracting the ring image 202 from the non-ring image 200. FIG. 2B The signal strength at the same cross-section of the bead in the non-ring (confocal) image 200 and the C-SIM image was compared. The width of the bead captured in the confocal image was 0.73 pm, and the width of the bead captured within the C-SIM image 204 was 0.45 pm, where the beam width was estimated based on the full-width-at-half-max of a Gaussian fit to the intensity distribution.

[0043] FIG. 3 A block diagram of an example microscopy system 300 is shown. The microscopy system 300 includes a light source 302 that emits a light beam 304 and a C-SIM optical component 306 that receives the light beam 304 from the light source 302. The C-SIM optical component 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, by a dichroic mirror).

[0044] The output beam 308 is transmitted through the dichroic filter 314 and into a 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 a sample 326. The scanner can cause the output beam 308 to scan 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 to a wavelength-selective element 315 via the dichroic filter 314. The wavelength-selective element 315 can receive the sample light 328 via an input port 317, such as a pinhole. The wavelength-selective element 315 selectively transmits a range of wavelengths 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 the spectrometer. Electrical signals are transmitted from the detector to a controller 330 that includes an electronic processor 313 and a memory 332. The controller 330 (i.e., by executing software stored in the memory 332 with the electronic processor 313) performs analysis on light detected from the sample, such as the sample light 328, based on the electrical signals from the detector 316.

[0045] Microscopy system 300 can be a structured illumination microscopy system in which output beam 308 from C-SIM optical components 306 can be a non-ring beam or a ring beam. Non-ring beams and ring beams are aligned. Input port 317 can be a confocal pinhole. Signals detected from the detector can include both fluorescence signals and Raman signals. Signals can be detected from the sample in any of the optical paths shown. FIG. 3 Optical fibers can be used as optical waveguides in any of the optical paths shown. For example, sample light 328 reflected from dichroic filter 314 can be coupled into one end of an optical fiber, and the other end of the optical fiber can be coupled with wavelength selective element 315. The end of the optical fiber that receives light from dichroic filter 314 can function as input port 317.

[0046] Controller 330 can have more or fewer components than shown. Controller 330 is suitable for the application and setup, and can include, for example, multiple electronic processors, multiple data storage devices (memory modules), or a combination thereof.

[0047] Memory 332 can include one or more local memory devices or remote memory devices, such as random access 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 (CBRAM) devices), hard disk drive-based memory devices, solid-state memory devices, networked drives, cloud drives, or any combination of memory devices.

[0048] In some implementations, microscopy system 300 includes more or fewer optical components than shown. For example, additional mirrors can be provided to direct the light beams. FIG. 3

[0049] FIGS. 4A-4B One example of C-SIM optical components 306 in different operating positions is shown. C-SIM optical components 306 include a first slider 400 and a second slider 402. A VPP 404 and a quarter waveplate 406 are positioned between first slider 400 and second slider 402. VPP 404 and quarter waveplate 406 are positioned along an optical path 408, and form, for example, a first set of optical components. Light beam 304 from light source 302 travels along first optical path 408.

[0050] ​The first slide 400 includes a first lens 410 and a linear polarizer 412 that form, for example, a second set of optical components. The second slide 402 includes a second lens 414. The first slide 400 and the second slide 402 move to place the first lens 410 and the second lens 414 within the optical path 408 or to place the linear polarizer 412 within the optical path such that the light beam 304 travels through the first lens 410 and the second lens 414 or through the linear polarizer 412. The linear polarizer 412 includes an optical device that converts incident light into a perfectly linearly polarized state.

