3D structured illumination microscope using a spinning disk

Abstract

A spinnable disk spatial filter for a confocal microscope comprises a plurality of semi-annular sections, each semi-annular section shaped to define a corresponding section pinhole group comprising a plurality of pinholes arranged in a lattice pattern, each pinhole permitting transmission of light through the disk. For each semi-annular section, the corresponding section pinhole group is located to have a corresponding section offset along a section offset direction orthogonal to the spinning axis and having at least a component that extends radially from the spinning axis. The section offset is different for at least two of the plurality of semi-annular sections.

Description

3D STRUCTURED ILLUMINATION MICROSCOPE USING A SPINNING DISKCross-Reference to Related Applications

[0001] This application claims priority from US application No. 63 / 533388 filed 18 August 2023 and entitled 3D STRUCTURED ILLUMINATION MICROSCOPE USING A SPINNING DISK which is hereby incorporated herein by reference for all purposes. For purposes of the United States of America, this application claims the benefit under 35 U.S.C. §119 of US application No. 63 / 533388 filed 18 August 2023 and entitled 3D STRUCTURED ILLUMINATION MICROSCOPE USING A SPINNING DISK.Field

[0002] This invention relates to apparatus and methods for structured illumination microscopy (SIM). Aspects of the invention provide spatial filters for use in SIM, and in particular embodiments, spinnable-disk spatial filters for use in SIM, methods of performing SIM using such spinnable-disk spatial filters and methods for facilitating transmission of light through the spatial filter.Background

[0003] Light microscopy has a wide range of applications. For example, fluorescence microscopy is a popular technique for visualizing cellular structures because of its high specificity and minimal invasiveness. However, the spatial resolution of conventional light microscopy is limited to about 250 nm laterally (in directions orthogonal to the axis of the light) and about 750 nm axially due to the diffraction limit of light.

[0004] Among other drawbacks, the diffraction limit hinders direct observation of subcellular structures, impeding the study of biological phenomena. In recent decades, various techniques have been proposed to improve the resolution of microscope systems beyond the diffraction limit. These techniques are generally referred to as “super-resolution microscopic techniques”.

[0005] Among the super-resolution microscopic techniques, structured illumination microscopy (SIM) has emerged as one of the most promising methods for high-speed super-resolution imaging, because SIM offers a relatively higher imaging speed and relatively lower phototoxicity in comparison to other super-resolution microscopic techniques.

[0006] Initially, SIM techniques were developed to improve resolution in two spatial dimensions (e.g. in x-and y-spatial directions orthogonal to the z-direction optical axis). This is referred to as “2D SIM”. Soon after, SIM techniques were improved to be able to improve resolution in three spatial dimensions (e.g. x-, y- and z-spatial directions, where the z-direction is parallel to the optical axis). This is referred to as “3D SIM”.

[0007] Despite conventional 3D SIM’s superior resolution in both lateral and axial directions compared to conventional light microscopy, conventional 3D SIM still has several limitations, particularly and without limitation, in relation to biological research. One limitation is imaging speed. For example, 2D SIM requires only 9 diffractionlimited images to digitally reconstruct one super-resolved image. On the other hand, conventional 3D SIM requires at least 15 images to digitally reconstruct one superresolved image. The requirement of additional images limits the imaging speed of a conventional 3D SIM and also makes conventional 3D SIMs more sensitive to sample motion, which can be an issue when observing biological samples, for example.

[0008] Another limitation of conventional 3D SIMs is the significant amount of out-of- focus signals in sample signals. Conventional 3D SIMs use wild-field illumination patterns created by the interference of 3 beams, which generate significant out-of- focus signals and make it difficult to extract high-frequency components desirable for improving image resolution. The out-of-focus signals are particularly problematic for imaging thick samples where the out-of-focus signals can overwhelm the in-focus signals, making it difficult to extract fine details from the final reconstructed images.

[0009] A further limitation of conventional 3D SIMs is that 3D SIM images are prone to reconstruction artifacts because 3D SIM images have less tolerance for distortion of illumination patterns compared to 2D SIM images. However, distortion of illumination patterns is often inevitable in practice because of mismatch between aberration and refractive index.

[0010] There is a desire for 3D SIM systems and methods that improve upon prior art 3D SIM techniques and / or ameliorate any of the drawbacks with prior art 3D SIM techniques.Summary

[0011] One aspect of the invention provides a spatial filter for a confocal microscope. The spatial filter comprises: a disk spinnable about a spinning axis in an optical path of the confocal microscope between a coherent light source providing an excitationlight and a target. The spinnable disk comprises: a plurality of semi-annular sections, each semi-annular section shaped to define a corresponding section pinhole group comprising a plurality of pinholes arranged in a lattice pattern, each pinhole permitting transmission of light through the disk. For each semi-annular section, the corresponding section pinhole group is located to have a corresponding section offset along a section offset direction orthogonal to the spinning axis and having at least a component that extends radially from the spinning axis. The section offset is different for at least two of the plurality of semi-annular sections.

[0012]

[0013] The section offset may be different for each of the plurality of semiannular sections.

[0014] The section offsets for the at least two of the plurality of semi-annular sections may be randomly selected.

[0015] The section offsets for the at least two of the plurality of semi-annular sections may be determined according to an optimization process which minimizes a cost function. The cost functional may have at least one term that attributes cost that is positively correlated with variation in scanning intensity.

[0016] The section offset direction for each semi-annular section may be radial.

[0017] The section offset direction may be a radial direction lying within the corresponding semi-annular section.

[0018] The section offset direction may be angularly spaced from a reference edge for each semi-annular section. The angular section offset direction relative to the corresponding reference edge may be the same for at least two semi-annular sections.

[0019] The angular section offset direction may extend along a radial direction that bisects the corresponding semi-annular section.

[0020] For each section pinhole group, the lattice pattern may comprise a 2D array of pinholes. The 2D array may extending in a pair of directions orthogonal to the spinning axis.

[0021] The pair of directions orthogonal to the spinning axis may be non-radial.

[0022] The 2D array of pinholes may be defined by polygonal lattice units, with pinholes located at vertices of the polygonal lattice units. The polygonal lattice units may be one of: triangular, quadrilateral, hexagonal.

[0023] The polygonal lattice units may have polygonal edge lengths that are the same for each polygonal lattice units.

[0024] The 2D array of pinholes may be defined by repeating lattice units, where neighboring lattice units share at least one edge.

[0025] For each section pinhole group, the lattice pattern may comprise a linear lattice of a plurality of linearly spaced apart pinholes. The linear lattice of a plurality of linearly spaced apart pinholes may comprise a radial lattice of pinholes that are radially spaced apart in a radial direction extending from the spinning axis.

[0026] The 2D array of pinholes may be defined by quadrilateral lattice units, with pinholes located at vertices of the quadrilateral lattice units.

[0027] The pair of directions orthogonal to the spinning axis may be orthogonal to one another.

[0028] The pinholes in the 2D array may be separated by a spatial period parameter ch in a first direction of the pair of directions and separated by a spatial period parameter d2 in a second direction of the pair of directions wherein di=d2.

[0029] The disk may comprise opaque material at locations away from the pinholes and may thereby block at least some out-of-focus signals in sampled light generated from an interaction between the excitation light and the target. An amount of the out- of-focus signals in the sampled light blocked by the disk may be greater than 50%.

[0030] Each pinhole in the plurality of pinholes in the corresponding section pinhole group of each of the plurality of semi-annular sections may have a common opening dimension w in a plane orthogonal to the spinning axis.

[0031] The spatial parameters di, d2 and the opening dimension w provide an opening ratio wd, wherein d=di=d2 and the opening ratio is in a range of 0.2-0.5, preferably in a range of 0.3-0.4.

[0032] The semi-annular sections and their corresponding section pinhole groups may be shaped to diffract the excitation light transmitted therethrough.

[0033] The semi-annular sections and their corresponding section pinhole groups may be shaped such that the diffracted excitation light transmitted through the disk interferes in a manner such that one Oth- order diffraction beam and a plurality of higher order diffraction beams impinge on a back pupil of an objective lens of the confocal microscope.

[0034] The plurality of 1st-order diffraction beams may comprise four 1st-order diffraction beams.

[0035] The semi-annular sections and their corresponding section pinhole groups may be shaped such that the Oth-order diffraction beam and the plurality of higherorder diffraction beams impinging on the back pupil of the objective lens interfere to produce a structured illumination pattern on a sampling surface of the target, the structured illumination pattern comprising spatially periodic illumination maxima.

[0036] The structured illumination pattern may comprise a 3D lattice illumination pattern comprising spatially periodic illumination maxima in both: one or more lateral directions orthogonal to the spinning axis; and in an axial direction that is parallel to the spinning axis.

[0037] The 3D lattice illumination pattern may have a contrast in a range of 0.7-0.95, preferably 0.75-0.9.

[0038] An autocorrelation of the Fourier transform of the structured illumination pattern may exhibit 17 delta functions in a spatial frequency space.

[0039] Each pinhole may be displaced from its nominal location by a jitter offset. The jitter offset may be oriented in a jitter offset direction orthogonal to the spinning axis. The jitter offset may have a jitter offset amount less than 10% of at least one of: a separation distance between neighboring (e.g. adjacent) pinholes; and a maximum dimension of a 2D unit cell of the lattice pattern of its corresponding section pinhole group.

[0040] For each pinhole, at least one of the jitter offset amount and the jitter offset direction may be randomly determined.

[0041] For each pinhole, at least one of the jitter offset amount and the jitter offset direction may be selected according to an optimization process. The optimization process may comprise a cost function having at least one term that attributes cost that is positively correlated with variation in scanning intensity.

[0042] The disk may be locatable in the optical path of the confocal microscope with the spinning axis co-axial with an optical axis of the confocal microscope. The disk may be locatable on a focal plane of an objective lens of the confocal microscope.

[0043] A scanning intensity resulting from the excitation light passing through the disk with section offsets may be relatively uniform along radial directions that extend orthogonally from the spinning axis compared to a scanning intensity resulting from the excitation light passing through a disk without the section offsets.

[0044] Another aspect of the invention provides a spatial filter for a confocal microscope. The spatial filter comprises a disk spinnable about a spinning axis in an optical path of the confocal microscope between a coherent light source providing an excitation light and a target. The spinnable disk comprises: a plurality of pinholesarranged a 2D-latice pattern wherein, for each 2-D lattice pattern, each pinhole is located at the vertices of a 2D unit cell and wherein the 2D-lattice pattern comprises a plurality of repeating 2D unit cells, each pinhole permitting transmission of light through the disk; and a mechanism for vibrating the disk in directions orthogonal to the spinning axis.

[0045] The vibrating disk may share any of the features of any of the other spatial filters described herein.

[0046] Another aspect of the invention provides a spatial filter for a confocal microscope. The spatial filter comprises a disk spinnable about a spinning axis in an optical path of the confocal microscope between a coherent light source providing an excitation light and a target. The spinnable disk comprises: a plurality of pinholes arranged in at least one 2D-latice pattern wherein, for each 2-D lattice pattern, each pinhole is nominally located at the vertices of a 2D unit cell and wherein the 2D-lattice pattern comprises a plurality of repeating 2D unit cells, each pinhole permitting transmission of light through the disk. Each pinhole is displaced from its nominal location by a jitter offset. The jitter offset is oriented in a jitter offset direction orthogonal to the spinning axis. The jitter offset has a jitter offset amount less than 10% of at least one of: a separation distance between neighboring (e.g. adjacent) pinholes; and a maximum dimension of the 2D unit cell.

