Patent Description:
Structured illumination microscopy (SIM) describes a technique by which spatially structured (i.e., patterned) light may be used to image a sample to increase the lateral resolution of the microscope by a factor of two or more. In some instances, during imaging of the sample, three images of fringe patterns of the sample are acquired at various pattern phases (e.g., <NUM>°, <NUM>°, and <NUM>°), so that each location on the sample is exposed to a range of illumination intensities, with the procedure repeated by rotating the pattern orientation about the optical axis to <NUM> separate angles (e.g. <NUM>°, <NUM>° and <NUM>°). The captured images (e.g., nine images) may be assembled into a single image having an extended spatial frequency bandwidth, which may be retransformed into real space to generate an image having a higher resolution than one captured by a conventional microscope.

In some implementations of current SIM systems, a linearly polarized light beam is directed through an optical beam splitter that splits the beam into two or more separate orders that may be combined and projected on the imaged sample as an interference fringe pattern with a sinusoidal intensity variation. Diffraction gratings are examples of beam splitters that can generate beams with a high degree of coherence and stable propagation angles. When two such beams are combined, the interference between them can create a uniform, regularly-repeating fringe pattern where the spacing is determined by factors including the angle between the interfering beams. If more than two beams are combined, the resulting pattern typically contains a mixture of fringe spacings, with the result that the difference between the maximum and minimum intensities (also known as the "modulation depth") is reduced, making it less suitable for SIM purposes.

In some implementations of current SIM systems, the orientation of the projected pattern is controlled by rotating the beam splitting element about the optic axis, and the phase of the pattern is adjusted by moving the element laterally across the axis. In such systems, a diffraction grating is typically mounted on a translation stage, which in turn is mounted on a rotation stage. Additionally, such systems often utilize a linear polarizer to polarize the light emitted by the light source before it is received at the grating.

<CIT> relates to an optical arrangement for positioning in a beam path of a light microscope and a light microscope. <CIT> relates to a method and an assembly for generating optical section images and serves the optical scanning of spatially extended objects, layer by layer. <CIT> A relates to a known spatial splitting-based optical Micro Electro-Mechanical Systems (MEMS) Interferometer including a spatial splitter for spatially splitting an input beam into two interferometer beams. <CIT> relates to a fluorescent microscope and a respective method for obtaining images of a sample labelled with at least one type fluorescent label by combining localization microscopy and structured illumination microscopy.

Implementations disclosed herein are directed to structured illumination systems and methods. The invention relates to system and corresponding method as defined by the independent claims. Preferred embodiments of the structured illumination system SIM and corresponding method according to the invention are the subject of the dependent claims.

The present disclosure, in accordance with one or more implementations, is described in detail with reference to the following figures. The figures are provided for purposes of illustration only and merely depict example implementations. Furthermore, it should be noted that for clarity and ease of illustration, the elements in the figures have not necessarily been drawn to scale.

Some of the figures included herein illustrate various implementations of the disclosed technology from different viewing angles. Although the accompanying descriptive text may refer to such views as "top," "bottom" or "side" views, such references are merely descriptive and do not imply or require that the disclosed technology be implemented or used in a particular spatial orientation unless explicitly stated otherwise.

The figures are not exhaustive and do not limit the present disclosure to the precise form disclosed.

As used herein to refer to light diffracted by a diffraction grating, the term "order" or "order number" is intended to mean the number of integer wavelengths that represents the path length difference of light from adjacent slits or structures of the diffraction grating for constructive interference. The interaction of an incident light beam on a repeating series of grating structures or other beam splitting structures can redirect or diffract portions of the light beam into predictable angular directions from the original beam. The term "zeroth order" or "zeroth order maximum" is intended to refer to the central bright fringe emitted by a diffraction grating in which there is no diffraction. The term "first-order" is intended to refer to the two bright fringes diffracted to either side of the zeroth order fringe, where the path length difference is ± <NUM> wavelengths. Higher orders are diffracted into larger angles from the original beam. The properties of the grating can be manipulated to control how much of the beam intensity is directed into various orders. For example, a phase grating can be fabricated to maximize the transmission of the ±<NUM> orders and minimize the transmission of the zeroth order beam.

As used herein to refer to a sample, the term "feature" is intended to mean a point or area in a pattern that can be distinguished from other points or areas according to relative location. An individual feature can include one or more molecules of a particular type. For example, a feature can include a single target nucleic acid molecule having a particular sequence or a feature can include several nucleic acid molecules having the same sequence (and/or complementary sequence, thereof).

As used herein, the term "xy plane" is intended to mean a <NUM>-dimensional area defined by straight line axes x and y in a Cartesian coordinate system. When used in reference to a detector and an object observed by the detector, the area can be further specified as being orthogonal to the beam axis, or the direction of observation between the detector and object being detected.

As used herein, the term "z coordinate" is intended to mean information that specifies the location of a point, line or area along an axis that is orthogonal to an xy plane. In particular implementations, the z axis is orthogonal to an area of an object that is observed by a detector. For example, the direction of focus for an optical system may be specified along the z axis.

As used herein, the term "optically coupled" is intended to refer to one element being adapted to impart light to another element directly or indirectly.

As noted above, pre-existing implementations of SIM systems mount a diffraction grating on a translation stage, which in turn is mounted on a rotation stage. Additionally, such systems often utilize a linear polarizer for polarizing the light source before it is received at the grating. This pre-existing design suffers from a number of drawbacks for use in a high-throughput microscopy system. First, because a rotation stage must rotate the grating several times during acquisition of an image set (e.g., three times), this slows down the instrument's speed and affects its stability. Typically, the fastest grating stages can rotate is on the order of tens of milliseconds (ms), which imposes a mechanical throughput limit on imaging speed. Second, the pre-existing design has poor repeatability because mechanical tolerances of the rotation stage limit the repeatability of the structured illumination patterns from one image acquisition set to the next. This also imposes a higher cost on the SIM system as it requires a very precise rotation stage.

Third, the pre-existing SIM design is not the most reliable for use in a high-throughput microscopy system because of the number of actuations that are made to rotate the grating. For example, if one SIM image set is acquired every second, the rotation stage may require millions to tens of millions actuations per year. Fourth, the pre-existing SIM design has low optical efficiency because the linear polarizer blocks at least <NUM>% of the light received at the grating.

To this end, implementations of the technology disclosed herein are directed to improved SIM systems and methods.

In accordance with a first set of implementations of the technology disclosed herein, a SIM imaging system may be implemented as a multi-arm SIM imaging system, whereby each arm of the system includes a light emitter and a beam splitter (e.g., a transmissive diffraction grating) having a specific, fixed orientation with respect to the optical axis of the system. In accordance with these implementations, the beam splitters in the SIM imaging system are rotatably fixed (i.e., do not require mechanical rotation), which may provide improved system speed, reliability, and repeatability. For systems where the objects being imaged are oriented primarily along <NUM> perpendicular axes (i.e. vertical and horizontal), it is possible to achieve enhanced spatial resolution using <NUM> pattern angles, instead of the <NUM> angles typically used for randomly-oriented objects. According to the invention, the system is implemented as a two-arm SIM imaging system including a fixed vertical grating and a fixed horizontal grating to project respective fringe patterns on an imaged sample. Other pairs of orthogonal grating and pattern angles can be used, provided they are aligned with the orientation of sample objects. Additionally, the system includes a mirror with holes to combine the two arms into the optical path in a lossless manner.

In accordance with a second set of implementations of the technology disclosed herein not in accordance with the claimed invention, a SIM imaging system may be implemented as a multiple beam splitter slide SIM imaging system, where one linear motion stage is mounted with a plurality of beam splitters (e.g., diffraction gratings) having a corresponding, fixed orientation with respect to the optical axis of the system. In particular implementations, the SIM imaging system may be implemented as a dual optical grating slide SIM imaging system whereby all phase shifts or rotations of the grating pattern projected on imaged sample may be made by linearly translating a motion stage along a single axis of motion, to select one of two gratings or to effect a phase shift of the pattern generated by a selected grating. In such implementations, only a single optical arm having a single emitter and single linear motion stage is needed to illuminate a sample, which may provide system advantages such as reducing the number of moving system parts to improve speed, complexity and cost. Additionally, in such implementations, the absence of a polarizer may provide the advantage of high optical efficiency.