[0051] In FIG. 4A , the second set of optical components of the C-SIM optical component 306 is in a first operational position. In particular, in the first operational position, the light beam 304 input to the C-SIM optical component 306 is expanded and output as a first output beam 411 (which can be, for example, the output beam 308) to the microscope 322 as a non-ring shaped beam. In the first operational position, the first slide 400 and the second slide 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 FIG. 3 ) travels along the first optical path 408, sequentially through the first lens 410, the VPP 404, the quarter waveplate 406, and the second lens 414. FIG. 3

[0052] When considered together, the first lens 410 and the second lens 414 form an expander 500, as shown in FIG. 5 . The dashed lines are ray traces of light passing through the expander. The expander 500 can be, for example, a Kepler expander that provides about 1x, 2x, or 3x beam expansion to the light beam 304. In FIG. 5 the example, the first lens 410 and the second lens 414 have a relatively long focal length such that the distance between the first lens 410 and the second lens 414 is between about ten inches and fourteen inches. In other embodiments, the distance between the first lens 410 and the second lens 414 can be greater than ten 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 a central region between the first lens 410 and the second lens 414. The vortex generator (in this example, the VPP 404 and the quarter waveplate 406) is located at the narrow space on the opposite side of the intersection 502 such that the beam diameter passing through the vortex generator is small so that the light beam 304 experiences minimal perturbation from the vortex generator. The intersection 502 is the intersection of the focal point of the first lens 410 and the focal point of the second lens 414. The expanded beam is provided as the output beam 308 and fills the back lens of the objective lens of the microscope 322.

[0053] In FIG. 4B ​In this configuration, the second set of optical components of the C-SIM optical component 306 is in a second operating position and forms a vortex (annular) 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 light beam 304 is not expanded, but instead is transformed by the VPP 404 and the quarter waveplate 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

[0054] The first slider 400 and the second slider 402 are mechanical sliding components that transition the second set of optical components between the first operating position and the second operating position. The first set of optical components, including the VPP 404 and the quarter waveplate 406, are fixedly aligned with the input light beam 304. The first set of optical components can be fastened to a base plate (as shown in FIG. 6

[0055] In some implementations, the C-SIM optical component 306 includes a beamsplitter 416 positioned between the quarter waveplate 406 and the second slider 402. The beamsplitter 416 can separate 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

[0056] FIG. 6 An example microscopy system 600 is shown having a C-SIM optical component 306 similar to the configuration of FIG. 4A and FIG. 4B The microscopy system 600 can be substantially similar to the microscopy system 300 shown in more detail. In FIG. 6 ​​​In the example shown, VPP 404 and quarter waveplate 406 are fixedly secured to base plate 602 along with other optical components. Additionally, microscopy system 600 includes a polarimeter 604 that receives a portion of light from beamsplitter 416. In microscopy system 600, focusing optics 608 are optically positioned after dichroic filter 314. Focusing optics 608 focus sample light 328 onto a fiber optic cable 606 that is connected to a spectrometer, such as spectrometer 312. Example microscopy system 600 can also include a plurality of mirrors 610 for guiding light to travel within microscopy system 600. For example, first mirror 610A and second mirror 610B direct output beam 308 from second slide 402 to dichroic filter 314. Third mirror 610 directs third light 318 from dichroic filter 314 to microscope 322 and directs scattered light 328 from microscope 322 to dichroic filter 314.

[0057] FIG. 7A First, second, and third C-SIM images 702, 704, and 706 of imaged 0.5 pm fluorescent beads are shown. C-SIM images 702-706 were captured using microscopy system 300. The size of the imaged beads was measured using a half-peak full width technique extracted from a line scan through the center of the beads. FIG. 7B A plot showing experimental (solid line) and theoretical (dashed line) signal strength along a cross-section of a fluorescent bead within a C-SIM image plotted against the theoretical signal strength is shown. The measured signal is Gaussian-like, indicating that there is no significant aberration. Further, the FWHM of the measured data is close to the theoretical signal strength, demonstrating the ability of the system for super-resolution imaging.

[0058] FIGS. 8A-8B Another example implementation of C-SIM optical components 306 is shown. In this example, the first set of optical components includes a vortex generator. The vortex generator includes vortex phase plate 404 and quarter waveplate 406. The first optical components can optionally include beamsplitter 416. The second set of optical components includes an expander 806. Expander 806 can be formed from two lenses, such as lenses 410 and 414, shown. The second set of optical components can optionally include one or more mirrors, such as mirrors 802, 804, 808, and 810, for directing input beam 304 through the expander when the second set of optical components is in the first operating position. Some of the optical components in the second set of optical components, such as mirrors 802 and 810, can be mounted on slides 400 and 402. Similar to the configuration of FIG. 4A and FIG. 4B slides 400 and 402, slides 400 and 402 can be driven individually or collectively by one or more actuators. FIG. 4A and FIG. 4B ​