[0047] for each pinhole, at least one of the jitter offset amount and the jitter offset direction may be randomly determined.

[0048] For each pinhole, at least one of the jitter offset amount and the jitter offset direction may be selected according to an optimization process. The optimization process may comprise a cost function having at least one term that attributes cost that is positively correlated with variation in scanning intensity.

[0049] The disk with jitter offsets may share any of the features of any of the other spatial filters described herein.

[0050] Another aspect of the invention provides a method for confocal microscopy. The method comprises: locating any of the spatial filters described herein in the optical path of a microscope system between a coherent light source and a target; spinning the disk about a spinning axis that is coaxial with an optical axis of the microscope; illuminating the target with the excitation light through the disk; and, receiving sampled light generated from an interaction between the excitation light and the target at a photodetector.

[0051] The microscope system may comprise an objective lens and the disk may be located on a focal plane of the objective lens.

[0052] Another aspect of the invention provides a confocal microscope comprising a spatial filter having the feature(s) of any of the spatial filters described herein.

[0053] Other aspects of the invention provide apparatus having any new and inventive feature, combination of features, or sub-combination of features as described herein.

[0054] Other aspects of the invention provide methods having any new and inventive steps, acts, combination of steps and / or acts or sub-combination of steps and / or acts as described herein.

[0055] It is emphasized that the invention relates to all combinations of the above features, even if these are recited in different claims.

[0056] Further aspects and example embodiments are illustrated in the accompanying drawings and / or described in the following description.Brief Description of the Drawings

[0057] The accompanying drawings illustrate non-limiting example embodiments of the invention.

[0058] FIG. 1 is a schematic diagram of a microscope system incorporating a spinnable disk spatial filter according to an example embodiment.

[0059] FIG. 2A is a plan view of a spinnable disk according to a non-limiting example embodiment.

[0060] FIG. 2B is a cross-sectional view of the disk shown in FIG. 2A taken along lines 2B-2B of FIG. 2A.

[0061] FIG. 2C shows an example semi-annular section of the disk shown in FIG. 2A in isolation.

[0062] FIG. 3A is a schematic diagram of the diffracted light on a back pupil of an objective lens of microscope systems disclosed herein using the FIG. 2A disk according to an example embodiment.

[0063] FIG. 3B is a schematic diagram of a perspective view of a Fourier transform of the beams shown in FIG. 3A in a 3D spatial frequency domain according to an example embodiment.

[0064] FIG. 3C is a schematic diagram of a perspective view of the autocorrelation of the frequency components shown in FIG. 3B in 3D spatial frequency domain according to an example embodiment.

[0065] FIG. 3D is a schematic depiction of a simulation of a portion of the normalized intensity of a structured illumination (e.g. 3D lattice) pattern generated by the microscope SIM systems disclosed herein in an x-y plane according to an example embodiment.

[0066] FIG. 3E is a schematic depiction of a cross-sectional view of the structured illumination (e.g. 3D lattice) pattern shown in FIG. 3D taken along a plane that includes the line 3E-3E of FIG. 3D.

[0067] FIG. 4 is a schematic diagram of an SIM microscope system incorporating a spinnable disk spatial filter according to an example embodiment.

[0068] FIG. 5 is a flowchart of a method for confocal microscopy according to an example embodiment.

[0069] FIG. 6A is an image of of a partial 3D view of an example optical transfer function (OTF) of a spinning-disk SIM system disclosed herein according to an example embodiment in the 3D spatial frequency domain showing a cross-section in the fy- fzplane (where fx=0).

[0070] FIG. 6B is an image of a partial 3D view of an example OTF of a conventional wide-field microscopy system 3D spatial frequency domain showing a cross-section fy- fzplane (where fx=0).

[0071] FIG. 7A is an example image of fluorescent beads in an x-y plane generated by a spinning-disk SIM system disclosed herein according to an example embodiment.

[0072] FIG. 7B is an example image of fluorescent beads in an x-y plane generated by a conventional wide-field microscopy system.

[0073] FIG. 7C is a cross-sectional view of fluorescent beads of FIG. 7A taken on a plane than includes the highlighted dashed line of FIG. 7A according to an example embodiment.

[0074] FIG. 7D is a cross-sectional view of fluorescent beads of FIG. 7B taken on a plane that includes the highlighted dashed line of FIG. 7B.

[0075] FIG. 8A is an example wide-field microscopy fluorescence image of ryanodine receptors in rat cardiac myocytes.

[0076] FIG. 8B is a cross-sectional view of fluorescence image of FIG. 8A taken on a plane that includes the highlighted dashed line of FIG. 8A.

[0077] FIG. 8C is an example fluorescence image of ryanodine receptors in a rat cardiac myocytes obtained using a spinning-disk SIM system disclosed hereinaccording to an example embodiment.

[0078] FIG. 8D is a cross-sectional view of fluorescence image of FIG. 8C taken on a plane that includes the highlighted dashed line of FIG. 80.

[0079] FIG. 8E shows enlarged views of regions 1 -4 shown respectively in FIGs. 8A- D.

[0080] FIG. 9 is a plot comparing an example scanning intensity curve of a disk with section offsets and a scanning intensity curve of a disk without section offsets along radial directions of the disks.Detailed Description

[0081] Throughout the following description, specific details are set forth in order to provide a more thorough understanding of the invention. However, the invention may be practiced without these particulars. In other instances, well known elements have not been shown or described in detail to avoid unnecessarily obscuring the invention. Accordingly, the specification and drawings are to be regarded in an illustrative, rather than a restrictive sense.

[0082] Aspects of the invention provide spatial filters for confocal microscope systems (e.g. structured illumination microscope (SIM) systems) and microscope systems incorporating same. The spatial filters comprise a disk spinnable about a spinning axis in an optical path of the confocal microscope between a coherent light source providing an excitation light and a target.

[0083] In some embodiments, the spinnable disk comprises: a plurality of semiannular sections, each semi-annular section shaped to define a corresponding section pinhole group comprising a plurality of pinholes arranged in a lattice pattern, each pinhole permitting transmission of light through the disk. For each semi-annular section, the corresponding section pinhole group is located to have a corresponding section offset along a section offset direction orthogonal to the spinning axis and having at least a component that extends radially from the spinning axis. The section offset is different for at least two of the plurality of semi-annular sections.

[0084] In some embodiments, the spinnable disk comprises a plurality of pinholes arranged a 2D-latice pattern wherein, for each 2-D lattice pattern, each pinhole is located at the vertices of a 2D unit cell and wherein the 2D-lattice pattern comprises a plurality of repeating 2D unit cells, each pinhole permitting transmission of light through the disk. A mechanism may be provided for vibrating the disk in directions orthogonal to the spinning axis.

[0085] In some embodiments, the spinnable disk comprises: a plurality of pinholes arranged in at least one 2D-latice pattern wherein, for each 2-D lattice pattern, each pinhole is nominally located at the vertices of a 2D unit cell and wherein the 2D-lattice pattern comprises a plurality of repeating 2D unit cells, each pinhole permitting transmission of light through the disk. Each pinhole is displaced from its nominal location by a jitter offset. The jitter offset is oriented in a jitter offset direction orthogonal to the spinning axis. The jitter offset has a jitter offset amount less than 10% of at least one of: a separation distance between neighboring (e.g. adjacent) pinholes; and a maximum dimension of the 2D unit cell.

[0086] Other aspects of the invention provide methods for confocal microscopy. Some such methods comprise: locating any of the spatial filters described herein in the optical path of a microscope system between a coherent light source and a target; spinning the disk about a spinning axis that is coaxial with an optical axis of the microscope; illuminating the target with the excitation light through the disk; and, receiving sampled light generated from an interaction between the excitation light and the target at a photodetector.

[0087] Another aspect of the invention provides a confocal microscope comprising a spatial filter having the feature(s) of any of the spatial filters described herein.

[0088] FIG. 1 is a schematic diagram showing a microscope system 100 incorporating a spinnable disk spatial filter 105 according to an example embodiment.

[0089] Microscope system 100 comprises a light source 102 configured to emit light 111. In some embodiments, light source 102 comprises a coherent light source and / or light from light source 102 is suitably filtered to provide coherent light 111. In some embodiments, light 111 may be referred to as excitation light 111 before interacting with target 101. In some embodiments, light source 102 comprises a laser or lasers. In some embodiments, light source 102 comprises one or more light emitting diodes (LEDs). In some embodiments, light 111 has a wavelength in the range of about 400 nm to 700 nm, although other wavelengths could additionally or alternatively be used. In a non-limiting example embodiment, light 111 has a wavelength of about 488 nm.

[0090] In an excitation path of system 100, excitation light 111 is directed by optical elements 104 to a spatial filter 105. In some embodiments, optical elements 104 may comprise any suitable light processing elements, such as, by way of non-limiting example, one or more lenses, one or more mirrors, a % wave plate (e.g. to circularlypolarize light 111 ), a beam splitter and / or the like. Spatial filter 105 is configured to diffract excitation light 111 transmitted through spatial filter 105. In some embodiments, spatial filter 105 comprises a spinnable disk 105A (often referred to herein as “disk” 105A, for brevity).

[0091] Excitation light 111 transmitted through spatial filter 105 is diffracted to propagate as diffracted light 111 D having a plurality of wave fronts. In some embodiments, interference among the plurality of wave fronts of diffracted light 11 1 D results in a plurality of amplitude maxima of diffracted light 111 D at a back pupil of objective lens 108 (the amplitude maxima at the back pupil resulting from the interference of diffracted light 111 D may also be referred to as “diffraction beams” herein). In some embodiments, the constructive interference of the plurality of wave fronts of diffracted light 111 D results in one Oth-order diffraction beam and a plurality of higher order diffraction beams at the back pupil of objective lens 108. In some embodiments, the plurality of higher order diffraction beams comprises four 1st-order diffraction beams.

[0092] In some embodiments, spatial filter 105 comprises a spinnable disk 105A locatable in an optical path of system 100 between light source 102 and a target 101 and spinnable about a spinning axis (which may be parallel to the optical axis (z-axis) of system 100).

[0093] In some embodiments, spinnable disk 105A comprises a plurality of pinholes arranged in at least one 2D-lattice pattern, each pinhole permitting transmission of light through disk 105A. For each 2-D lattice pattern, each pinhole may nominally be located at the vertices of a 2D unit cell. The 2D-lattice pattern may comprise a plurality of repeating 2D unit cells. Spatial filter 105 may comprise a mechanism for vibrating the disk in directions orthogonal to the spinning axis.