In accordance with a third set of implementations of the technology disclosed herein not in accordance with the claimed invention, a SIM imaging system may be implemented as a pattern angle spatial selection SIM imaging system, whereby a fixed two-dimensional diffraction grating is used in combination with a spatial filter wheel to project one-dimensional diffraction patterns on a sample. In such implementations, the primary optical components of the imaging system may remain stationary, which may improve the stability of the optical system (and of the illumination pattern) and minimize the weight, vibration output, and cost of the moving elements of the system.

Before describing various implementations of the systems and methods disclosed herein, it is useful to describe an example environment with which the technology disclosed herein can be implemented. One such example environment is that of a structured illumination imaging system <NUM>, illustrated in <FIG>, that illuminates a sample with spatially structured light. For example, system <NUM> may be a structured illumination fluorescence microscopy system that utilizes spatially structured excitation light to image a biological sample.

In the example of <FIG>, a light emitter <NUM> is configured to output a light beam that is collimated by collimation lens <NUM>. The collimated light is structured (patterned) by light structuring optical assembly <NUM> and directed by dichroic mirror <NUM> through objective lens <NUM> onto a sample of a sample container <NUM>, which is positioned on a motion stage <NUM>. In the case of a fluorescent sample, the sample fluoresces in response to the structured excitation light, and the resultant light is collected by objective lens <NUM> and directed to an image sensor of camera system <NUM> to detect fluorescence.

Light structuring optical assembly <NUM> in various implementations, further described below, includes one or more optical diffraction gratings or other beam splitting elements (e.g., a beam splitter cube or plate) to generate a pattern of light (e.g., fringes, typically sinusoidal) that is projected onto samples of a sample container <NUM>. The diffraction gratings may be one-dimensional or two-dimensional transmissive or reflective gratings. The diffraction gratings may be sinusoidal amplitude gratings or sinusoidal phase gratings.

As further described below with reference to particular implementations, in system <NUM> the diffraction gratings do not require a rotation stage like the typical structured illumination microscopy system of preexisting systems discussed above. According to the invention, the diffraction gratings are fixed during operation of the imaging system (i.e., not require rotational or linear motion). In the particular implementation, in accordance with the claimed invention and further described below, the diffraction gratings include two fixed one-dimensional transmissive diffraction gratings oriented perpendicular to each other (e.g., a horizontal diffraction grating and vertical diffraction grating).

As illustrated in the example of <FIG>, light structuring optical assembly <NUM> outputs the first orders of the diffracted light beams (e.g., m = ± <NUM> orders) while blocking or minimizing all other orders, including the zeroth orders. However, in alternative implementations, additional orders of light may be projected onto the sample.

During each imaging cycle, imaging system <NUM> utilizes light structuring optical assembly <NUM> to acquire a plurality of images at various phases, with the fringe pattern displaced laterally in the modulation direction (e.g., in the x-y plane and perpendicular to the fringes), with this procedure repeated one or more times by rotating the pattern orientation about the optical axis (i.e., with respect to the x-y plane of the sample). The captured images may then be computationally reconstructed to generate a higher resolution image (e.g., an image having about twice the lateral spatial resolution of individual images).

In system <NUM>, light emitter <NUM> may be an incoherent light emitter (e.g., emit light beams output by one or more excitation diodes), or a coherent light emitter such as emitter of light output by one or more lasers or laser diodes. As illustrated in the example of system <NUM>, light emitter <NUM> includes an optical fiber <NUM> for guiding an optical beam to be output. However, other configurations of a light emitter <NUM> may be used. In implementations utilizing structured illumination in a multi-channel imaging system (e.g., a multi-channel fluorescence microscope utilizing multiple wavelengths of light), optical fiber <NUM> may optically couple to a plurality of different light sources (not shown), each light source emitting light of a different wavelength. Although system <NUM> is illustrated as having a single light emitter <NUM>, in some implementations multiple light emitters <NUM> may be included. For example, multiple light emitters may be included in the case of a structured illumination imaging system that utilizes multiple arms, further discussed below.

In some implementations, system <NUM> may include a projection lens <NUM> that may include a lens element to articulate along the z-axis to adjust the structured beam shape and path. For example, a component of the projection lens may be articulated to account for a range of sample thicknesses (e.g., different cover glass thickness) of the sample in container <NUM>.

In the example of system <NUM>, fluid delivery module or device <NUM> may direct the flow of reagents (e.g., fluorescently labeled nucleotides, buffers, enzymes, cleavage reagents, etc.) to (and through) sample container <NUM> and waste valve <NUM>. Sample container <NUM> can include one or more substrates upon which the samples are provided. For example, in the case of a system to analyze a large number of different nucleic acid sequences, sample container <NUM> can include one or more substrates on which nucleic acids to be sequenced are bound, attached or associated. The substrate can include any inert substrate or matrix to which nucleic acids can be attached, such as for example glass surfaces, plastic surfaces, latex, dextran, polystyrene surfaces, polypropylene surfaces, polyacrylamide gels, gold surfaces, and silicon wafers. In some applications, the substrate is within a channel or other area at a plurality of locations formed in a matrix or array across the sample container <NUM>. System <NUM> also may include a temperature station actuator <NUM> and heater/cooler <NUM> that can optionally regulate the temperature of conditions of the fluids within the sample container <NUM>.

In particular implementations, the sample container <NUM> may be implemented as a patterned flow cell including a translucent cover plate, a substrate, and a liquid contained therebetween, and a biological sample may be located at an inside surface of the translucent cover plate or an inside surface of the substrate. The flow cell may include a large number (e.g., thousands, millions, or billions) of wells or regions that are patterned into a defined array (e.g., a hexagonal array, rectangular array, etc.) into the substrate. Each region may form a cluster (e.g., a monoclonal cluster) of a biological sample such as DNA, RNA, or another genomic material which may be sequenced, for example, using sequencing by synthesis. The flow cell may be further divided into a number of spaced apart lanes (e.g., eight lanes), each lane including a hexagonal array of clusters. Example flow cells that may be used in implementations disclosed herein are described in <CIT>.

Sample container <NUM> can be mounted on a sample stage <NUM> to provide movement and alignment of the sample container <NUM> relative to the objective lens <NUM>. The sample stage can have one or more actuators to allow it to move in any of three dimensions. For example, in terms of the Cartesian coordinate system, actuators can be provided to allow the stage to move in the X, Y and Z directions relative to the objective lens. This can allow one or more sample locations on sample container <NUM> to be positioned in optical alignment with objective lens <NUM>. Movement of sample stage <NUM> relative to objective lens <NUM> can be achieved by moving the sample stage itself, the objective lens, some other component of the imaging system, or any combination of the foregoing. In some implementations, movement of sample stage <NUM> may be implemented during structured illumination imaging to move structured illumination fringes with respect to the sample to change phases. Further implementations may also include moving the entire imaging system over a stationary sample. Alternatively, sample container <NUM> may be fixed during imaging.

In some implementations, a focus (z-axis) component <NUM> may be included to control positioning of the optical components relative to the sample container <NUM> in the focus direction (typically referred to as the z axis, or z direction). Focus component <NUM> can include one or more actuators physically coupled to the optical stage or the sample stage, or both, to move sample container <NUM> on sample stage <NUM> relative to the optical components (e.g., the objective lens <NUM>) to provide proper focusing for the imaging operation. For example, the actuator may be physically coupled to the respective stage such as, for example, by mechanical, magnetic, fluidic or other attachment or contact directly or indirectly to or with the stage. The one or more actuators can be configured to move the stage in the z-direction while maintaining the sample stage in the same plane (e.g., maintaining a level or horizontal attitude, perpendicular to the optical axis). The one or more actuators can also be configured to tilt the stage. This can be done, for example, so that sample container <NUM> can be leveled dynamically to account for any slope in its surfaces.