[0059] In FIG. 8A , the second set of optical components of the C-SIM optical component 306 is in the first operational position. In FIG. 8A , the first operational position of the C-SIM optical component 306, the light beam 304 is deflected away from the optical path 408 and thus bypasses the vortex generator. The light beam 304 is deflected 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 positioned 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 FIG. 3 ) is directed by the first mirror 802 to the beam expander 806, which expands the beam to generate an expanded output beam 411, which is a non-ring shaped beam. As FIG. 8A illustrated, in some cases, the C-SIM optical component 306 also includes a third mirror 804 for directing light from the first mirror 802 towards the beam expander 806, and a fourth mirror 808 for directing the expanded output beam 411 from the beam expander 806 to the 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.

[0060] In FIG. 8B , the second set of optical components of the C-SIM optical component 306 is in the second operational position. In particular, in the second operational position, the C-SIM optical component 306 is configured to generate a vortex beam, such as a ring shaped beam, such as a circular ring shaped focal spot, at the sample plane. In the second operational position, the first slider 400 and the second slider 402 are positioned such that the first mirror 802 and the second mirror 810 are removed from the optical path 408. Thus, in this position, the light beam 304 is not expanded, but instead transformed by the VPP 404 and the quarter wave plate 406 into a narrow vortex beam 418. 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 light beam can pass through the first slider and the second slider via apertures on the sliders.

[0061] The microscopy system 300 and the microscopy system 600 are merely examples, and further microscopy systems including a fixed or static vortex phase plate are also possible. For example, FIGS. 9-10 a microscopy system 900 is shown. In FIG. 9 , the microscopy system 900 is in the first operational position. In FIG. 10 , the microscopy system 900 is in the second operational position. The optical components found within the microscopy system 900 can operate similarly to their counterparts in the microscopy system 300, but are arranged differently.

[0062] 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 an aperture, and a reflective portion, such as a mirror. In a first operational position, the reflective portion of the first slider 906 directs the light through a beam expander 926 (see FIG. FIG. 9 After expansion, light 904 is reflected by a steering mirror 928 onto a second slider 908. Second slider 908 also has a transmissive portion and a reflective portion. The reflective portion of second slider 908 directs the expanded light 904 toward a dichroic filter 918 and a detector 920. Light 904 is reflected by dichroic filter 918 onto a third mirror 922 and toward a microscope 924.

[0063] 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 changes. FIG. 10 As shown, the empty portion of first slider 906 is positioned along the optical path of light 904, and light 904 travels through first slider 906 (bypassing beam expander 926). Light 904 travels through first slider 906 and is reflected by first mirror 910 toward VPP 912 and quarter-wave plate 914. Light 904 travels through VPP 912 and quarter-wave plate 914 to second slider 908. In the second operational position, the empty portion of second slider 908 is positioned along the optical path of light 904, and light 904 travels through second slider 908. Light 904 traveling through second slider 908 is reflected by second mirror 916 toward dichroic filter 918 and detector 920. Light 904 is reflected by dichroic filter 918 toward third mirror 922 and toward microscope 924.

[0064] Thus, in microscopy system 900, both beam expander 926 and VPP 912 are fixedly secured and do not require adjustment during measurement. Instead, additional mirrors are moved in or out of the optical path of light 904 to direct the light along the appropriate path.

[0065] FIG. 11 is a flow chart illustrating an example method 1100 for generating a C-SIM image. Method 1100 can be executed by controller 330 and can be performed using any of the example microscopy systems described herein. For descriptive purposes, the steps of method 1100 are described in an iterative manner. The various steps described herein with respect to method 1100 can be performed simultaneously, in parallel, or in an order different from the serial and iterative execution illustrated. In some cases, individual steps can be omitted from method 1100.