[0094] In some embodiments, spinnable disk 105A comprises a plurality of pinholes arranged in at least one 2D-lattice pattern, each pinhole permitting transmission of light through disk 105A. For each 2-D lattice pattern, each pinhole may nominally be located at the vertices of a 2D unit cell. The 2D-lattice pattern may comprise a plurality of repeating 2D unit cells. Each pinhole may be displaced from its nominal location in the 2D-lattice pattern by a jitter offset oriented in a jitter offset direction orthogonal to the spinning axis and a jitter offset amount that is less than 10% of at least one of: a separation distance between neighboring (e.g. adjacent) pinholes; and a maximum dimension of the 2D unit cell. In some embodiments, for each pinhole, atleast one of the jitter offset amount and the jitter offset direction is randomly determined. In some embodiments, the jitter offset and jitter offset direction in the 2D- lattice can follow a pattern where the pattern is randomly determined, provided that the pattern has sufficient (e.g. greater than 25 or greater than 100 or greater than 200) distinct displacement amounts and / or directions. In some embodiments, for each pinhole, the offset jitter and / or the jitter offset direction is determined according to an optimization process which minimizes a cost function having at least one term that attributes cost that is positively correlated with variation in scanning intensity.

[0095] In some embodiments, the 2D unit cells of the at least one 2D-lattice pattern repeat in a pair of directions orthogonal to the spinning axis. In some embodiments, the pair of directions orthogonal to the spinning axis are non-radial. In some embodiments, the repeating 2D unit cells are arranged to share at least one edge with one another. In some embodiments, for each 2D-lattice pattern, the 2D unit cells have the same size and shape. In some embodiments, the 2D unit cell comprises a 2D polygonal lattice unit, with pinholes located at vertices of the 2D polygonal lattice unit. The 2D polygonal lattice unit may be defined by any suitable polygonal lattice unit, including but not limited to: triangular, quadrilateral, hexagonal. In some embodiments, the polygonal lattice units have the same polygonal edge lengths, although this is not necessary. In some embodiments, the polygonal lattice units have a plurality of edges where at least two of the edges have different polygonal edge lengths.

[0096] In some embodiments and as discussed in more detail below, spinnable disk 105A may comprise a plurality of semi-annular sections, each of the plurality of semiannular sections shaped to define a section pinhole group comprising a section plurality of pinholes arranged in a section lattice pattern, each pinhole permitting transmission of light (e.g. excitation light 111 and sampled light 113 that interacts with target 101 ) through disk 105A. The section lattice pattern is similar to the 2D-lattice pattern as described elsewhere herein and disclosure related to the 2D-lattice pattern also applies to the section lattice pattern. In some embodiments, for each semiannular section, the corresponding section pinhole group is located to have a section offset along a corresponding section offset direction orthogonal to the spinning axis and having at least a component that extends radially from the spinning axis. In some embodiments, the section offset is different for at least two of the plurality of semiannular sections.

[0097] In some embodiments, the section offsets for at least some of the plurality of semi-annular sections are randomly selected. In some embodiments, the section offsets for the at least two of the plurality of semi-annular sections 204 is determined according to an optimization process which minimizes a cost function having at least one term that attributes cost that is positively correlated with variation in scanning intensity.

[0098] In some embodiments, the section lattice pattern comprises a linear lattice of a plurality of linearly spaced apart pinholes. In some embodiments, the linear lattice of a plurality of linearly spaced apart pinholes comprises a radial lattice of pinholes that are radially spaced apart in a radial direction extending from the spinning axis.

[0099] In some embodiments, in addition to section offsets for at least two of the plurality of semi-annular sections, each pinhole can be additionally displaced from its nominal location in its corresponding section lattice patterns by a jitter offset oriented in a jitter offset direction orthogonal to the spinning axis and a jitter offset amount that is less than 10% of at least one of: a separation distance between neighboring (e.g. adjacent) pinholes; and a maximum dimension of a 2D unit cell of the section lattice pattern of the corresponding section pinhole group. At least one of the jitter offset amount and the jitter offset direction can be randomly determined or optimized as described elsewhere herein.

[0100] Diffracted light 111 D is then directed by optical elements 106 to an objective lens 108. Diffracted light 111 D is projected onto target 101 (e.g. a sample) by objective lens 108. In some embodiments, spatial filter 105 is located on a focal plane of objective lens 108. In some embodiments, diffracted light 111 D is projected onto target 101 in an illumination pattern. In some embodiments, the illumination pattern projected onto target 101 comprises a structured illumination pattern. In some embodiments, the structured illumination pattern comprises a 3-dimensional (3D) lattice illumination pattern comprising spatially periodic illumination maxima in both lateral directions (e.g. in x and y directions that are orthogonal to the z-direction optical axis of system 100) and axial directions (e.g. in directions parallel to the z- direction optical axis of system 100). In some embodiments, the illumination pattern projected onto target 101 is generated from the interference of the Oth-order diffraction beam and a plurality of 1st-order diffraction beams of diffracted light 111 D. In a nonlimiting example embodiment, the illumination pattern projected onto target 101 is generated from the interference of the Oth-order diffraction beam and four 1st-orderdiffraction beams of diffracted light 111 D.

[0101] Diffracted light 111 D interacts with target 101 to generate sampled light 113. As is known to those of skill in the art, sampled light 113 typically comprises in-focus signals and out-of-focus signals. In a collection path of system 100, sampled light 113 is collected by objective lens 108 and then directed by optical elements 106 back to spatial filter 105. Spatial filter 105 is configured to filter sampled light 113 in the collection path of system 100. Since sampled light 113 is incoherent, the image formed by sampled light on detector 110 (described in more detail below) is not significantly impacted by the diffraction caused by spatial filter 105. In some embodiments, spatial filter 105 filters sampled light 113 by rejecting out-of-focus signals of sampled light 113 and transmitting in-focus signals. In some embodiments, at least a portion of the surface of spatial filter 105 is coated with a suitable material of sufficient opacity (e.g. aluminum, gold, silver, etc.) which rejects out-of-focus signals in sampled light 113 by physically blocking at least portions of the out-of-focus signals. In some embodiments, this portion of the surface of spatial filter 105 occupies more than 50% of the surface area of spatial filter 105 and spatial filter 105 rejects more than 50% of the out-of-focus signals of sample light 113. In some embodiments, this portion of the surface of spatial filter 105 occupies more than 75% of the surface area of spatial filter 105 and spatial filter 105 rejects more than 75% of the out-of- focus signals of sampled light 113. In a non-limiting example embodiment, spatial filter 105 rejects about 90% of the out-of-focus signals of sampled light 113.

[0102] Filtered sampled light 113F is then directed by optical elements 104 to a light detector 110 configured to collect filtered sampled light 113F and, optionally, convert collected filtered sampled light 113F into suitable electronic signals and / or image data. In some embodiments, light detector 110 comprises a camera. Collected filtered sampled light 113F may be further optically, electronically and / or digitally processed by any suitable processing techniques for any suitable purposes, for example, to generate images (e.g. fluorescence images) as is known in the art.

[0103] FIG. 2A is a plan view of a disk 200 according to a non-limiting example embodiment. FIG. 2B is a cross-sectional view of disk 200 taken along lines 2B-2B of FIG. 2A. FIG. 2C shows a semi-annular section 204-1 of disk 200 of FIG. 2A in isolation. FIGs. 2A, 2B and 2C (collectively, FIG. 2) are presented for illustrative purposes only and are not drawn to scale. Disk 200 may be incorporated as a spinnable disk into any spatial filter disclosed herein (e.g. spatial filters 105, 405,etc.).

[0104] Disk 200 comprises a body 202. Body 202 of the illustrated embodiment extends in orthogonal transverse directions 213 and 215 from spinning axis 209 and spans an area 202S in a plane orthogonal to a spinning axis 209 and extends longitudinally in a direction parallel to spinning axis 209 to define a thickness 202T (shown in FIG. 2B). In some embodiments, spinning axis 209 is co-axial with or parallel with an optical axis (z-axis) of a microscope system incorporating disk 200 (e.g. microscope systems 100, 400 described herein). While the thickness 202T of disk 200 is shown as being uniform in the FIG. 2B embodiment, this is not necessary and the thickness of disk 200 may vary with its transverse extension (e.g. in directions 213 and 215) from spinning axis 209. Spinning axis 209 extends into and out of the page in FIG. 2A.

[0105] Disk 200 of the illustrated FIG. 2 embodiment is shaped to define a through- bore 203 extending through body 202 in a direction parallel to spinning axis 209. In the illustrated embodiment, bore 203 is defined by bore-defining surface 203A which extends generally through body 202 in a direction parallel to spinning axis 209. A shaft (not shown in FIG. 2) may extend into and through through-bore 203 such that an outer (e.g. circumferential or keyed circumferential) surface of the shaft bears against bore-defining surface 203A to facilitate engagement between the shaft and disk 200. In some embodiments, disk 200 is rotatable about spinning axis 209, for example, by rotation of the shaft which, through engagement between the shaft and disk 200, causes corresponding rotation of disk 200 about axis 209. In other embodiments, any other suitable mechanism(s) (e.g. clamp(s), vacuum chuck(s) and / or the like) may be used to rotatably support body 202 of disk 200 and to cause rotation of disk 200 about spinning axis 209. In some such embodiments, bore 203 may not be required.

[0106] As shown best in FIG. 2A, disk 200 comprises a body 202 that is circularly shaped in a cross-sectional plane orthogonal to spinning axis 209 having an outer circumference (or circumferential surface) 202A. However, this circular shape is not necessary. Body 202 may be of any suitable shapes. In some embodiments, body 202 has a polygonal shape in a cross-sectional plane orthogonal to spinning axis 209.

[0107] Disk 200 of the FIG. 2 embodiment comprises a plurality of semi-annular sections 204 (e.g. semi-annular sections 204-1 , 204-2, 204-3...204-8 shown in FIG. 2A). Each semi-annular section 204 of disk 200 is defined by: two straight edges 210which extend in directions (e.g. radial directions) orthogonal to spinning axis 209, a corresponding segment (e.g. an arcuate segment) of bore-defining surface 203A and a corresponding segment of outer circumferential surface 202A. For example, Fig. 2C shows semi-annular section 204-1 in isolation. Semi-annular section 204-1 is defined by straight edges 210-1 , 210-2 which extend in directions (e.g. radial directions) orthogonal to spinning axis 209 and corresponding segments 203A-1 and 202A-1 of bore-defining surface 203A and outer circumferential surface 202A.

[0108] Each straight edge 210 defines a boundary separating a pair of angularly adjacent semi-annular sections 204. For example, edge 210-1 defines the boundary between semi-annular sections 204-1 and 204-8 and edge 210-2 defines the boundary between semi-annular sections 204-1 and 204-2, so on and so forth. While each semi-annular section 204 is defined in part by a corresponding pair of straight edges 210, one edge 210 that defines each semi-annular section 204 may be referred to herein as a “reference edge” for that semi-annular section. For example, in the embodiment of disk 200 of FIG. 2, edge 210-1 may serve as a reference edge for semi-annular section 204-1 ; edge 210-2 may serve as a reference edge for semiannular section 204-2; and so on.

[0109] Straight edges 210 shown in FIG. 2A and described herein are schematic / conceptual in nature and are used to describe the defining boundaries of semi-annular sections 204. Other than for defining the boundaries of semi-annular sections 204 and the features of semi-annular sections 204, straight edges 210 need not be actually physically present (e.g. visual, tactile, or otherwise) on disk 200. Disk 200 of the FIG. 2 embodiment is shown to have eight semi-annular sections 204-1 , 204-2, 204-3,... 204-8 and eight straight edges 210-1 , 210-2, 201 -3,... 210-8. However, this is not necessary. Disk 200 may comprise any suitable number of semiannular sections 204. In some embodiments, the number of semi-annular sections 204 of disk 200 is between 2 and 360, and preferably between 4 and 36.