It should be appreciated that although <FIG> illustrates the use of an objective lens <NUM> for combining and projecting the two beam orders on the imaged sample as an interference fringe pattern, other suitable means may be used to combine the two beams and/or project the interference pattern on the sample. Any means of redirecting the beams may suffice (e.g., using mirrors), provided the path length traversed by the beams is within a temporal coherence length of the beams. Additionally, in some implementations, the two beam orders may automatically overlay for a distance beyond the beam splitter (e.g., diffraction grating). In such implementations, an interference pattern may appear near the grating, removing the requirement of an additional projection system if the diffraction grating is placed sufficiently close to the sample. As such, it should be appreciated that implementations for SIM described herein may apply to systems that do not rely on objective lens systems to project interference patterns.

The structured light emanating from a test sample at a sample location being imaged can be directed through dichroic mirror <NUM> to one or more detectors of camera system <NUM>. In some implementations, a filter switching assembly <NUM> with one or more emission filters may be included, where the one or more emission filters can be used to pass through particular emission wavelengths and block (or reflect) other emission wavelengths. For example, the one or more emission filters may be used to switch between different channels of the imaging system. In a particular implementation, the emission filters may be implemented as dichroic mirrors that direct emission light of different wavelengths to different image sensors of camera system <NUM>.

Camera system <NUM> can include one or more image sensors to monitor and track the imaging (e.g., sequencing) of sample container <NUM>. Camera system <NUM> can be implemented, for example, as a charge-coupled device (CCD) image sensor camera, but other image sensor technologies such as active pixel sensors (e.g., complementary metal-oxide-semiconductor (CMOS) image sensors) can be used. In some implementations, structured illumination imaging system <NUM> may utilize an image sensor (e.g., active pixel sensor) in an active plane of the sample. In such implementations, the imaged sample may be patterned and/or aligned over the image sensor.

Output data (e.g., images) from camera system <NUM> may be communicated to a real-time analysis module (not shown) that may be implemented as a software application that, as further described below, may reconstruct the images captured during each imaging cycle to create an image having a higher spatial resolution. Alternatively, the output data may be stored for reconstruction at a later time.

Although not illustrated, a controller can be provided to control the operation of structured illumination imaging system <NUM>, including synchronizing the various optical components of system <NUM>. The controller can be implemented to control aspects of system operation such as, for example, configuration of light structuring optical assembly <NUM> (e.g., selection and/or linear translation of diffraction gratings), movement of projection lens <NUM>, focusing, stage movement, and imaging operations. In various implementations, the controller can be implemented using hardware, algorithms (e.g., machine executable instructions), or a combination of the foregoing. For example, in some implementations the controller can include one or more CPUs or processors with associated memory. As another example, the controller can comprise hardware or other circuitry to control the operation, such as a computer processor and a non-transitory computer readable medium with machine-readable instructions stored thereon. For example, this circuitry can include one or more of the following: field programmable gate array (FPGA), application specific integrated circuit (ASIC), programmable logic device (PLD), complex programmable logic device (CPLD), a programmable logic array (PLA), programmable array logic (PAL) or other similar processing device or circuitry. As yet another example, the controller can comprise a combination of this circuitry with one or more processors.

In accordance with some implementations of the technology disclosed herein, the SIM imaging system may be implemented as a multi-arm SIM imaging system, where each arm of the system includes a light emitter and a grating having a specific, fixed orientation with respect to the optical axis of the system.

<FIG> is an optical diagram illustrating one example optical configuration of a two-arm SIM imaging system <NUM> in accordance with the claimed invention. The first arm of system <NUM> includes a light emitter 210A, an optical collimator 220A to collimate light output by light emitter 210A, a diffraction grating 230A in a first orientation with respect to the optical axis, a rotating window 240A, and a projection lens 250A. The second arm of system <NUM> includes a light emitter 210B, an optical collimator 220B to collimate light output by light emitter 210B, a diffraction grating 230B in a second orientation with respect to the optical axis, a rotating window 240B, and a projection lens 250B. Although diffraction gratings are illustrated in this example, in other implementations, other beam splitting elements such as a beam splitter cube or plate may be used to split light received at each arm of SIM imaging system <NUM>.

Each light emitter 210A-210B may be an incoherent light emitter (e.g., emit light beams output by one or more light emitting diodes(LEDs)), or a coherent light emitter such as emitter of light output by one or more lasers or laser diodes. In the example of system <NUM>, each light emitter 210A-210B is an optical fiber that outputs an optical beam that is collimated by a respective collimator 220A-220B.

In some implementations, each optical fiber may be optically coupled to a corresponding light source (not shown) such as a laser. During imaging, each optical fiber may be switched on or off using a high-speed shutter (not shown) positioned in the optical path between the fiber and the light source, or by pulsing the fiber's corresponding light source at a predetermined frequency during imaging. In some implementations, each optical fiber may be optically coupled to the same light source. In such implementations, a beam splitter or other suitable optical element may be used to guide light from the light source into each of the optical fibers. In such examples, each optical fiber may be switched on or off using a high-speed shutter (not shown) positioned in the optical path between the fiber and beam splitter.

In example SIM imaging system <NUM>, the first arm includes a fixed vertical grating 230A to project a grating pattern in a first orientation (e.g., a vertical fringe pattern) onto the sample, and the second arm includes a fixed horizontal grating 230B to project a grating pattern in a second orientation (e.g., a horizontal fringe pattern) onto the sample <NUM>. Unlike in pre-existing SIM imaging systems, the gratings of SIM imaging system <NUM> do not need to be mechanically rotated or translated, which may provide improved system speed, reliability, and repeatability.

As illustrating in the example of <FIG>, gratings 230A-230B are transmissive diffraction gratings, including a plurality of diffracting elements (e.g., parallel slits or grooves) formed into a glass substrate or other suitable surface. The gratings may be implemented as phase gratings that provide a periodic variation of the refractive index of the grating material. The groove or feature spacing may be chosen to diffract light at suitable angles and tuned to the minimum resolvable feature size of the imaged samples for operation of SIM imaging system <NUM>. In other implementations, the gratings may be reflective diffraction gratings.

In the example of SIM imaging system <NUM>, the vertical and horizontal patterns are offset by about <NUM> degrees. In other implementations, other orientations of the gratings may be used to create an offset of about <NUM> degrees. For example, the gratings may be oriented such that they project images that are offset ±<NUM> degrees from the x or y plane of sample <NUM>. The configuration of example SIM imaging system <NUM> may be particularly advantageous in the case of a regularly patterned sample <NUM> with features on a rectangular grid, as structured resolution enhancement can be achieved using only two perpendicular gratings (e.g., vertical grating and horizontal grating).

Gratings 230A-230B, in the example of system <NUM>, are configured to diffract the input beams into a number of orders (e.g., <NUM> order, ± <NUM> orders, ± <NUM> orders, etc.) of which the ± <NUM> orders may be projected on the sample <NUM>. As shown in this example, vertical grating 230A diffracts a collimated light beam into first order diffracted beams (± <NUM> orders), spreading the first orders on the plane of the page, and horizontal grating 230B diffracts a collimated light beam into first order diffracted beams, spreading the orders above and below the plane of the page (i.e., in a plane perpendicular to the page). To improve efficiency of the system, the zeroth order beams and all other higher order beams (i.e., ± <NUM> orders or higher) may be blocked (i.e., filtered out of the illumination pattern projected on the sample <NUM>). For example, a beam blocking element (not shown) such as an order filter may be inserted into the optical path after each diffraction grating to block the <NUM>-order beam and the higher order beams. In some implementations, diffraction gratings 230A-230B may configured to diffract the beams into only the first orders and the <NUM>-order (undiffracted beam) may be blocked by some beam blocking element.