[0066] At block 1102, the method 1100 includes calibrating the microscopy system 300. Calibrating the microscopy system can include aligning first and second output beams generated at first and second operating positions, respectively, of the second set of optical components. The microscopy system can be calibrated with a reference sample. For example, prior to measurement, a toroidal image of 0.5 pm diameter fluorescent beads can be collected using the vortex phase plate 404 in the optical path. In other words, the toroidal image can be obtained with the C-SIM components 306 of the microscopy system in the second operating position. Examples of such images are shown in FIG. 12A , FIG. 12B , FIG. 12C and FIG. 12D . In each image, a toroidal morphology is visible, and a non-uniform intensity in the intensity of the outer toroidal region is a result of photobleaching. These images can be used to ensure that the C-SIM optical components 306 are aligned prior to detailed imaging. In some cases, the operations of block 1102 are optional, and calibration can not be performed for each sample or prior to each scan operation.

[0067] At block 1104, the method 1100 includes loading a sample into the microscope 322. As one non-limiting example, after verifying alignment (block 1102), a sulfur sample is loaded into the microscope 322.

[0068] 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 optical components 306 are placed in the first operating position, such as shown in FIG. 4A , FIG. 8A or FIG. 9 . An example image of a sample (e.g., sulfur) acquired by scanning a non-toroidal scan spot having a pixel size of 0.2 pm is shown in FIG. 13A .

[0069] 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 optical components 306 are placed in the second operating position, as shown in FIG. 4B , FIG. 8B or FIG. 10 . An example image of a sulfur sample acquired by scanning a toroidal scan spot having a pixel size of 0.2 pm is shown in FIG. 13B .

[0070] At block 1110, the method 1100 includes generating a C-SIM image based on the non-toroidal image (acquired at block 1106) and the toroidal image (acquired at block 1108). For example, FIG. 13C the image in FIG. 13A andFIG. 13B weighted subtraction of the annular image of 1002 and represents a super-resolution image. In FIGS. 13A-13C In the example of 1002, the annular image of 1000 is subtracted from 1002. As compared to the images of 1000 and 1004, FIG. 13A the super-resolution image of 1002 contains significantly more surface and edge detail. In 1002, a complex set of intensity inhomogeneities can be seen. In some examples, block 1108 is performed before block 1106. That is, the annular image is acquired before the non-annular image. FIG. 13B FIG. 13A FIG. 13B FIG. 13C FIG. 13C In 1002, a complex set of intensity inhomogeneities can be seen. In some examples, block 1108 is performed before block 1106. That is, the annular image is acquired before the non-annular image.

[0071] FIG. 14A Another example C-SIM image 1400 of a matrix of 1.0 pm diameter polystyrene beads obtained via the methods and systems described herein is shown. The polystyrene beads were dried on a coverslip and images were collected with a 100x 0.95 NA objective. As shown in the confocal image 1400, the beads are tightly packed together in a matrix-like fashion and are symmetric and regular in size. A line scan 1405 was taken across the center of one bead to capture a cross-section of that bead, as shown in FIG. 14B

[0072] While the examples described herein primarily involve both a VPP and a quarter waveplate positioned adjacent to one another, in some cases the quarter waveplate can be omitted such that light passes through the VPP rather than the quarter waveplate while the image is being captured. Additionally, in some implementations, the VPP can be replaced by other optical devices that can produce a vortex beam, such as a spatial light modulator.

[0073] Additionally, while the sliders described herein with respect to microscopy system 300, microscopy system 600, and microscopy system 900 are shown 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 can also be implemented.

[0074] Accordingly, the implementations described herein provide systems, methods, computing and storage devices, and computer readable media for capturing C-SIM microscopy images with fixed optical components. As described above, the implementations described herein provide for tuning between confocal imaging mode and annular imaging mode without tuning the vortex phase plate. The implementations described herein improve the reliability and reproducibility of super-resolution imaging. Accordingly, the implementations disclosed herein provide improvements to super-resolution imaging.

[0075] ​​​​​As described above in the detailed description, reference was made to the drawings, which form a part thereof, and in which are shown by way of illustration various embodiments by which the principles of the disclosure can be practiced. The embodiments illustrated are intended to be examples only and are not limiting in any way. As such, the disclosure is to be accorded the widest scope of the principles as disclosed and equivalent aspects.