[0110] As discussed elsewhere herein, in some embodiments, bore 203 through disk 200 is not required. In such embodiments, disk 200 may comprise a plurality of sectors defined by a pair of straight edges 210 which extend in directions (e.g. radial directions) orthogonal to spinning axis 209 and which intersect at spinning axis 209 and a corresponding segment of outer circumferential surface 202A. Such sectors may have the functionality and features of semi-annular sections 204 described herein. References herein to semi-annular sections 204 should be understood toinclude such sectors, except where the context clearly indicates otherwise.

[0111] Each semi-annular section 204 is shaped to define a section pinhole group 206A comprising a section plurality of pinholes 206 arranged in a section lattice pattern, each pinhole 206 permitting transmission of light (e.g. excitation light 111 and sampled light 113) through disk 200. Pinholes 206 extend generally in directions parallel to or substantially parallel to spinning axis 209 to extend between a first and second sides 207A, 207B (shown in FIG. 2B) of disk 200. Each pinhole 206 provides a corresponding optical path 205 (illustrated as double-headed arrow in FIG. 2B) between first and second sides 207A, 207B of disk 200 to facilitate light transmission through disk 200. Disk 200 of the FIG. 2 embodiment is shown to have eight semiannular sections 204-1 , 204-2, 204-3,... 204-8 having corresponding section pinhole groups 206A-1 , 206A-2, 206A-3,... 206A-8. However, this is not necessary. Disk 200 may comprise any suitable number of semi-annular sections 204 and any suitable number of section pinhole groups 206A. Excitation light 111 transmitted through a pinhole 206 is diffracted, i.e. bending around the portion of body 202 of sufficient opacity in the corresponding section pinhole group 206A of the corresponding semiannular section 204 to spread out from the optical path provided by pinhole 206 to propagate as a wave front.

[0112] In the illustrated FIG. 2 embodiment, the section lattice pattern of pinholes 206 in the section pinhole groups 206A is the same across semi-annular sections 204 - i.e. the section lattice pattern of pinholes 206 is the same for each semi-annular section 204 of disk 200, although this is not necessary. Disk 200 of the FIG. 2 embodiment is shown to have section lattice pattern of 2D array of pinholes defined by quadrilateral lattice units with pinholes located at vertices of the quadrilateral lattice units and having polygonal edge lengths that are the same for each polygonal lattice units. However, this is not necessary. It is to be understood that disk 200 of the FIG. 2 embodiment is a specific non-limiting embodiment. The 2D array of pinholes may be defined by any repeating lattice units. Neighboring lattice units may share at least one edge. The 2D array of pinholes of disk 200 may be defined by any suitable polygonal lattice units, including but not limited to: triangular, quadrilateral, hexagonal. It is also not necessary that the polygonal lattice units have the same polygonal edge lengths. In some embodiments, the polygonal lattice units have a plurality of edges where at least two of the edges have different polygonal edge lengths. It is also not necessary that the section lattice pattern comprises a 2D array of pinholes 206. In someembodiments, for each section pinhole group 206A, the section lattice pattern comprises a linear lattice of a plurality of linearly spaced apart pinholes 206. In some embodiments, the linear lattice of a plurality of linearly spaced apart pinholes 206 comprises a radial lattice of pinholes 206 that are radially spaced apart in a radial direction extending from spinning axis 209.

[0113] In some embodiments, the 2D array of pinholes in each section pinhole group 206A is spatially periodic in at least a first direction. The first direction of spatial periodicity of section pinhole groups 206A may be same across (e.g. for each of the plurality of) semi-annular section 204. In some embodiments, the first direction of spatial periodicity of section pinhole groups 206A is orthogonal to an optical axis (e.g. z-axis) of a microscope system (e.g. microscope system 100 or any other microscope systems described herein) and / or orthogonal to spinning axis 209. For example, in the non-limiting example embodiment of the FIG. 2 disk 200, the pinholes 206 in each section pinhole group 206A are periodically spaced apart (e.g. center-to-center) by a distance di in direction 215 (y-axis). As discussed above, direction 215 may be generally orthogonal to spinning axis 209 and / or to the optical axis (z-axis) of any optical system in which disk 200 is used.

[0114] In some embodiments, each section pinhole group 206A is spatially periodic in a second direction. The second direction of spatial periodicity of section pinhole groups 206A may be same across (e.g. for each of the plurality of) semi-annular section 204. In some embodiments, the second direction of spatial periodicity of section pinhole groups 206A is orthogonal to an optical axis (e.g. z-axis) of a microscope system (e.g. microscope system 100 or any other microscope systems described herein) and / or orthogonal to spinning axis 209. For example, in the nonlimiting example embodiment of the FIG. 2 disk 200, the pinholes 206 in each section pinhole group 206A are periodically spaced apart (e.g. center-to-center) by a distance d2 in direction 213 (x-axis). As discussed above, direction 213 may be generally orthogonal to spinning axis 209 and / or to the optical axis (z-axis) of any optical system in which disk 200 is used.

[0115] In currently preferred embodiments, the directions 213, 215 of spatial periodicity of pinholes 206 and the spatial period parameters di, d2 are the same across (e.g. for all or for some subset of) the plurality of section pinhole groups 206A, although neither of these characteristics is necessary. In currently preferred embodiments, the directions 213, 215 of spatial periodicity of pinholes 206 in eachsection pinhole group 206A are also orthogonal to one another, although this is not necessary. In currently preferred embodiments, the spatial period parameters di, d2 are equal to one another (i.e. di=d2), although this is not necessary. In an alternative embodiment, each section pinhole group 206A is spatially periodic in a sectionspecific direction (e.g. in a radial direction between the straight edges 210 that define the semi-annular section 204) and in a second direction that is orthogonal to both the section-specific direction and the spinning axis 209 (or optical z-axis). In some embodiments, the value of spatial period parameter d (or spatial period parameters di, d2) is / are selected such that one Oth-order diffraction beam and four 1st-order diffraction beams impinge on a back pupil of an objective lens (e.g. objective lens 108) of a confocal microscope.

[0116] In disk 200 of the FIG. 2A illustrated embodiment, pinholes 206 are illustrated as being circularly shaped in a cross-sectional plane orthogonal to spinning axis 209. However, this is not necessary. Pinholes 206 may be of any suitable shape in the plane orthogonal to spinning axis 209. In some embodiments, pinholes 206 are of polygonal cross-sectional shapes or other cross-sectional shapes.

[0117] In some embodiments, each pinhole 206 in the section plurality of pinholes in the corresponding section pinhole group 206A of each of the plurality of semi-annular sections 204 has a size defined by a common opening dimension factor w (shown in FIG. 2A) in a plane orthogonal to spinning axis 209, although this is not required. For example, in embodiments where pinholes 206 are circularly shaped in a cross- sectional plane orthogonal to spinning axis 209, dimension factor w may comprise a diameter of the circle. A ratio between the dimension factor w and the spatial period parameter di=d2=d, i.e., w / d, may be referred to herein as the “opening ratio”. In some embodiments, the opening ratio is in a range of about 0.2 to about 0.5, and preferably in a range of 0.3 to 0.4. In one non-limiting example embodiment, the dimension factor w is about 28 / zm and the spatial period parameter d is about 84 / zm and the opening ratio is about 0.33. A disk with an opening ratio of about 0.33 is able to reject about 90% of the out-of-focus signals. In some embodiments, the parameters wand d may be selected to provide a smaller opening ratio (w / d) to filter a greater percentage of the out-of-focus signals from sampled light 113. In some embodiments, the parameters wand d may be selected to provide a larger opening ratio (w / d) to increase the amount of detected signals and possibly to increase the signal to noise ratio (SNR).

[0118] In some embodiments, section pinhole group 206A of each semi-annular section 204 is located to have a section offset relative to spinning axis 209 along a corresponding section offset direction where the section offset is different for at least two of the plurality of semi-annular sections 204. The section offset in disk 200 of the FIG. 2 embodiment is different for each of the plurality of semi-annular sections 204, although this is not necessary. In some embodiments, the section offsets for the at least two of the plurality of semi-annular sections 204 is determined according to an optimization process which minimizes a cost function having at least one term that attributes cost that is positively correlated with variation in scanning intensity. For each semi-annular section 204, the corresponding section offset direction may be radial. In some embodiments, the section offset direction is a radial direction lying within the corresponding semi-annular section 204. For example, for each semiannular section 204, the corresponding section offset direction may be a radial direction that bisects (e.g. is in a center of) the semi-annular section 204. In the nonlimiting example embodiment of disk 200 shown in FIG. 2, the section offsets to section pinhole groups 206A-1 , 206A-2, 206A-3,... 206A-8 for corresponding semiannular sections 204-1 , 204-2, 204-3,... 204-8 are respectively: 19.5 / zm, 216.1 / zm, 27.2 zm, 51 .4 / zm, 4.8 / zm, 26.5 / zm, 221 .0 / zm, and 58.8 / zm as measured along corresponding section offset directions that bisect the corresponding semi-annular sections 204-1 , 204-2, 204-3,... 204-8, although this is not necessary. In some embodiments, the section offsets for the at least two of the plurality of semi-annular sections 204 is randomly selected.

[0119] In some embodiments, in addition to or in the alternative to section offsets for at least two of the plurality of semi-annular sections, each pinhole is displaced from its nominal location by a jitter offset, the jitter offset oriented in a jitter offset direction orthogonal to the spinning axis and a jitter offset amount less than 10% of at least one of: a separation distance between neighboring (e.g. adjacent) pinholes; and a maximum dimension of a 2D unit cell of the lattice pattern of its corresponding section pinhole group. In some embodiments, for each pinhole, at least one of the jitter offset amount and the jitter offset direction is randomly determined. In some embodiments, for each pinhole, at least one of the jitter offset amount and the jitter offset direction is selected according to an optimization process, wherein the optimization process comprises a cost function having at least one term that attributes cost that is positively correlated with variation in scanning intensity.

[0120] For each semi-annular section 204, the corresponding section offset direction may be may be angularly spaced apart from the corresponding reference edge 210 for each semi-annular section 204 and the angular section offset direction relative to the corresponding reference edge 210 is the same for at least two semi-annular sections 204. For example, in disk 200 of the FIG. 2 embodiment, the section pinhole group 206A-1 of semi-annular section 204-1 is offset along a section offset direction 214-1 that extends from spinning axis 209 (illustrated as a double-headed arrow in FIG. 2A). In the embodiment of disk 200 shown in FIG. 2, section offset direction 214- 1 is along a central radial direction of semi-annular section 204-1 (i.e. section offset direction 214-1 bisects semi-annular section 204-1 ). However, this is not necessary. The section offset direction by which the section pinhole group 206A-1 is offset may be any suitable direction orthogonal to spinning axis 209 and having at least a component that extends radially from spinning axis 209. For example, for illustrative purposes, section offset directions 214-2 and 214-3 (shown in FIG. 2A) are also suitable section offset directions within semi-annular section 204-1 . The section offset directions for the at least two of the plurality of semi-annular sections 204 are preferably consistently angularly offset from the reference edges 210 of their corresponding semi-annular sections 204. For example, in the case of the FIG. 2 embodiment, each semi-annular section 204 subtends an angle of 45° and section offset directions that are 22.5° from the reference edges 210 of the semi-annular sections 204 would bisect the semi-annular sections 204 (as is the case in the illustrated FIG. 2 embodiment). However, section offset directions that are 10° from the reference edges of the semi-annular sections 204 would also be suitable, provided that such angular offsets from the reference edges are consistent for the at least two of the plurality of semi-annular sections 204.