Each arm includes an optical phase modulator or phase shifter 240A-240B to phase shift the diffracted light output by each of gratings <NUM>. For example, during structured imaging, the optical phase of each diffracted beam may be shifted by some fraction (e.g., <NUM>/<NUM>, <NUM>/<NUM>, <NUM>/<NUM>, etc.) of the pitch (λ) of each fringe of the structured pattern. In the example of <FIG>, phase modulators 240A and 240B are implemented as rotating windows that may use a galvanometer or other rotational actuator to rotate and modulate the optical path-length of each diffracted beam. For example, window 240A may rotate about the vertical axis to shift the image projected by vertical grating 230A on sample <NUM> left or right, and window 240B may rotate about the horizontal axis to shift the image projected by horizontal grating 230B on sample <NUM> up or down.

In other implementations, further described below, other phase modulators that change the optical path length of the diffracted light (e.g. linear translation stages, wedges, etc.) may be used. Additionally, although optical phase modulators 240A-240B are illustrated as being placed after gratings 230A-230B, in other implementations they may be placed at other locations in the illumination system. In some implementations, a single phase modulator may be operated in two different directions for the different fringe patterns, or a single phase modulator may use a single motion to adjust both of the path lengths, as described below.

In example system <NUM>, a mirror <NUM> with holes <NUM> combines the two arms into the optical path in a lossless manner (e.g., without significant loss of optical power, other than a small absorption in the reflective coating). Mirror <NUM> can be located such that the diffracted orders from each of the gratings are spatially resolved, and the unwanted orders can be blocked. Mirror <NUM> passes the first orders of light output by the first arm through holes <NUM>. Mirror <NUM> reflects the first orders of light output by the second arm. As such, the structured illumination pattern may be switched from a vertical orientation (e.g., grating 230A) to a horizontal orientation (e.g., grating 230B) by turning each emitter on or off or by opening and closing an optical shutter that directs a light source's light through the fiber optic cable. In other implementations, the structured illumination pattern may be switched by using an optical switch to change the arm that illuminates the sample.

Also illustrated in example imaging system <NUM> are a projection lens <NUM>, a semi-reflective mirror <NUM>, objective <NUM>, and camera <NUM>. The projection lens <NUM> may be utilized in conjunction with lens 250A to project the Fourier transform of grating 230A into the entrance pupil of the objective lens <NUM>. Similarly, the projection lens <NUM> may be utilized in conjunction with lens 250B to project the Fourier transform of grating 230B into the entrance pupil of the objective lens <NUM>. The projection lens <NUM> may also be implemented to articulate along the z-axis to adjust the grating focus on the sample plane. Semi-reflective mirror <NUM> may be a dichroic mirror to reflect structured illumination light received from each arm down into objective <NUM> for projection onto sample <NUM>, and to pass through light emitted by sample <NUM> (e.g., fluorescent light, which is emitted at different wavelengths than the excitation) onto camera <NUM>.

It is worth noting that the example of system <NUM> may provide a high optical efficiency due to the absence of a polarizer. Additionally, the use of unpolarized light may not have a significant impact on pattern contrast depending on the numerical aperture setting of the objective <NUM>.

It should be noted that, for the sake of simplicity, optical components of SIM imaging system <NUM> may have been omitted from the foregoing discussion. Additionally, although system <NUM> is illustrated in this example as a single channel system, in other implementations, it may be implemented as a multi-channel system (e.g., by using two different cameras and light sources that emit in two different wavelengths).

<FIG> is an optical diagram illustrating another example optical configuration of a two-arm SIM imaging system <NUM> in accordance with the claimed invention. In system <NUM>, a large, rotating optical window <NUM> may be placed after mirror <NUM> with holes <NUM>. In this case, window <NUM> may be used in place of windows 240A and 240B to modulate the phases of both sets of diffracted beams output by the vertical and horizontal diffraction gratings. Instead of being parallel with respect to the optical axis of one of the gratings, the axis of rotation for the rotating window <NUM> may be offset <NUM> degrees (or some other angular offset) from the optical axis of each of the vertical and horizontal gratings to allow for phase shifting along both directions along one common axis of rotation of window <NUM>. In some implementations, the rotating window <NUM> may be replaced by a wedged optic rotating about the nominal beam axis.

<FIG> is an optical diagram illustrating another example optical configuration of a two-arm SIM imaging system <NUM> not in accordance with the claimed invention. In system <NUM>, gratings 230A and 230B are mounted on respective linear motion stages 410A and 410B that may be translated to change the optical path length (and thus the phase) of light emitted by gratings 230A and 230B. The axis of motion of linear motion stages 410A-410B may be perpendicular or otherwise offset from the orientation of their respective grating to realize translation of the grating's pattern along a sample <NUM>. In implementations, stages 410A and 410B may each utilize crossed roller bearings, a linear motor, a high-accuracy linear encoder, and/or other technologies to provide precise linear translations of the gratings to phase shift the projected images.

<FIG> is an operational flow diagram illustrating an example method <NUM> that may be performed by a multi-arm SIM imaging system during one imaging cycle to use structured light to create a high-resolution image in accordance with some implementations described herein. In implementations, method <NUM> may be performed to image an entire sample or a location of a larger sample. Method <NUM> will be described in conjunction with <FIG>, which illustrates simplified illumination fringe patterns that may be projected onto the plane of a sample <NUM> by a vertical grating and horizontal grating of a two-arm SIM imaging system during image capture. For example, SIM imaging system <NUM> may use vertical grating 230A and horizontal grating 230B to generate the horizontal and vertical illumination patterns shown in <FIG>, while phase modulators 230A and 230B may be set to three different positions to produce the three phase shifts shown.

At operation <NUM>, a first arm corresponding to a first grating orientation is turned on to begin generating illumination patterns using the first arm. For instance, in the implementation of imaging system <NUM>, a high-speed shutter positioned in the path between optical fiber 210A and a light source may be opened or otherwise actuated such that the light source is not blocked. Alternatively, one or more light sources may be turned on or off (e.g., pulsed), or an optical switch may be used to direct a light source through the optical path of the first arm (e.g., through one of the first or second emitter). In some instances, operation <NUM> may also include turning on the light source (e.g., in the case of the first imaging cycle).

Once the first arm is turned on, at operation <NUM> a first grating pattern may be projected on the sample and an image may be captured. For example, as illustrated by <FIG>, vertical grating 230A may project first-order illumination fringes on sample <NUM>. Any light emitted by the sample may be captured by camera <NUM> and a first phase image of the first pattern (e.g., vertical pattern) may be captured. For instance, fluorescent dyes situated at different features of the sample <NUM> may fluoresce and the resultant light may be collected by the objective lens <NUM> and directed to an image sensor of camera <NUM> to detect the florescence.

If additional phase shifted images need to be captured (decision <NUM>), at operation <NUM> the pattern projected by the grating may be phase shifted to capture the next phase image of the pattern. For example, in the implementation of system <NUM>, the phase of the pattern projected by vertical grating 230A may be phase shifted by rotating optical window 240A. Alternatively, other optical phase modulators such as translation stages or rotating optical wedges may be used to shift the phase. For instance, as illustrated in the example of <FIG>, the phase may be shifted by <NUM>/<NUM> of the pitch (λ) of the fringe pattern such that the pattern projected on the sample is offset by <NUM>/3λ from the prior image that was captured. In some implementations, the pattern projected by the grating may be phase shifted by moving the sample (e.g., using a motion stage) while the projected fringes remain stationary. In some implementations, the pattern projected by the grating may be phase shifted by moving both the sample and the projected fringes. Operations <NUM>-<NUM> may iterate until all phase images of a first pattern are captured (e.g., three phase-shifted images of the vertical pattern in the case of <FIG>.