[0076] Various operations can be described as multiple discrete actions or operations in turn, in a manner that is most helpful in understanding the disclosed subject matter. However, the order of description should not be construed as to imply that these operations are necessarily order dependent. In particular, these operations can not be performed in the order of presentation. Operations described can be performed in a different order than the described embodiment. Various additional operations can be performed and / or described operations can be omitted.

[0077] 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 case of conflict, the present document, including definitions, controls. Although methods and systems similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the exemplary methods and systems described below are

[0078] The terms “comprise,” “comprising,” “have,” “has,” “can,” “contain,” “containing,” “include,” “including,” and variations thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the presence of an additional item or step. Unless otherwise defined, singular forms “a,” “an” and “the” include plural referents.

[0079] 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., it includes the degree of error associated with the measurement of the particular quantity). The modifier “about” should also be considered in its normal sense as disclosing a range inclusive of the absolute value of the two endpoints. For example, the expression “about 2 to about 4” also discloses the range “2 to 4.” The term “about” can mean plus or minus 10% of the indicated number. For example, “about 10%” can mean a range of 9% to 11%, and “about 1” can mean 0.9 to 1.1. Other meanings of “about” can be apparent from the context, such as rounding, so, for example, “about 1” can also mean 0.5 to 1.4.

[0080] As used herein, the term "or" is intended to mean an inclusive "or" rather than an exclusive "or". That is, unless specified otherwise, or clear from context, "X employs A or B" is intended to mean any of the 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" is satisfied under any of the foregoing instances. Moreover, articles "a" and "an" as used in the subject specification and annexed drawings should generally be construed to mean "one or more" unless specified otherwise or clear from context to be directed to a singular form.

[0081] For recitations of numerical ranges of values herein, it is specifically intended that every intervening value, to the exclusion of none, be expressly set forth. For example, the recitation of a range of values from 6 to 9 is intended to include the values 7 and 8, unless the context clearly dictates otherwise. For recitations of numerical ranges of values herein, it is specifically intended that every intervening value, to the exclusion of none, be expressly set forth. For example, the recitation of a range of values from 6.0 to 7.0 is intended to include the values 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0.

[0082] The disclosure is now 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 a better understanding of the present disclosure. It will be apparent, however, that systems and methods of the present disclosure can be practiced in other ways without these specific details. In other instances, well-known structures and devices are shown in block diagram form in order to facilitate describing the systems and methods of the present disclosure.

[0083] Clause

[0084] The following clauses disclose specific implementations of the present disclosure:

[0085] Clause 1 : An optical system for structured illumination, the optical system comprising: a first set of optical components fixedly aligned with respect to a light beam entering the optical system; and a second set of optical components adjustable between a first operational position and a second operational position, wherein, when the second set of optical components is in the first operational position, the light beam is expanded and output as a first output beam, and wherein, when the second set of optical components is in the second operational position, the light beam passes through the first set of optical components and forms a vortex beam, the vortex beam output as a second output beam.

[0086] Clause 2: The optical system of clause 1, wherein each optical component in the first set of optical components is aligned along a first optical axis.

[0087] Clause 3: The optical system of any one of clauses 1-2, wherein the first set of optical components comprises a vortex generator.

[0088] Clause 4: The optical system of clause 3, wherein the second set of optical components includes at least one beam expander.

[0089] Clause 5: The optical system of clause 4, wherein the optical beam passes through the first set of optical components when the second set of optical components is in the first operational position.

[0090] Clause 6: The optical system of claim 5, wherein the beam expander includes a first lens and a second lens, and the optical beam sequentially passes through the first lens, the vortex generator, and the second lens when the second set of optical components is in the first operational position.

[0091] Clause 7: The optical system of clause 6, wherein at the first operational position, the optical beam entering the optical system is focused on an intersection between the first lens and the second lens, and a cross-section of the beam passing through the vortex generator is no more than 20 mm to 200 mm.

[0092] Clause 8: The optical system of clause 7, wherein the vortex generator includes a vortex phase plate and a quarter wave plate, and wherein the intersection is between the vortex phase plate and the quarter wave plate.