[0121] In some embodiments, a scanning intensity resulting from light (e.g. excitation light 111) passing through a disk 200 with section offsets for the section pinhole group 206A of at least two of the plurality of semi-annular sections 204 is relatively uniform along radial directions that extend orthogonally from spinning axis 209 or from the optical axis (z-axis) of the optical system in which disk 200 is employed compared to a scanning intensity resulting from light (e.g. excitation light 111 ) passing through a disk without the section offsets. FIG. 9 is a plot 900 comparing an example scanning intensity curve 904 of a disk with section offsets (e.g. disk 200) and scanning intensity curve 902 of a disk without section offsets along a radial direction that extends fromthe optical axis (z-axis) of a microscope system. Plot 900 shows the intensity values in a range of 3.50 cm radial distance from the optical axis and 3.60 cm radial distance from the optical axis. As can be seen from plot 900, scanning intensity curve 904 is significantly more uniform in magnitude of scanning intensity compared to scanning intensity curve 902. Given a definition of uniformity being the minimum intensity divided by the maximum intensity, scanning intensity curve 904 has a uniformity of 94.4% and scanning intensity curve 902 has a uniformity of only 72.7%.

[0122] It is to be understood that disk 200 of the FIG. 2 embodiment is a specific embodiment comprising semi-annular sections 204 with section offsets for at least two of the semi-annular sections 204. However, it is not necessary that a spinnable disk disclosed herein comprises semi-annular sections with section offsets. In some embodiments, spinnable disk 200 comprises a plurality of pinholes arranged in at least one 2D-lattice pattern, each pinhole permitting transmission of light through the disk. For each 2-D lattice pattern, each pinhole is nominally located at the vertices of a 2D unit cell. The 2D-lattice pattern may comprise a plurality of repeating 2D unit cells. For example, the 2D unit cell may comprise a polygonal lattice unit which may be, but are not limited to, triangular, quadrilateral, hexagonal polygons. In some embodiments, a spatial filter comprising the disk comprises a mechanism for vibrating the disk in directions orthogonal to spinning axis 209. In some embodiments, each pinhole is displaced from its nominal location by a jitter offset, the jitter offset oriented in a jitter offset direction orthogonal to the spinning axis and a jitter offset amount less than 10% of at least one of: a separation distance between neighboring (e.g. adjacent) pinholes; and a maximum dimension of the 2D unit cell. At least one of the jitter offset amount and the jitter offset direction may be randomly determined or optimized as described elsewhere herein.

[0123] Referring again to Figure 1 , in some embodiments, excitation light 111 transmitted through disk 200 (e.g. diffracted light 111 D) has an interference resulting in five diffraction beams at a back pupil of an objective lens (e.g. objective lens 108). Light propagating through disk 200 (e.g. diffracted light 11 1 D) may be steered, directed, focused or otherwise optically manipulated by any suitable optical elements (e.g. optical elements 106) onto the back pupil of the objective lens.

[0124] FIG. 3A is a schematic diagram of the diffracted light (e.g. diffracted light 111 D) impinging on a back pupil 300 of an example objective lens (e.g. objective lens 108) in microscope systems disclosed herein (e.g. systems 100, 400, etc.) using disk200 according to an example embodiment. FIG. 3A is shown for illustrative purposes only and is not drawn to scale. The diffraction introduced by pinholes 206 with spatial period parameter d (or spatial period parameters di, d2) of disk 200 and the subsequent interference among propagating wave fronts of the diffracted light 111 D result in Oth-order diffraction beam 301 and four 1st-order diffraction beams 303, 305, 307 and 309 (all illustrated as black dots in FIG. 3A) being projected within aperture 302 of back pupil 300 of an objective lens. In some embodiments, the one Oth-order diffraction beam 301 and the four 1st-order diffraction beams 303, 305, 307 and 309 are of equal or approximately equal intensities. In some embodiments, the value of spatial period parameter d (or spatial period parameters di , d2) is / are selected such that the four 1st-order diffraction beams 303, 305, 307 and 309 are as close as possible to an edge of aperture 302 (e.g. within a distance, from the edge, of 20% of a cross-sectional dimension of aperture 302 or within 10% in some embodiments) while remaining within aperture 302.

[0125] FIG. 3B is a schematic diagram 350B showing a perspective view of the distribution of the frequency components based on a Fourier transform of the five beams 301 , 303, 305, 307 and 309 shown in FIG. 3A in a 3D spatial frequency space defined by fx, fyand fz. FIG. 3C is a schematic diagram 350C showing a perspective view of an autocorrelation of the frequency components shown in FIG. 3B exhibiting 17 delta functions 311 -1 , 311-2, 311 -3,... 311 -17 in the 3D frequency space defined by fx, fy and fz. FIGs. 3B and 3C are drawn for illustrative purposes only and are not drawn to scale.

[0126] As shown in FIG. 3B, the frequency component 301 F of Oth-order diffraction beam 301 has a distinct axial frequency along fzdirection different from the axial frequencies of the frequency components 303F, 305F, 307F, and 309F of the four 1st- order diffraction beams 303, 305, 307 and 309. Consequently, interference among the one Oth-order diffraction beam 301 and the four 1st-order diffraction beams 303, 305, 307 and 309 as a result of excitation light 111 passing through disk 200 produce structured illumination patterns in both lateral directions (i.e. x - y) orthogonal to an optical axis of microscope 100 and in the axial directions (i.e. z) parallel to the optical axis of microscope 100. 0 is the half-angle subtended by the frequency components 303F, 305F, 307F, and 309F of the four 1st-order diffraction beams 303, 305, 307 and 309 with respect to the optical axis of the back pupil (e.g. optical (z) axis of the microscope systems disclosed herein). The value of 0 depends at least in part on thevalue of spatial period parameter d (or parameters di, d2 of pinholes 206. In a nonlimiting example embodiment, where the spatial period parameter cf has a value of 84 m, 9 is about 50°.

[0127] FIG. 3C exhibits 17 delta functions 311 -1 to 311 -17 in the 3D spatial frequency space. The 17 delta functions 311 -1 to 311 -17 are the autocorrelation of the frequency components 301 F, 303F, 305F, 307F, and 309F as shown in FIG. 3B of the 5 beams 301 , 303, 305, 307 and 309 shown in FIG. 3A. The 17 delta functions 311 -1 to 311 -17 shift the object spectrum shown in FIG. 3B to 17 different positions, allowing the frequencies that are beyond the diffraction limit of the conventional optical transfer function (OTF) to be captured. Since the frequency component 301 F of Oth-order diffraction beam 301 has a distinct axial frequency different from the axial frequencies of the frequency components 303F, 305F, 307F, and 309F of the four 1st- order diffraction beams 303, 305, 307 and 309, the resulting 17 delta functions 311 -1 to 311 -17 expand the OTF both laterally (i.e. fx- fy) and axially (i.e. fz).

[0128] In some embodiments, the interference among the Oth-order diffraction beam and the four 1st-order diffraction beams as projected by an objective lens on a target (e.g. sample 101 ) results in a structured illumination pattern. In some embodiments, the structured illumination pattern projected onto the target comprises a 3D lattice illumination pattern comprising spatially periodic illumination maxima in both lateral directions (i.e. x - y) and in the axial directions (i.e. z).

[0129] FIG. 3D is a schematic depiction of a simulation of a portion of the normalized intensity of a 3D lattice pattern 350D generated by microscope systems disclosed herein (e.g. systems 100, 400, etc.) on a sample x-y plane (a plane orthogonal to the optical (z) axis)) of a target (e.g. target 101 , 401 , etc.) according to an example embodiment. FIG. 3E is a cross-sectional view of 3D lattice pattern 350D taken along lines 3E-3E of FIG. 3D.

[0130] For the example embodiment shown in FIG. 3D, 3D lattice illumination pattern 350D comprises a spatially periodic plurality of illuminating spots 322 in the x-y plane, each illuminating spot 322 being the result of constructive interference among the 5 beams shown in FIG. 3A (i.e. one Oth-order diffraction beam (e.g. beam 301 ) and four 1st-order diffraction beams (e.g. beams 303, 305, 307 and 309)). As shown in FIG. 3E, 3D lattice illumination pattern 350D also comprises a spatially periodic plurality of illuminating spots 322 in the axial (z) direction, each illuminating spot 322 being the result of constructive interference among the 5 beams shown in FIG. 3A (i.e. one 0th-order diffraction beam (e.g. beam 301 ) and four 1st-order diffraction beams (e.g. beams 303, 305, 307 and 309)). In some embodiments, 3D lattice illumination pattern 350D has a lateral period in a range of 200nm to 1500nm and an axial period in a range of 400nm to 3000nm. In a non-limiting example embodiment, with a pinhole spatial frequency period of di=d2=d=84 pm and a magnification of 167 times, 3D lattice illumination pattern 350D has a lateral period lxyof about 500 nm (e.g. in x and y in directions orthogonal to the optical axis) and an axial period lzof about 1072 nm (e.g. in the axial (z) direction). A contrast of illumination pattern 350D can be defined as the ratio between: the difference between intensity maximum and intensity minimum; and a sum of the intensity maximum and intensity minimum. In some embodiments, 3D lattice illumination pattern 350D has a contrast in a range of about 0.75 to about 0.95. In a non-limiting example embodiment, using circularly polarized excitation light, 3D lattice illumination pattern 350D has a contrast of about 88%.

[0131] FIG. 4 is a schematic diagram showing an SIM system 400 incorporating a spatial filter which includes a spinnable disk spatial filter 405 with section offsets according to any of the embodiments disclosed herein (e.g. spinnable disk 200) according to an example embodiment. In many respects, SIM system 400 is similar to SIM system 100 described elsewhere herein and, unless explicitly described as being different or the context indicates a difference, SIM system 400 may comprise any of the features of SIM system 100 and vice versa.

[0132] SIM system 400 comprises a light source 402 configured to emit excitation light 411 (illustrated as solid line arrows in FIG. 4). In some embodiments, light source 402 comprises a coherent light source. In some embodiments, light source 402 comprises a laser or lasers. In some embodiments, light source 402 comprises light emitting diodes (LEDs). In some embodiments, excitation light 411 has a wavelength in the range of about 400 nm to 700 nm. In a non-limiting example embodiment, excitation light 411 has a wavelength of about 488 nm.

[0133] In an excitation path of system 400, excitation light 411 first passes through a quarter-wave plate 422 configured to polarize (e.g. circularly polarize) excitation light 411 . Excitation light 411 is then directed by lens 424 to a beam splitter 426 configured to direct excitation light onto an optical (z) axis 409 of system 400. In some embodiments, beam splitter 426 comprises a dichroic mirror.

[0134] Light 411 propagating from beam splitter 426 is directed by a relay lens 428 to a spatial filter 405. Spatial filter 405 may comprise any spinnable disk disclosedherein (e.g. spinnable disk 200, which may be referred to herein as a disk 200 for brevity). Spatial filter 405 is configured to cause light 411 transmitted therethrough to diffract in the excitation path of system 400.