Once all phase images of a pattern have been captured, at operation <NUM> the second arm corresponding to a second grating orientation of the SIM imaging system may be turned on. For instance, in the implementation of imaging system <NUM>, a high-speed shutter positioned in the path between optical fiber 210B and a light source may be opened or otherwise actuated such that the light source is not blocked. Alternatively, one or more light sources may be turned on or off (e.g., pulsed), or an optical switch may be used to direct a light source through the optical path of the second arm. Additionally, the other arm may be turned off. A series of phase images may then be captured for the next arm by repeating operations <NUM>-<NUM>. For instance, as illustrated by <FIG>, horizontal grating 230B may project first-order illumination fringes on sample <NUM>, and the projected fringes may be shifted in position by <NUM>/3λ to capture three phase images of the horizontal pattern. As another example, the pattern projected by the grating may be phase shifted by moving the sample (e.g., using a motion stage) while the projected fringes remain stationary, or by moving both the sample and the projected fringes.

Once all images have been captured for the imaging cycle, at operation <NUM>, a high resolution image may be constructed from the captured images. For example, a high resolution image may be reconstructed from the six images shown in <FIG>. Suitable algorithms may be used to combine these various images to synthesize a single image of the sample with significantly better spatial resolution than any of the individual component images.

It should be noted that although method <NUM> has been primarily described in the context of single channel imaging (e.g., imaging a sample using a light source having a single wavelength), in some implementations method <NUM> may be adapted for multi-channel imaging (e.g., imaging a sample using light sources having different wavelengths). In such implementations, method <NUM> may be repeated for each channel of the imaging system (e.g., sequentially, or in parallel) to generate high resolution images for each channel.

Although implementations of the two-arm SIM imaging system <NUM> described herein have so far been described in the context of system <NUM> that utilizes a mirror <NUM> with holes <NUM> to losslessly combine the optical paths of the two arms, in an alternative implementation, the two images of the horizontal and vertical gratings 230A-230B may be losslessly combined by using a polarizing beam splitter in place of the mirror with holes and to illuminate the vertical grating with vertically-polarized light and the horizontal grating with horizontally-polarized light. In such implementations, the structured illumination pattern can be switched from horizontal to vertical by turning the corresponding polarized illumination sources on and off.

By way of example, <FIG> illustrates an example experimental design of a two-arm SIM imaging system <NUM> that uses a polarizing beam splitter not in accordance with the claimed invention to combine the optical paths of the arms, and that illuminates a vertical grating with vertically-polarized light and a horizontal grating with horizontally-polarized light. In the implementation of <FIG>, the horizontal and vertical gratings are G1 and G2, the horizontal and vertical rotating windows are W1 and W2, and the polarizing beam splitter for combining the horizontal and vertical grating images is PBS2. The output of a fiber-coupled mode-scrambled multi-mode laser is Fiber1.

<FIG> illustrates an afocal mirror image and fluorescent slide captured using example SIM imaging system <NUM>, using a 20x/<NUM> NA microscope. The afocal mirror image has fringe visibility of <NUM>%. The fluorescent slide image has fringe visibility of <NUM>%.

<FIG> illustrates fringe modulation measurements acquired using system <NUM> with a beaded flowcell. The graph illustrates typical feature image intensity changes during a phase adjustment cycle, as the angle of parallel plate W2 of <FIG> is changed.

<FIG> illustrates another example optical configuration of a two-arm SIM imaging system <NUM> in accordance with some implementations described herein. The first arm of system <NUM> includes a light emitter 910A (e.g., optical fiber), an optical collimator 920A to collimate light output by light emitter 910A, a diffraction grating 930A in a first orientation with respect to the optical axis, and a relay lens 940A. The second arm of system <NUM> includes a light emitter 910B, an optical collimator 920B to collimate light output by light emitter 910B, a diffraction grating 930B in a second orientation with respect to the optical axis, and a relay lens 940B.

System <NUM> also includes a beam combining element <NUM> for combining the two arms of the optical system. As illustrated, beam combining element <NUM> includes a <NUM>° prism with holes to pass through structured light from the second arm of the system and a mirrored surface for reflecting structured light received from the first arm. Before entering beam combining element <NUM>, each structured beam of light passes through a spatial filter having a pair of apertures to pass the ±<NUM> orders and block other orders. Structured light emanating from the first arm in a first plane may be directed by reflective optic <NUM> into beam combing element <NUM>. In system <NUM>, parallel plate optical element <NUM> serves as a phase adjuster and may be rotated to shift structured light in either orientation after beam combining element <NUM>.

Implementations described herein have so far been described in the context of a two-arm structured illumination imaging system that includes two gratings oriented at two different angles, wherein in the case of a regularly patterned sample with features on a rectangular grid, resolution enhancement can be achieved with only two perpendicular angles (e.g., vertical grating and horizontal grating) as described above. On the other hand, for image resolution enhancement in all directions for other samples (e.g., hexagonally patterned samples), three grating angles may be used. For example, a three-arm system may include three light emitters and three fixed diffraction gratings (one per arm), where each diffraction grating is oriented around the optical axis of the system to project a respective pattern orientation on the sample (e.g., a <NUM>° pattern, a <NUM>° pattern, or a <NUM>° pattern). In such systems, additional mirrors with holes may be used to combine the additional images of the additional gratings into the system in a lossless manner. Alternatively, such systems may utilize one or more polarizing beam splitters to combine the images of each of the gratings.

In accordance with some implementations of the technology disclosed herein, the SIM imaging system may be implemented as a multiple optical grating slide SIM imaging system, where one linear motion stage is mounted with a plurality of diffraction gratings (or other beam splitting optical elements) having a corresponding, fixed orientation with respect to the optical axis of the system.

<FIG> are schematic diagrams illustrating an example optical configuration of a dual optical grating slide SIM imaging system <NUM> not in accordance with the claimed invention. As further described below, in the configuration of system <NUM>, all changes to the grating pattern projected on sample <NUM> (e.g., pattern phase shifts or rotations) may be made by linearly translating a motion stage <NUM> along a single axis of motion, to select a grating <NUM> or <NUM> (i.e., select grating orientation) or to phase shift one of gratings <NUM>-<NUM>.

System <NUM> includes a light emitter <NUM> (e.g., optical fiber optically coupled to a light source), a first optical collimator <NUM> (e.g., collimation lens) to collimate light output by light emitter <NUM>, a linear motion stage <NUM> mounted with a first diffraction grating <NUM> (e.g., horizontal grating) and a second diffraction grating <NUM> (e.g. vertical grating), a projection lens <NUM>, a semi-reflective mirror <NUM> (e.g., dichroic mirror), an objective <NUM>, a sample <NUM>, and a camera <NUM>. For simplicity, optical components of SIM imaging system <NUM> may be omitted from <FIG>. Additionally, although system <NUM> is illustrated in this example as a single channel system, in other implementations, it may be implemented as a multi-channel system (e.g., by using two different cameras and light sources that emit in two different wavelengths).

As illustrated by <FIG>, a grating <NUM> (e.g., a horizontal diffraction grating) may diffract a collimated light beam into first order diffracted light beams (on the plane of the page). As illustrated by <FIG>, a diffraction grating <NUM> (e.g., a vertical diffraction grating) may diffract a beam into first orders (above and below the plane of the page). In this configuration only a single optical arm having a single emitter <NUM> (e.g., optical fiber) and single linear motion stage is needed to image a sample <NUM>, which may provide system advantages such as reducing the number of moving system parts to improve speed, complexity and cost. Additionally, in system <NUM>, the absence of a polarizer may provide the previously mentioned advantage of high optical efficiency. The configuration of example SIM imaging system <NUM> may be particularly advantageous in the case of a regularly patterned sample <NUM> with features on a rectangular grid, as structured resolution enhancement can be achieved using only two perpendicular gratings (e.g., vertical grating and horizontal grating).