[0093] Clause 9: The optical system of clause 8, wherein the first lens and the second lens are separated by 10 inches to 14 inches.

[0094] Clause 10: The optical system of any one of clauses 3 to 9, wherein the second set of optical components further includes a linear polarizer, and the optical beam passes through the linear polarizer before passing through the vortex generator when the second set of optical components is in the second operational position.

[0095] Clause 11: The optical system of clause 3, wherein the optical beam bypasses the first set of optical components when the second set of optical components is in the first operational position.

[0096] Clause 12: The optical system of clause 11, wherein the second set of optical components includes at least one mirror, and the optical 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 operational position.

[0097] Clause 13: The optical system of any one of clauses 1 to 12, wherein the first output beam and the second output beam are aligned.

[0098] Clause 14: The optical system of any one of clauses 1 to 13, wherein the optical components in the second set of optical components are fixedly mounted relative to each other.

[0099] Clause 15: The optical system of clause 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 the first operational position and the second operational position by operating the movable structure.

[0100] Clause 16: A structured illumination microscopy system, the structured illumination microscopy system comprising: a light source configured to generate a light beam; an optical system for structured illumination according to any one of clauses 1-15 to receive the light beam and generate the first output beam or the second output beam; at least one actuator to adjust the second set of optical components between the first operational position and the second operational position; an objective lens to direct the first output beam or the second output beam to a sample; a detector to receive light from the sample; a controller comprising a processor and a non-transitory memory to store computer readable instructions that, when executed by the processor, configure the microscopy system to: adjust the second set of optical components in the first operational position via the actuator; direct the first output beam toward the sample and generate a first image based on light received from the detector; adjust the second set of optical components in the second operational position via the actuator; direct the second output beam toward the sample and generate a second image based on light received from the detector; and generate a sample image based on the first image and the second image.

[0101] Clause 17: The microscopy system of clause 16, wherein the light from the sample comprises a Raman shift.

[0102] Clause 18: The microscopy system of any one of clauses 16-17, wherein at least one optical component of the second set of optical components is fixedly mounted on a movable structure, wherein the movable structure comprises a slide, and the actuator is configured to slide the slide to move the second set of optical components between the first operational position and the second operational position.

[0103] Clause 19: The microscopy system of any one of clauses 16-18, further comprising a scanner to scan the first output beam or the second output beam over a sample region to acquire a first sample image and a second sample image.

[0104] Clause 20: The microscopy system of clause 19, wherein the microscopy system is configured to scan the second output beam over the sample region prior to scanning the first output beam over the sample region.

Claims

1. An optical system for structured lighting, the optical system comprising: a first set of optical components fixedly aligned relative to a beam of light entering the optical system; and a second set of optical components, the second set of optical components being adjustable between a first operative position and a second operative 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 Wherein, when the second group of optical components is in the second operating position, the light beam passes through the first group of optical components and forms a vortex beam, and the vortex beam 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 comprises at least one beam expander.

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

6. The optical system of claim 5, wherein at the first operating position, the light beam entering the optical system is focused on an intersection between the first lens and the second lens, and a cross-section of the beam passing through the vortex generator is no greater than 20 mm to 200 mm.

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

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

9. The optical system of any one of claims 2-8, wherein the second set of optical components further comprises a linear polarizer, and when the second set of optical components is in the second operating position, the light beam passes through the linear polarizer before passing through the vortex generator.

10. The optical system of any of claims 2-8, 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. The optical system of claim 10, wherein the second set of optical components includes at least one mirror, and when the second set of optical components is in the first operating position, the light beam is deflected by the mirror to bypass the first set of optical components.

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

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

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

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

16. The microscopy system of claim 14 , wherein at least one optical component of the second set of optical components is fixedly mounted on a movable structure, wherein the movable structure comprises a slider, and the actuator is configured to slide the slider to move the second set of optical components between the first operating position and the second operating position.

17. The microscopy system of claim 14, further comprising a scanner configured to scan the first output beam or the second output beam over a sample area to acquire a first sample image and a second sample image.

18. The microscopy system of claim 17, 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.

Images