[0135] In some embodiments, spatial filter 405 causes light 411 to diffract such that light 411 transmitted through spatial filter 405 has one Oth-order diffraction beam and a plurality of higher order diffraction beams at back pupil 300 of aperture 302 of objective lens 408. In some embodiments, spinnable disk 200 is supported to spin about a spinning axis 209 and locatable in an optical path of system 400 between light source 402 and a target 401 , such that spinning axis 209 is parallel to, or coaxial with, optical axis 409.

[0136] As discussed above, disk 200 may comprise a plurality of semi-annular sections 204, wherein each of the plurality of semi-annular sections 204 is shaped to define a corresponding section pinhole group 206A comprising a corresponding section plurality of pinholes 206 permitting transmission of light (e.g. excitation light 411 and sampled light 413 from interaction with target 401 ) through disk 200. The section lattice pattern of pinholes 206 in the section pinhole groups 206A may be the same across semi-annular sections 204 - i.e. the section lattice pattern of pinholes 206 may be the same for each semi-annular section 204. The section lattice pattern of pinholes 206 in section pinhole groups 206A may be spatially periodic (e.g. with periods di, d?) in a pair of directions, both of which are orthogonal to spinning (z) axis 209. In some embodiments, the pair of directions may be orthogonal to one another (e.g. x, y). In some embodiments, the pair of directions are non-radial. The two orthogonal directions of spatial periodicity of section pinhole groups 206A may be same across (e.g. for each of the plurality of) semi-annular section 204. In some embodiments, di=d2=d- i.e. the spatial period parameters of the section lattice pattern of pinholes 206 in section pinhole groups 206A are the same along the pair of directions. In some embodiments, each pinhole 206 has a size defined by a common dimension factor w in a plane orthogonal to spinning axis 209. The parameters di, d2 and w may be in ranges of those discussed above in relation to disk 200. In some embodiments, the parameters wand d may be selected to provide a smaller opening ratio (w / d) to filter a greater percentage of the out-of-focus signals from sampled light 413. In some embodiments, the parameters wand d may be selected to provide a larger opening ratio (w / d) to increase the amount of detected signals and possibly to increase the signal to noise ratio (SNR). In some embodiments, the value of spatialperiod parameter d (or spatial period parameters di, d2) is / are selected such that one Oth-order diffraction beam 301 and four 1st-order diffraction beams 303, 305, 307 and 309 impinge on aperture 302. In some embodiments, the value of spatial period parameter d (or spatial period parameters di , d2) is / are selected such that the four 1st- order diffraction beams 303, 305, 307 and 309 are as close as possible to an edge of aperture 302 (e.g. within a distance, from the edge, of 20% of a cross-sectional dimension of aperture 302 or within 10% in some embodiments) while remaining within aperture 302.

[0137] In some embodiments, for each semi-annular section 204, the corresponding section pinhole group 206A is located to have a section offset relative to spinning axis 209 along a corresponding section offset direction. The section offset is different for the section pinhole group 206A in at least two of the plurality of semi-annular sections 204. For each semi-annular section 204, the corresponding section offset direction of the section offset is orthogonal to spinning axis 209 having at least a component that extends radially from spinning axis 209. Each section offset direction may be a radial direction that bisects its corresponding semi-annular section 204 or may be a radial direction that is otherwise angularly offset from the reference edge 210 of its corresponding semi-annular section 204 by an angular offset that is consistent across the plurality of semi-angular sections 204.

[0138] Diffracted light 411 D is directed by a lens 430 towards an objective lens 408. In some embodiments, lens 430 comprises a tube lens. In a non-limiting example embodiment, lens 430 has a focal length of 300 mm. Objective lens 408 projects diffracted light 411 D onto target 401 . Target 401 may comprise a sample of tissue from an organism, by way of non-limiting example.

[0139] In some embodiments, spatial filter 405 diffracts excitation light 411 such that diffracted light 411 D forms structured illumination pattern on target 401 . In some embodiments, one Oth-order diffraction beam (e.g. beam 301) and four 1st-order diffraction beams (e.g. beams 303, 305, 307 and 309) admitted by aperture 302 of objective lens 408 interfere to produce a structured illumination pattern (e.g. 3D lattice illumination pattern 350D) on a sampling surface of target 401 .

[0140] Diffracted excitation light 411 D interacts with target 401 to generate sampled light 413. Sampled light 413 is incoherent. In the collection path of system 400, sampled light 413 is collected by objective lens 408 and then directed by lens 430 back to spatial filter 405. Spatial filter 405 is configured to filter sampled light 413 inthe collection path of system 400 to generate filtered sampled light 413F. In some embodiments, spatial filter 405 filters sampled light 413 by rejecting out-of-focus signals of sampled light 413. In some embodiments, spatial filter 405 rejects about 90% of the out-of-focus signals of sampled light 413. In some embodiments, spatial filter 405 comprises a layer that is shaped (e.g. by the parameters w and d discussed herein) to filter the out-of-focus signals from sampled light 413 to thereby provide filtered sample light 413F. In some embodiments, target 401 fluoresces upon being struck by excitation light 411 and thus generates fluorescent light as sampled light 413.

[0141] Filtered sampled light 413F is then directed by relay lens 428 to beam splitter 426. Beam splitter 426 allows filtered sampled light 413F to continue propagating on the collection path of system 400. Filtered sampled light 413F is then further filtered by a spectral filter 432 configured to transmit only a selected range of wavelengths. Filtered sampled light 413F is then directed by relay lens 434 to a light detector 410 configured to collect filtered sampled light 413F. In some embodiments, light detector 410 comprises a camera. In some embodiments, filtered sampled light 413F is processed to generate images. In some embodiments, fluorescent light is collected as sampled light 413 and SIM system 400 is configured to generate fluorescence images based on fluorescent sampled light 413. In some embodiments, SIM system 400 is capable of generating super-resolved images at a frequency greater than 60Hz. In some embodiments, this super-resolved image-generating frequency may be greater than 100 Hz or 200 Hz.

[0142] Collected filtered sampled light 413F may be further optically, electronically and / or digitally processed by any suitable processing techniques for any suitable purposes. In some embodiments, filtered sample light 413F is processed using, inter alia, a Wiener filtering technique or other suitable noise-reduction technique.

[0143] FIG. 5 is a flowchart of a method 500 for confocal microscopy according to an example embodiment. Method 500 may be applied using any systems described herein (e.g. systems 100, 400, etc.)

[0144] Method 500 begins with step 501 of locating a spatial filter (e.g. spatial filters 105, 405 described herein) in an optical path of a microscope system (e.g. microscope systems 100, 400 disclosed herein). Method 500 then facilitates confocal microscopy by step 503 of rotating the spatial filter about a rotating axis (e.g. the spinning axis of disk 200) that is parallel with the optical path of the microscopesystem. In some embodiments, the spatial filter is rotated up to 7200 revolutions per minute. Method 500 then proceeds to step 505 of illuminating a sample (e.g. target 101 , 401 ) through the rotating spatial filter (e.g. spatial filters 105, 405 described herein) with an excitation light (e.g. excitation light 111 , 411 ) and concludes with step 507 of receiving sampled light (e.g. sampled light 113, 413 resulting from interaction between the excitation light and the sample) at a photodetector to carry out confocal microscopy.

[0145] In some embodiments, the spatial filter is configured (as described herein by suitable section offsets of corresponding section pinhole groups along corresponding section offset directions for each of a plurality of semi-annular sections) such that a scanning intensity resulting from light passing through the spatial filter with section offsets is relatively uniform along radial directions of the spatial filter (i.e. disk) compared to a scanning intensity resulting from light passing through a disk without the section offsets (see FIG. 9). In some embodiments, the spatial filter is configured (as described herein) to diffract the excitation light to provide one Oth-order diffraction beam and four 1st-order diffraction beams at the back pupil of an objective lens of the microscope system. In some embodiments, the spatial filter is configured (e.g. by providing an opaque layer and suitable selection of the parameters di, cfe, w) to filter sampled light in a light collection path. In some embodiments, the spatial filter is configured to reject about 80% of out-of-focus signals in the sampled light in the light collection path.

[0146] FIG. 6A is an image 600A of a partial 3D view of an example optical transfer function (OTF) of a spinning-disk 3D SIM as described herein (e.g. systems 100, 400, etc.) in the 3D spatial frequency domain showing a cross-section in the fy- fzplane (where fx=0), where the ^-direction is the direction of the axial spatial frequency and the ^ directions are the directions of spatial frequency in a y-direction and x- direction that are orthogonal to one another and to the axial (z) direction. FIG. 6B is an image 600B a partial 3D view of an example OTF of a conventional wide-field microscopy in the 3D spatial frequency domain showing a cross-section fy- fzplane. FIGs. 6A and 6B are presented in a logarithmic intensity scale with values between 0 and -6 as reflected in the grey scale bar.

[0147] As can be seen from FIG. 6B, with conventional wide-field microscopy, there is a region of missing frequencies known as the “missing cone”. The missing cone indicates that there is little transmission of axial spatial frequencies through theimaging system in the convention wide-field microscopy system shown in FIG. 6B. For this reason, conventional wide-field microscopy systems lack optical sectioning capability. FIG. 6A shows the OTF of a spinning-disk SIM system disclosed herein. As can be seen in FIG. 6A, the “missing cone” is filled, demonstrating the improved optical sectioning capability of spinning-disk SIM systems disclosed herein.

[0148] The spinning-disk SIM systems also expand the cut-off frequency of the OTF by a factor of 2 relative to conventional wide-field microscopy in all directions, which is reflected in the difference in size of OTFs shown in FIGs. 6A and 6B. The expansion of cut-off frequency may lead to a corresponding increase in both the lateral and the axial resolution of spinning-disk SIM systems disclosed herein compared to that of conventional wide-field microscopy. In some embodiments, images detected by the microscope systems described herein may be Wiener filtered or processed by any suitable processes to further enhance the details and / or contrast of the resultant images.

[0149] Spinning-disk SIM systems disclosed herein also have the advantages of being less prone to artifacts compared to conventional SIM systems (e.g. wide-field microscopy systems). A relatively small opening ratio (w / d) can be used to increase the relative intensity of the OTF of the spinningOdisk SIM system at high frequencies at the potential cost of reducing the amount of detected signal and decreased SNR.

[0150] FIG. 7A is an example image 700A of fluorescent beads with a diameter of 100 nm in the x-y plane generated by a spinning-disk SIM system of the type disclosed herein (e.g. system 100, 400) according to an example embodiment. FIG. 7B is an example image 900B of fluorescent beads with a diameter of 100 nm in the x-y plane generated by conventional wide field microscopy systems. FIG. 7C is an example enlarged cross-sectional view 700C of fluorescent beads of FIG. 7A in a plane along the highlighted dashed line of FIG. 7A generated by a spinning-disk SIM system disclosed herein (e.g. system 100, 400) according to an example embodiment. FIG. 7D is an example enlarged cross-sectional view 700D of fluorescent beads of FIG. 7B in a plane along the highlighted dashed line of FIG. 7B generated by a conventional wide-field microscopy system.