To improve efficiency of the system, the zeroth order beams and all other higher order diffraction beams (i.e., ± <NUM> orders or higher) output by each grating may be blocked (i.e., filtered out of the illumination pattern projected on the sample <NUM>). For example, a beam blocking element (not shown) such as an order filter may be inserted into the optical path after motion stage <NUM>. In some implementations, diffraction gratings <NUM>-<NUM> may configured to diffract the beams into only the first orders and the <NUM>-order (undiffracted beam) may be blocked by some beam blocking element.

In the example of system <NUM>, the two gratings may be arranged about ±<NUM>° from the axis of motion (or other some other angular offset from the axis of motion such as about +<NUM>°/-<NUM>°, about +<NUM>°/-<NUM>°, etc.) such that a phase shift may be realized for each grating <NUM>-<NUM> along a single axis of linear motion. In some implementations, the two gratings may be combined into one physical optical element. For example, one side of the physical optical element may have a grating pattern in a first orientation, and an adjacent side of the physical optical element may have a grating pattern in a second orientation orthogonal to the first orientation.

Single axis linear motion stage <NUM> may include one or more actuators to allow it to move along the X-axis relative to the sample plane, or along the Y-axis relative to the sample plane. During operation, linear motion stage <NUM> may provide sufficient travel (e.g., about <NUM>-<NUM>) and accuracy (e.g., about less than <NUM> micrometer repeatability) to cause accurate illumination patterns to be projected for efficient image reconstruction. In implementations where motion stage <NUM> is utilized in an automated imaging system such as a fluorescence microscope, it may be configured to provide a high speed of operation, minimal vibration generation and a long working lifetime. In implementations, linear motion stage <NUM> may include crossed roller bearings, a linear motor, a high-accuracy linear encoder, and/or other components. For example, motion stage <NUM> may be implemented as a high-precision stepper or piezo motion stage that may be translated using a controller.

<FIG> is an operational flow diagram illustrating an example method <NUM> that may be performed by a multiple optical grating slide SIM imaging system during one imaging cycle to use structured light to create a high resolution image in accordance with some implementations described herein. Depending on the implementation, method <NUM> may be performed to image an entire sample or a location of a larger sample. Method <NUM> will be described in conjunction with <FIG>, which illustrates simplified illumination fringe patterns that may be projected onto the plane of a sample <NUM> by a first diffraction grating and a second diffraction grating of a dual optical grating slide SIM imaging system during image capture. For example, a SIM imaging system <NUM> may use a first diffraction grating <NUM> and second diffraction grating <NUM> to generate the illumination patterns shown in <FIG>. As illustrated in the example of <FIG>, the two gratings project perpendicular fringe patterns on the surface of sample <NUM> and are arranged about ±<NUM>° from the axis of motion of linear motion stage <NUM>.

At operation <NUM>, the light source is turned on. For example, an optical shutter may be actuated to optically couple the optical fiber of light emitter <NUM> to a light source. As another example, a light source may be pulsed or an optical switch may be used to direct a light source through the optical path of the light emitter. At operation <NUM>, a first grating pattern may be projected on the sample and an image may be captured. For example, as illustrated by <FIG>, a first grating (e.g., grating <NUM>), may project first-order illumination fringes on sample <NUM>. Any light emitted by the sample may be captured by camera <NUM> and a first phase image of the first pattern (e.g., +<NUM>° pattern) may be captured. For instance, fluorescent dyes situated at different features of the sample <NUM> may fluoresce and the resultant light may be collected by the objective lens <NUM> and directed to an image sensor of camera <NUM> to detect the florescence.

To capture additional phase shifted images, at operation <NUM> the pattern projected by the grating may be phase shifted by translating the linear motion stage. In the example of <FIG>, these phase shift motions are illustrated as steps <NUM> and <NUM>. The phase shift motions may provide small (e.g., about <NUM> to <NUM> micrometers or smaller) moves of the gratings to slightly shift the fringe pattern projected on the grating.

By way of particular example, consider the case where the pitch λ of the fringe at the sample of <FIG> is <NUM>. In this case, three phase shifted images are captured in the sample, requiring phase shifts of the projected fringes of λ/<NUM>, or <NUM>. Assuming an objective illumination magnification of 10X, the phase shift steps (linear translations) required of the single axis linear motion stage may be calculated as <NUM> * <NUM> * sqrt(<NUM>), or about <NUM>. In this case, the sqrt(<NUM>) factor accounts for the <NUM> degree offset between the orientation of the grating and the axis of motion of the motion stage. More generally, the translation distance of the linear motion stage during each phase shift step in this example configuration may be described by <MAT> where MAG is the illumination magnification.

Following capture of all phase shifted images for a diffraction grating (decision <NUM>), at operation <NUM> the system may switch diffraction gratings by translating the linear motion stage to optically couple another diffraction grating to the light source of the imaging system (e.g., transition from <FIG>). This motion is illustrated as step <NUM> in the example of <FIG>. In the case of diffraction grating changes, the linear motion stage may provide a relatively large translation (e.g., on the order of <NUM>-<NUM>).

A series of phase images may then be captured for the next grating by repeating operations <NUM>-<NUM>. For instance, as illustrated by <FIG>, a second diffraction grating may project first-order illumination fringes on sample <NUM>, and the projected fringes may be shifted in position by λ/<NUM> by translating the linear motion stage to capture three phase images of the grating's pattern (e.g., steps <NUM> and <NUM> of <FIG>).

Once all images have been captured for the imaging cycle, at operation <NUM>, a high resolution image may be constructed from the captured images. For example, a high resolution image may be reconstructed from the six images shown schematically in <FIG>. As the foregoing example illustrates, a multiple optical grating slide SIM imaging system advantageously may switch between fringe angles and phases with a single linear actuator, thereby saving on cost and complexity of the structured illumination imaging system.

<FIG> is a diagram illustrating an example dual optical grating slide SIM imaging configuration <NUM>. As illustrated, the configuration <NUM> may include an optical fiber <NUM> to emit light, a collimator <NUM>, a linear motion stage <NUM> mounted with first and second diffraction gratings <NUM>-<NUM>, a projection lens <NUM>, and a turning mirror <NUM>. In this example, gratings <NUM>-<NUM> are embedded in the same object, adjacent to each other along the axis of motion of stage <NUM>. Other components not shown may be similar to those in <FIG>, such as dichroic mirror <NUM>, objective <NUM> and sample <NUM>.

In some implementations, the linear motion stage or slide of the dual optical grating slide SIM imaging system may be mounted with one or more additional lower frequency patterns to aid with alignment of the fringe pattern that is projected on the sample by the imaging gratings (e.g., the two gratings arranged at about ±<NUM>° from the axis of motion of linear motion stage). For example, linear motion stage <NUM> of <FIG> may be mounted with the additional alignment pattern, or linear motion stage <NUM> of <FIG> may be mounted with the additional alignment pattern. In instances where the two imaging gratings are embedded in the same substrate as depicted in <FIG>, the alignment grating may also be embedded in that substrate, or it may be incorporated in a separate substrate. The alignment pattern may be placed between the two imaging gratings or in some other suitable position on the motion stage.

The alignment pattern, when illuminated, may be configured to project a pattern having a lower frequency and/or greater pitch on a sample. By virtue of these characteristics, coarse alignment of the gratings with respect to the sample may be facilitated. The alignment pattern may be implemented as parallel lines, orthogonal lines, and/or a grating having a lower frequency of slits than the other gratings. In some implementations, multiple alignment patterns may be used. <FIG> shows one example of an alignment pattern that may be used in implementations of the disclosure. As illustrated in this example, an alignment pattern mark <NUM> is implemented on the same substrate as a grating <NUM>, outside of clear aperture <NUM>. In this example, the alignment pattern is implemented as two sets of orthogonal lines. By virtue of this implementation, grating tilt may be checked. In some implementations, the illustrated alignment pattern may be implemented in multiple areas (e.g., four corners of a substrate).