[0151] As can be seen in a comparison of FIG. 7A and FIG. 7B (images 700A, 700B), the resolution of the spinning-disk SIM image is superior to the resolution of the conventional wide-field microscope image. Comparing FIG. 7C and FIG. 7D (images 700C, 700D), it can be seen that the spatial resolution of the spinning-disk SIM imageis about 2 times more precise than the resolution of the conventional wide-field microscope image. For example, as can be seen in FIG. 70, spinning-disk SIM reduces the full width at half maximum (FWHM) of the beads from 280 nm to 140 nm in the lateral direction (orthogonal to the axial (z) direction) and from 815 nm to 407 nm in the axial (z) direction. Furthermore, as can be seen from image 700C in FIG. 7C, spinning-disk SIM is able to distinguish a plurality of closely located beads which could not be resolved in the conventional wide-field microscopy image 700D shown in FIG. 7D.

[0152] In an experiment conducted by the inventors, cardiac myocytes of rats were used to evaluate the ability of spinning-disk SIM systems to reduce out-of-focus signals. A typical rat cardiac myocytes has a thickness of 10-20 zm, which is a relatively thick sample. For the experiment, the inventors imaged the ryanodine receptors in a rat cardiac myocyte with a thickness of around 12 / zm. Therefore, imaging by wide-field microscopy produces a notable out-of-focus background. FIG. 8A is an example conventional wide-field microscopy fluorescence image 800A of ryanodine receptors in a rat cardiac myocytes at a depth of 9 / zm in the x-y plane. FIG. 8B is a cross-sectional view of image 800B in a plane that includes the highlighted dashed line of FIG. 8A. As can be seen from the images 800A, 800B of FIG. 8A and FIG. 8B, the fluorescence image appears hazy and blurry.

[0153] On the other hand, spinning-disk SIM systems are able to significantly reduce the out-of-focus signals for the cardiac myocytes sample. FIG. 8C is an example spinning-disk SIM fluorescence image 800C of ryanodine receptors in a rat cardiac myocytes at a depth of 9 / zm in the x-y plane using a spinning-disk SIM system of the type disclosed herein (e.g. system 100, 400) according to an example embodiment. FIG. 8D is a cross-sectional view of image 800D in a plane that includes the highlighted dashed line of FIG. 8C. As can be seen from the images 800C, 800D of FIG. 8C and FIG. 8D, compared to the conventional wide-field microscopy fluorescence images 800A, 800B shown in FIG. 8A and FIG. 8B, the spinning-disk SIM fluorescence images 8000, 800D shown in FIG. 80 and FIG. 8D provide better contrast and more clearly identify ryanodine receptors that are obscured by background signal in the images 800A, 800B of FIG. 8A and FIG. 8B.

[0154] FIG. 8E shows enlarged views 800E of regions 1 -4 respectively shown in FIGs. 8A-8D. As can be seen from FIG. 8E, in each of regions 1 -4, spinning-disk SIM fluorescence images 8000, 800D in FIGs. 80 and 8D provide better contrast andmore clearly identify ryanodine receptors that are obscured by background signal in the images 800A, 800B of FIGs. 8A and 8B.

[0155] Systems and methods disclosed herein provide an approach for 3D SIM that increases the contrast for optical sectioning, is less prone to artifacts induced by sample movement and optical imperfections, and is able to be performed at a frequency that enables “real-time” reconstruction of super-resolution images.

[0156] Where a component (e.g. a software module, processor, assembly, device, circuit, etc.) is referred to herein, unless otherwise indicated, reference to that component (including a reference to a “means”) should be interpreted as including as equivalents of that component any component which performs the function of the described component (i.e. , that is functionally equivalent), including components which are not structurally equivalent to the disclosed structure which performs the function in the illustrated exemplary embodiments of the invention.

[0157] Embodiments of the invention may be implemented using specifically designed hardware, configurable hardware, programmable data processors configured by the provision of software (which may optionally comprise “firmware”) capable of executing on the data processors, special purpose computers or data processors that are specifically programmed, configured, or constructed to perform one or more steps in a method as explained in detail herein and / or combinations of two or more of these. Examples of specifically designed hardware are: logic circuits, application-specific integrated circuits (“ASICs”), large scale integrated circuits (“LSIs”), very large scale integrated circuits (“VLSIs”), and the like. Examples of configurable hardware are: one or more programmable logic devices such as programmable array logic (“PALs”), programmable logic arrays (“PLAs”), and field programmable gate arrays (“FPGAs”). Examples of programmable data processors are: microprocessors, digital signal processors (“DSPs”), embedded processors, graphics processors, math coprocessors, general purpose computers, server computers, cloud computers, mainframe computers, computer workstations, and the like. For example, one or more data processors in a control circuit for a device may implement methods as described herein by executing software instructions in a program memory accessible to the processors.

[0158] Processing may be centralized or distributed. Where processing is distributed, information including software and / or data may be kept centrally or distributed. Such information may be exchanged between different functional units by way of acommunications network, such as a Local Area Network (LAN), Wide Area Network (WAN), or the Internet, wired or wireless data links, electromagnetic signals, or other data communication channel.

[0159] The invention may also be provided in the form of a program product. The program product may comprise any non-transitory medium which carries a set of computer-readable instructions which, when executed by a data processor, cause the data processor to execute a method of the invention. Program products according to the invention may be in any of a wide variety of forms. The program product may comprise, for example, non-transitory media such as magnetic data storage media including floppy diskettes, hard disk drives, optical data storage media including CD ROMs, DVDs, electronic data storage media including ROMs, flash RAM, EPROMs, hardwired or preprogrammed chips (e.g., EEPROM semiconductor chips), nanotechnology memory, or the like. The computer-readable signals on the program product may optionally be compressed or encrypted.

[0160] In some embodiments, the invention may be implemented in software. For greater clarity, “software” includes any instructions executed on a processor, and may include (but is not limited to) firmware, resident software, microcode, code for configuring a configurable logic circuit, applications, apps, and the like. Both processing hardware and software may be centralized or distributed (or a combination thereof), in whole or in part, as known to those skilled in the art. For example, software and other modules may be accessible via local memory, via a network, via a browser or other application in a distributed computing context, or via other means suitable for the purposes described above.

[0161] Software and other modules may reside on servers, workstations, personal computers, tablet computers, and other devices suitable for the purposes described herein.Interpretation of Terms

[0162] Unless the context clearly requires otherwise, throughout the description and the claims:• “comprise”, “comprising”, and the like are to be construed in an inclusive sense, as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”;• “connected”, “coupled”, or any variant thereof, means any connection or coupling, either direct or indirect, between two or more elements; the couplingor connection between the elements can be physical, logical, or a combination thereof;• “herein”, “above”, “below”, and words of similar import, when used to describe this specification, shall refer to this specification as a whole, and not to any particular portions of this specification;• “or”, in reference to a list of two or more items, covers all of the following interpretations of the word: any of the items in the list, all of the items in the list, and any combination of the items in the list;• the singular forms “a”, “an”, and “the” also include the meaning of any appropriate plural forms. These terms (“a”, “an”, and “the”) mean one or more unless stated otherwise;• “and / or” is used to indicate one or both stated cases may occur, for example A and / or B includes both (A and B) and (A or B);• “approximately” when applied to a numerical value means the numerical value ± 10%;• where a feature is described as being “optional” or “optionally” present or described as being present “in some embodiments” it is intended that the present disclosure encompasses embodiments where that feature is present and other embodiments where that feature is not necessarily present and other embodiments where that feature is excluded. Further, where any combination of features is described in this application this statement is intended to serve as antecedent basis for the use of exclusive terminology such as "solely," "only" and the like in relation to the combination of features as well as the use of "negative" limitation(s)” to exclude the presence of other features; and• “first” and “second” are used for descriptive purposes and cannot be understood as indicating or implying relative importance or indicating the number of indicated technical features.

[0163] Words that indicate directions such as “vertical”, “transverse”, “horizontal”, “upward”, “downward”, “forward”, “backward”, “inward”, “outward”, “left”, “right”, “front”, “back”, “top”, “bottom”, “below”, “above”, “under”, and the like, used in this description and any accompanying claims (where present), depend on the specific orientation of the apparatus described and illustrated. The subject matter described herein may assume various alternative orientations. Accordingly, these directional terms are notstrictly defined and should not be interpreted narrowly.

[0164] Where a range for a value is stated, the stated range includes all sub-ranges of the range. It is intended that the statement of a range supports the value being at an endpoint of the range as well as at any intervening value to the tenth of the unit of the lower limit of the range, as well as any subrange or sets of sub ranges of the range unless the context clearly dictates otherwise or any portion(s) of the stated range is specifically excluded. Where the stated range includes one or both endpoints of the range, ranges excluding either or both of those included endpoints are also included in the invention.

[0165] Certain numerical values described herein are preceded by "about". In this context, "about" provides literal support for the exact numerical value that it precedes, the exact numerical value ±5%, as well as all other numerical values that are near to or approximately equal to that numerical value. Unless otherwise indicated a particular numerical value is included in “about” a specifically recited numerical value where the particular numerical value provides the substantial equivalent of the specifically recited numerical value in the context in which the specifically recited numerical value is presented. For example, a statement that something has the numerical value of “about 10” is to be interpreted as: the set of statements:• in some embodiments the numerical value is 10;• in some embodiments the numerical value is in the range of 9.5 to 10.5; and if from the context the person of ordinary skill in the art would understand that values within a certain range are substantially equivalent to 10 because the values with the range would be understood to provide substantially the same result as the value 10 then “about 10” also includes:• in some embodiments the numerical value is in the range of C to D where C and D are respectively lower and upper endpoints of the range that encompasses all of those values that provide a substantial equivalent to the value 10

[0166] Specific examples of systems, methods and apparatus have been described herein for purposes of illustration. These are only examples. The technology provided herein can be applied to systems other than the example systems described above. Many alterations, modifications, additions, omissions, and permutations are possiblewithin the practice of this invention. This invention includes variations on described embodiments that would be apparent to the skilled addressee, including variations obtained by: replacing features, elements and / or acts with equivalent features, elements and / or acts; mixing and matching of features, elements and / or acts from different embodiments; combining features, elements and / or acts from embodiments as described herein with features, elements and / or acts of other technology; and / or omitting combining features, elements and / or acts from described embodiments.

[0167] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any other described embodiment(s) without departing from the scope of the present invention.

[0168] Any aspects described above in reference to apparatus may also apply to methods and vice versa.

[0169] Any recited method can be carried out in the order of events recited or in any other order which is logically possible. For example, while processes or blocks are presented in a given order, alternative examples may perform routines having steps, or employ systems having blocks, in a different order, and some processes or blocks may be deleted, moved, added, subdivided, combined, and / or modified to provide alternative or subcombinations. Each of these processes or blocks may be implemented in a variety of different ways. Also, while processes or blocks are at times shown as being performed in series, these processes or blocks may instead be performed in parallel, simultaneously or at different times.

[0170] Various features are described herein as being present in “some embodiments”. Such features are not mandatory and may not be present in all embodiments. Embodiments of the invention may include zero, any one or any combination of two or more of such features. All possible combinations of such features are contemplated by this disclosure even where such features are shown in different drawings and / or described in different sections or paragraphs. This is limited only to the extent that certain ones of such features are incompatible with other ones of such features in the sense that it would be impossible for a person of ordinary skill in the art to construct a practical embodiment that combines such incompatible features. Consequently, the description that “some embodiments” possess feature A and “some embodiments” possess feature B should be interpreted as an expressindication that the inventors also contemplate embodiments which combine features A and B (unless the description states otherwise or features A and B are fundamentally incompatible).This is the case even if features A and B are illustrated in different drawings and / or mentioned in different paragraphs, sections or sentences.

[0171] It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions, omissions, and sub-combinations as may reasonably be inferred. The scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

Claims

WHAT IS CLAIMED IS:1 . A spatial filter for a confocal microscope, the spatial filter comprising: a disk spinnable about a spinning axis in an optical path of the confocal microscope between a coherent light source providing an excitation light and a target, the spinnable disk comprising: a plurality of semi-annular sections, each semi-annular section shaped to define a corresponding section pinhole group comprising a plurality of pinholes arranged in a lattice pattern, each pinhole permitting transmission of light through the disk; wherein, for each semi-annular section, the corresponding section pinhole group is located to have a corresponding section offset along a section offset direction orthogonal to the spinning axis and having at least a component that extends radially from the spinning axis; wherein the section offset is different for at least two of the plurality of semi-annular sections.

2. The spatial filter according to claim 1 or any other claim herein wherein the section offset is different for each of the plurality of semi-annular sections.

3. The spatial filter according to any one of claims 1 to 2 wherein the section offsets for the at least two of the plurality of semi-annular sections is randomly selected.

4. The spatial filter according to any one of claims 1 to 2 wherein the section offsets for the at least two of the plurality of semi-annular sections is determined according to an optimization process which minimizes a cost function having at least one term that attributes cost that is positively correlated with variation in scanning intensity.

5. The spatial filter according to any one of claims 1 to 4 or any other claim herein wherein the section offset direction for each semi-annular section is radial.

6. The spatial filter according to claim 5 or any other claim herein wherein the section offset direction is a radial direction lying within the corresponding semiannular section.

7. The spatial filter according to claim 6 or any other claim herein wherein the section offset direction is angularly spaced from a reference edge for each semi-annular section and the angular section offset direction relative to the corresponding reference edge is the same for at least two semi-annular sections.

8. The spatial filter according to claim 7 or any other claim herein wherein the angular section offset direction extends along a radial direction that bisects the corresponding semi-annular section.

9. The spatial filter according to any of one of claims 1 to 8 or any other claim herein wherein, for each section pinhole group, the lattice pattern comprises a 2D array of pinholes, the 2D array extending in a pair of directions orthogonal to the spinning axis.

10. The spatial filter according to claim 9 or any other claim herein, the pair of directions orthogonal to the spinning axis are non-radial.11 . The spatial filter according to any one of claims 9 to 10 or any other claim herein wherein the 2D array of pinholes is defined by polygonal lattice units, with pinholes located at vertices of the polygonal lattice units.

12. The spatial filter according to claim 11 or any other claim herein wherein the polygonal lattice units are one of: triangular, quadrilateral, hexagonal.

13. The spatial filter according to any one of claims 11 to 12 or any other claim herein wherein the polygonal lattice units have polygonal edge lengths that are the same for each polygonal lattice units.

14. The spatial filter according to any one of claims 9 to 13 or any other claimherein wherein the 2D array of pinholes is defined by repeating lattice units, where neighboring lattice units share at least one edge.

15. The spatial filter according to any one of claims 1 to 9 or any other claim herein wherein, for each section pinhole group, the lattice pattern comprises a linear lattice of a plurality of linearly spaced apart pinholes.

16. The spatial filter according to claim 15 or any other claim herein wherein the linear lattice of a plurality of linearly spaced apart pinholes comprises a radial lattice of pinholes that are radially spaced apart in a radial direction extending from the spinning axis.

17. The spatial filter according to any one of claims 9 to 10 or any other claim herein wherein the 2D array of pinholes is defined by quadrilateral lattice units, with pinholes located at vertices of the quadrilateral lattice units.

18. The spatial filter according to any one of claims 9 to 10 or 15 or any other claim herein wherein the pair of directions orthogonal to the spinning axis are orthogonal to one another.

19. The spatial filter according to any one of claims 9-14, 17 or 18 or any other claim herein wherein the pinholes in the 2D array are separated by a spatial period parameter di in a first direction of the pair of directions and separated by a spatial period parameter c in a second direction of the pair of directions wherein di = d2.

20. The spatial filter according to any one of claims 1 to 19 or any other claim herein wherein the disk comprise opaque material at locations away from the pinholes and thereby blocks at least some out-of-focus signals in sampled light generated from an interaction between the excitation light and the target.21 . The spatial filter according to claim 20 or any other claim herein wherein an amount of the out-of-focus signals in the sampled light blocked by the disk is greater than 50%.

22. The spatial filter according to claim 19 or any other claim herein wherein each pinhole in the plurality of pinholes in the corresponding section pinhole group of each of the plurality of semi-annular sections has a common opening dimension w in a plane orthogonal to the spinning axis.

23. The spatial filter according to claim 22 or any other claim herein wherein the spatial parameters d1 , d2 and the opening dimension w provide an opening ratio w / d, wherein d = ch = cfe and the opening ratio is in a range of 0.2-0.5, preferably in a range of 0.3-0.4.

24. The spatial filter according to any one of claims 1 to 23 or any other claim herein wherein the semi-annular sections and their corresponding section pinhole groups are shaped to diffract the excitation light transmitted therethrough.

25. The spatial filter according to claim 24 or any other claim herein wherein the semi-annular sections and their corresponding section pinhole groups are shaped such that the diffracted excitation light transmitted through the disk interferes in a manner such that one 0th- order diffraction beam and a plurality of higher order diffraction beams impinge on a back pupil of an objective lens of the confocal microscope.

26. The spatial filter according to claim 25 or any other claim herein wherein the plurality of 1st-order diffraction beams comprises four 1st-order diffraction beams.

27. The spatial filter according to any one of claims 25 to 26 or any other claim herein wherein the semi-annular sections and their corresponding section pinhole groups are shaped such that the Oth-order diffraction beam and the plurality of higher order diffraction beams impinging on the back pupil of the objective lens interfere to produce a structured illumination pattern on a sampling surface of the target, the structured illumination pattern comprising spatially periodic illumination maxima.

28. The spatial filter according to claim 26 or any other claim herein wherein the structured illumination pattern comprises a 3D lattice illumination pattern comprising spatially periodic illumination maxima in both: one or more lateral directions orthogonal to the spinning axis; and in an axial direction that is parallel to the spinning axis.

29. The spatial filter according to claim 28 or any other claim herein wherein the 3D lattice illumination pattern has a contrast in a range of 0.7-0.95, preferably 0.75-0.9.

30. The spatial filter according to any one of claims 27 to 29 or any other claim herein wherein an autocorrelation of the Fourier transform of the structured illumination pattern exhibits 17 delta functions in a spatial frequency space.31 . The spatial filter according to any one of claims 1 -30 wherein each pinhole is displaced from its nominal location by a jitter offset, the jitter offset oriented in a jitter offset direction orthogonal to the spinning axis and a jitter offset amount less than 10% of at least one of: a separation distance between neighboring (e.g. adjacent) pinholes; and a maximum dimension of a 2D unit cell of the lattice pattern of its corresponding section pinhole group.

32. The spatial filter according to claim 31 or any other claim herein wherein, for each pinhole, at least one of the jitter offset amount and the jitter offset direction is randomly determined.

33. The spatial filter according to any one of claims 31 to 32 or any other claim herein wherein, for each pinhole, at least one of the jitter offset amount and the jitter offset direction is selected according to an optimization process, wherein the optimization process comprises a cost function having at least one term that attributes cost that is positively correlated with variation in scanning intensity.

34. The spatial filter according to any one of claims 1 to 33 or any other claimherein wherein the disk is locatable in the optical path of the confocal microscope with the spinning axis co-axial with an optical axis of the confocal microscope.

35. The spatial filter according to claim 34 or any other claim herein wherein the disk is locatable on a focal plane of an objective lens of the confocal microscope.

36. The spatial filter according to any one of claims 1 to 35 or any other claim herein wherein a scanning intensity resulting from the excitation light passing through the disk with section offsets is relatively uniform along radial directions that extend orthogonally from the spinning axis compared to a scanning intensity resulting from the excitation light passing through a disk without the section offsets.

37. A spatial filter for a confocal microscope, the spatial filter comprising: a disk spinnable about a spinning axis in an optical path of the confocal microscope between a coherent light source providing an excitation light and a target, the spinnable disk comprising: a plurality of pinholes arranged a 2D-latice pattern wherein, for each 2-D lattice pattern, each pinhole is located at the vertices of a 2D unit cell and wherein the 2D-lattice pattern comprises a plurality of repeating 2D unit cells, each pinhole permitting transmission of light through the disk; a mechanism for vibrating the disk in directions orthogonal to the spinning axis.

38. A spatial filter according to claim 37 comprising any of the features, combinations of features and / or sub-combinations of features of any of the other claims recited herein.

39. A spatial filter for a confocal microscope, the spatial filter comprising: a disk spinnable about a spinning axis in an optical path of the confocal microscope between a coherent light source providing an excitation light and a target, the spinnable disk comprising:a plurality of pinholes arranged in at least one 2D-latice pattern wherein, for each 2-D lattice pattern, each pinhole is nominally located at the vertices of a 2D unit cell and wherein the 2D-lattice pattern comprises a plurality of repeating 2D unit cells, each pinhole permitting transmission of light through the disk; and wherein each pinhole is displaced from its nominal location by a jitter offset, the jitter offset oriented in a jitter offset direction orthogonal to the spinning axis and a jitter offset amount less than 10% of at least one of: a separation distance between neighboring (e.g. adjacent) pinholes; and a maximum dimension of the 2D unit cell.

40. The spatial filter according to claim 39 or any other claim herein wherein, for each pinhole, at least one of the jitter offset amount and the jitter offset direction is randomly determined.41 . The spatial filter according to any one of claims 39 to 40 or any other claim herein wherein, for each pinhole, at least one of the jitter offset amount and the jitter offset direction is selected according to an optimization process, wherein the optimization process comprises a cost function having at least one term that attributes cost that is positively correlated with variation in scanning intensity.

42. A spatial filter according to any one of claims 49 to 41 comprising any of the features, combinations of features and / or sub-combinations of features of any of the other claims recited herein.

43. A method for confocal microscopy, the method comprising: locating the spatial filter of any one of claims 1 to 42 or any other claim herein in the optical path of a microscope system between a coherent light source and a target; spinning the disk about a spinning axis that is coaxial with an optical axis of the microscope; illuminating the target with the excitation light through the disk; and, receiving sampled light generated from an interaction between theexcitation light and the target at a photodetector.

44. The method of claim 43 or any other claim herein, wherein the microscope system comprises an objective lens and the disk is located on a focal plane of the objective lens.

45. The method of any one of claims 43 to 44 comprising method steps corresponding to any feature, combination of features or sub-combination of features described or claimed herein.

46. A confocal microscope comprising the spatial filter of any one of claims 1 to 42.

47. Apparatus having any new and inventive feature, combination of features, or sub-combination of features as described herein.

48. Methods having any new and inventive steps, acts, combination of steps and / or acts or sub-combination of steps and / or acts as described herein.

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