During use, the alignment pattern may be illuminated to project a pattern. The alignment pattern may be utilized during SIM imaging system manufacture, after field installation, or during a field service engineer check. In some implementations, the alignment pattern may be utilized during operation of the dual optical grating slide SIM imaging system. For example, the alignment pattern may be illuminated to project an alignment pattern before imaging of a sample begins.

In some implementations of the dual optical grating slide SIM imaging system, an optical phase modulator (e.g., a rotating window) that is a separate component than the linear motion stage may be utilized for phase tuning. In such implementations, the optical phase modulator may be used for phase tuning instead of the linear motion stage (e.g., the linear motion stage may only be used for switching between the two gratings). By virtue of such implementations, the speed, accuracy, and/or reliability of the system may potentially be improved by substantially decreasing the number of translations required over time by the motion stage and by obviating the need to use a motion stage to make fine translations (e.g., on the order of µm) to select a phase.

The optical phase modulator may be placed in the light path between the light source and sample, after the gratings (e.g., directly after the motion stage). <FIG> illustrates some components of one example dual optical grating slide SIM imaging system <NUM> in accordance with such implementations. As shown, system <NUM> includes a light emitter <NUM> (e.g., optical fiber optically coupled to a light source), a first optical collimator <NUM> (e.g., collimation lens) to collimate light output by light emitter <NUM>, a linear motion stage <NUM> mounted with a first diffraction grating <NUM> (e.g., horizontal grating) and a second diffraction grating <NUM> (e.g. vertical grating), and an optical phase modulator <NUM> to phase shift the diffracted light output by each grating.

In accordance with some implementations of the technology disclosed herein, the SIM imaging system may be implemented as a pattern angle spatial selection SIM imaging system, whereby a fixed two dimensional diffraction grating is used in combination with a spatial filter wheel to project one-dimensional diffraction patterns on the sample.

<FIG> is a schematic diagram illustrating an example optical configuration of a pattern angle spatial selection SIM imaging system <NUM> in accordance with some implementations described herein. For simplicity, optical components of SIM imaging system <NUM> may be omitted from <FIG>. Additionally, although system <NUM> is illustrated in this example as a single channel system, in other implementations, it may be implemented as a multi-channel system (e.g., by using two different cameras and light sources that emit in two different wavelengths).

As illustrated, system <NUM> includes a light emitter <NUM> (e.g., optical fiber), a collimator <NUM> to collimate light emitted by emitter <NUM>, a two-dimensional grating <NUM>, a zero order beam blocker <NUM>, an optical phase modulator <NUM>, a projection lens <NUM>, a spatial filter wheel <NUM>, a dichroic mirror <NUM>, an objective <NUM>, a sample <NUM>, and a camera <NUM>.

In this example configuration, grating <NUM> is a two-dimensional transmission diffraction grating configured to diffract an input beam into a number of orders (e.g., <NUM> order, ± <NUM> orders, ± <NUM> orders, etc.) in two perpendicular directions. To improve the efficiency and performance of the system, the zeroth order beams and all other higher order beams (i.e., ± <NUM> orders or higher) may be blocked (i.e., filtered out of the illumination pattern projected on the sample <NUM>). While higher orders may be diffracted out at wide angles where they may be filtered using a variety of filtering elements, the <NUM>-order component pass through straight through the grating in the beam path toward the sample. To block the <NUM>-order component, a beam blocking element <NUM> may be inserted into the optical path after two-dimensional diffraction grating <NUM>. For example, beam blocking element <NUM> may be a Volume Bragg Grating (VBG), a diffractive optical element that can be patterned to reflect light normal to the element (e.g., <NUM>-order light) and pass through light at other angles, such as the +<NUM> & -<NUM> orders. With the <NUM>-order removed, smaller and more compact optics can be used to focus the +<NUM> & -<NUM> orders down to the objective lens.

Optical phase modulator <NUM> (e.g., a rotating window) may be used to change the phase of the incident light to adjust the pattern phase position on the sample <NUM>. For example, optical phase modulator <NUM> may include a variety of moving optical elements, including a parallel plate optic tilted at a variable angle to the optical axis, a wedged optic rotated about the optical axis, a mirror tilted to translate the beam, electro-optical elements, or acousto-optical elements. In one particular implementation, optical phase modulator <NUM> may be implemented as a parallel plate optic tilted in two perpendicular directions to adjust the phase of two different grating angle patterns. Alternatively, in some implementations, the pattern phase position may be adjusted by moving the sample (e.g., using a motion stage) while the projected pattern remains stationary, or by moving both the sample and the projected pattern.

In the example of system <NUM>, a rotatable spatial filter wheel <NUM> may include a plurality of holes oriented in two perpendicular directions (e.g., a vertical set of holes <NUM> and a horizontal set of holes <NUM>) for selecting a vertical grating image or a horizontal grating image for projection on the sample <NUM>. For example, by rotating the spatial filter wheel, the +/- <NUM> orders of one of the grating patterns may pass through one of the set of holes to generate a horizontal or vertical fringed pattern on sample <NUM>. In implementations, spatial filter wheel <NUM> may be implemented as a lightweight mask or spatial filter (e.g., a rotating disk including a plurality of ports or apertures).

In the configuration of system <NUM>, the primary optical components of system <NUM> may remain stationary, which may improve the stability of the optical system (and of the illumination pattern) and minimize the weight, vibration output and cost of the moving elements. As some of the beam intensity (e.g., up to <NUM>%) may need to be filtered out in either orientation of spatial filter wheel <NUM>, in some implementations the spatial filter may be configured to reflect the unneeded beams (e.g., orders of diffraction grating pattern that is not passed through) into a beam dump for proper heat management.

<FIG> is a schematic diagram illustrating another example optical configuration of a pattern angle spatial selection SIM imaging system <NUM> in accordance with some implementations described herein. In example imaging system <NUM>, the functions of the two-dimensional transmission grating and beam blocking element may be integrated into a solid optic <NUM>. Additionally, the function of a projection lens may be integrated into solid optic <NUM>. In this example implementation, a two-dimensional transmission grating <NUM> is fabricated on or otherwise disposed over a face of optic <NUM> that receives collimated light from emitter <NUM> (the input of optic <NUM>). The dispersion angles of the grating <NUM> may be arranged such that the <NUM>-order light can be blocked on the far side of the optic. The desired +<NUM> & -<NUM> orders, in both directions, may exit from optic <NUM> through angled faces <NUM> (the output of optic <NUM>) that diffract the +<NUM> & -<NUM> orders in an optically desirable direction. These output faces may include diffractive focusing lenses. Alternatively, a separate optic may be used as a projection lens to focus the beams onto the objective <NUM>. In system <NUM>, a phase shifter <NUM> and rotating spatial filter mask <NUM> may be used as described above.

<FIG> is a schematic diagram illustrating another example optical configuration of a pattern angle spatial selection SIM imaging system <NUM> in accordance with some implementations described herein. In example imaging system <NUM>, a solid optic <NUM> again may be used to integrate the functions of a two-dimensional grating and a beam blocking element. Additionally it may integrate the function of a projection lens. In contrast to example imaging system <NUM>, the input of solid optic <NUM> is an inlet window or aperture <NUM> that guides received light to a two-dimensional reflective grating <NUM>. As grating <NUM> is reflective in this example, the <NUM>-order light may be reflected back out through inlet window <NUM>. The desired +<NUM> & -<NUM> orders of diffracted light, in each of the perpendicular directions, may reflect off of respective reflectively-coated internal faces <NUM> of the optic <NUM>, and exit through outlet faces <NUM>. In implementations, these outlet faces may include diffractive focusing lenses. Alternatively, a separate projection lens optic <NUM> may be used to focus the beams onto the objective <NUM>. In system <NUM>, a phase shifter <NUM> and rotating spatial filter mask <NUM> may be used as described above.

Although some implementations of the disclosure have been illustrated in the figures in the context of SIM imaging systems that use one or more optics to reimage collected excitation light (e.g., light recollected by the objective) onto an image sensor (e.g., a CCD camera sensor), it should be appreciated that the various implementations described herein may apply to SIM imaging systems that utilize an image sensor (e.g., a CMOS sensor) that is in an active plane of an imaged sample. By way of illustrative example, <FIG>, illustrates a sample <NUM> that may be formed over an image sensor assembly <NUM> of a SIM imaging system, in accordance with some implementations described herein. For example, features of the sample may be photolithographically aligned with pixels of the image sensor. Any light emitted by patterned sample <NUM> in response to structured illumination is collected by image sensor assembly <NUM>, which is positioned directly below sample <NUM> in this example. Forming sample <NUM> over image sensor assembly <NUM> may provide the advantage of ensuring that patterned features <NUM> of the sample <NUM> remain aligned relative to particular photosites (e.g., pixels) of image sensor assembly <NUM> during imaging.

Sample <NUM> may be patterned and aligned with image sensor assembly <NUM> such that each light sensor (e.g., pixel) of image sensor <NUM> has one or more features <NUM> formed and/or mounted above it. As illustrated in the example of <FIG>, sample <NUM> is patterned over image sensor assembly <NUM> such that one feature <NUM> is formed over each pixel of the pixel array of image sensor assembly <NUM>. In other implementations, more than one feature may be formed over each pixel.

In the case of a fluorescent sample, for instance, illuminated features <NUM> of the sample may fluoresce in response to the structured excitation light <NUM>, and the resultant light <NUM> emitted by features <NUM> may be collected by photosites (e.g., pixels) of image sensor assembly <NUM> to detect fluorescence. For example, as illustrated by <FIG>, pixels (<NUM>,<NUM>) and (<NUM>,<NUM>) of image sensor assembly <NUM> may collect light <NUM> that is emitted by the feature <NUM> of the sample that is positioned or patterned over it. In some implementations, a layer (not shown) may provide isolation between sample <NUM> and image sensor assembly <NUM> (e.g., to shield the image sensor assembly from a fluidic environment of the sample). In other implementations, sample <NUM> may be mounted and aligned over image sensor assembly <NUM>.

It should be noted that although <FIG> illustrates an example representation of a SIM imaging system where the SIM fringes line up with the features of the sample in the correct orientation, in practice this is not necessarily or typically the case for SIM imaging. For example, over time and/or space, there may be drift in the spacing between adjacent fringes, the phase or angle of the structured illumination pattern, and/or the orientation of the fringe pattern relative to the illuminated sample. Owing to these variations in SIM parameters, in some instances some illuminated features may be <NUM>% "on" while other features may be <NUM>% "on" and yet other features may be <NUM>% "on. " As such, it should be appreciated that in such systems, SIM imaging algorithms may be utilized to take into account these process variations during image reconstruction. For example, variations in structured illumination parameters may be estimated and/or predicted over time to account for these variations.

As used herein, the term module might describe a given unit of functionality that can be performed in accordance with one or more implementations of the present application. As used herein, a module might be implemented utilizing any form of hardware, software, or a combination thereof. For example, one or more processors, controllers, ASICs, PLAs, PALs, CPLDs, FPGAs, logical components, software routines or other mechanisms might be implemented to make up a module. In implementation, the various modules described herein might be implemented as discrete modules or the functions and features described can be shared in part or in total among one or more modules. In other words, as would be apparent to one of ordinary skill in the art after reading this description, the various features and functionality described herein may be implemented in any given application and can be implemented in one or more separate or shared modules in various combinations and permutations. Even though various features or elements of functionality may be individually described or claimed as separate modules, one of ordinary skill in the art will understand that these features and functionality can be shared among one or more common software and hardware elements, and such description shall not require or imply that separate hardware or software components are used to implement such features or functionality.

In this document, the terms "computer readable medium", "computer usable medium" and "computer program medium" are used to generally refer to non-transitory media, volatile or non-volatile, such as, for example, a memory, storage unit, and media. These and other various forms of computer program media or computer usable media may be involved in carrying one or more sequences of one or more instructions to a processing device for execution. Such instructions embodied on the medium, are generally referred to as "computer program code" or a "computer program product" (which may be grouped in the form of computer programs or other groupings).

Although described above in terms of various example implementations and implementations, it should be understood that the various features, aspects and functionality described in one or more of the individual implementations are not limited in their applicability to the particular implementation with which they are described, but instead can be applied, alone or in various combinations, to one or more of the other implementations of the application, whether or not such implementations are described and whether or not such features are presented as being a part of a described implementation. Thus, the breadth and scope of the present application should not be limited by any of the above-described example implementations.

The terms "substantially" and "about" used throughout this disclosure, including the claims, are used to describe and account for small fluctuations, such as due to variations in processing. For example, they can refer to less than or equal to ±<NUM>%, such as less than or equal to ±<NUM>%, such as less than or equal to ±<NUM>%, such as less than or equal to ±<NUM>%, such as less than or equal to ±<NUM>%, such as less than or equal to ±<NUM>%, such as less than or equal to ±<NUM>%.

To the extent applicable, the terms "first," "second," "third," etc. herein are merely employed to show the respective objects described by these terms as separate entities and are not meant to connote a sense of chronological order, unless stated explicitly otherwise herein.

Terms and phrases used in this document, and variations thereof, unless otherwise expressly stated, should be construed as open ended as opposed to limiting. As examples of the foregoing: the term "including" should be read as meaning "including, without limitation" or the like; the term "example" is used to provide some instances of the item in discussion, not an exhaustive or limiting list thereof; the terms "a" or "an" should be read as meaning "at least one," "one or more" or the like; and adjectives such as "conventional," "traditional," "normal," "standard," "known" and terms of similar meaning should not be construed as limiting the item described to a given time period or to an item available as of a given time, but instead should be read to encompass conventional, traditional, normal, or standard technologies that may be available or known now or at any time in the future.

The presence of broadening words and phrases such as "one or more," "at least," "but not limited to" or other like phrases in some instances shall not be read to mean that the narrower case is intended or required in instances where such broadening phrases may be absent. The use of the term "module" does not imply that the components or functionality described or claimed as part of the module are all configured in a common package. Indeed, any or all of the various components of a module, whether control logic or other components, can be combined in a single package or separately maintained and can further be distributed in multiple groupings or packages or across multiple locations.

Additionally, the various implementations set forth herein are described in terms of example block diagrams, flow charts and other illustrations. As will become apparent to one of ordinary skill in the art after reading this document, the illustrated implementations and their various alternatives can be implemented without confinement to the illustrated examples. For example, block diagrams and their accompanying description should not be construed as mandating a particular architecture or configuration.

Claim 1:
A structured illumination imaging system (<NUM>), comprising:
a first optical arm, comprising:
a first light emitter (210A) configured to emit light;
a first transmissive diffraction grating (230A) configured to split light emitted by the
first light emitter (210A) configured to project a first plurality of fringes on a plane of a sample;
one or more optical elements (240A, 240B, <NUM>, 410A, 410B) configured to phase shift the first plurality of fringes and the second plurality of fringes;
a second optical arm, comprising:
a second light emitter (210B) configured to emit light; and
a second transmissive diffraction grating (230B) configured to split light emitted by the second light emitter (210B) configured to project a second plurality of fringes on the plane of the sample, wherein the first plurality of fringes are angularly offset from the second plurality of fringes on the sample plane by about <NUM> degrees; and
an optical element configured to combine the optical paths of the first optical arm and the second optical arm,
wherein the optical element to combine an optical path of the first plurality of fringes and the second plurality of fringes comprises a mirror (<NUM>) with holes (<NUM>), with the mirror (<NUM>) arranged to reflect light diffracted by the first transmissive diffraction grating (230A) and with the holes (<NUM>) arranged to pass through at least first orders of light diffracted by the second transmissive diffraction grating (230B);
wherein the structured illumination system (<NUM>) is a two-arm structured illumination system and the first and second transmissive diffraction gratings (230A, 230B) are mechanically fixed oriented perpendicular to each other.