Apparatus and method for performing confocal microscopy

Abstract

According to embodiments of the present invention, an apparatus for performing confocal microscopy is provided. The apparatus includes an optical arrangement including one or more optical components configured to direct light reflected from a sample under observation; and an imaging sensor configured to capture an image of the sample based on at least part of the reflected light. The optical arrangement further includes a pinhole element configured to spatially filter the reflected light. The imaging sensor and the pinhole element are configured to simultaneously tilt away from a plane perpendicular to an optical axis of the optical arrangement in a matched manner such that the captured image has optimal resolution and image quality in terms of glare or reflection suppression and reduced imaging distortion. According to further embodiments of the present invention, a method for performing confocal microscopy is also provided.

Description

APPARATUS AND METHOD FOR PERFORMING CONFOCAL MICROSCOPYCross-Reference To Related Application

[0001] This application claims the benefit of priority of Singapore patent application No. 10202500783Q, filed 26 March 2025, the content of it being hereby incorporated by reference in its entirety for all purposes.Technical Field

[0002] Various embodiments relate to an apparatus and a method for performing confocal microscopy.Background

[0003] Confocal microscopy is an advanced imaging technique that can provide three-dimensional surface profile of a specimen. The 3D optical section capability is achieved using a small pinhole, as a spatial filter, to block the unwanted stray light or reflection from upper or lower layers of targeted sample surface enter the optical sensor. Confocal microscopes scan the sample along all x, y, and z dimensions, to construct the 3D surface point cloud. To increase the scanning speed, a spinning disk with thousands of pinholes arranged in 12 to 15 spirals pattern may be introduced. It is equivalent to using multiple pinholes scan the large sample surface in thex-y plane simultaneously. As the disk spinning speed may be very high, e g. more than 10000 revolutions per second, a spinning disk confocal microscope may obtain much higher, up to 10 to 100 times, imaging speed compared to confocal microscopes using other scanning mechanism such as galvanometer. Scanning in the z direction is achieved by vertical stacking of the images. Furthermore, in chromatic confocal microscopy case, dispersion of white light may also be used to implement z-axis scanning.

[0004] A typical chromatic confocal microscope structure may include a laser source configured to emit light through a focusing lens for focusing the light through at least afirst confocal pinhole towards an objective lens that direct the light onto an in-focus plane and several out-of-focus planes. Due to the in-focus plane and out-of-focus planes, the light is reflected along different paths through the objective lens and directed, by a beam splitter, towards at least a second confocal pinhole before reaching a detector or a camera The laser, the first confocal pinhole, the beam splitter, the objective lens, the in-focus plane and out-of-focus planes may be arranged along an optical axis. The second confocal pinhole and the detect or / camera may be placed perpendicularly to the optical axis

[0005] In a chromatic confocal microscope, a tilted spinning Nipkow disk is typically used to reduce glare and back-reflection but restricts the Depth of field (DOF) of the camera sensor.

[0006] For example in one publication, a tilted disk configuration was explored, but as in traditional confocal microscopy, the camera remained perpendicular to the optical axis. To address the back reflection issue, wedge prisms were used to redirect the stray light away from the path back to the sensor. Although this may be a valid way to reduce the tilt of the disk and increase the portion of disk that falls within the focal plane of the camera, this is an imperfect solution. If needed to increase the scale of capture (Field-of-View, FOV) and / or a use case with a sensor with smaller DOF, this solution falls apart as the edges of the disk remain out of the focal plane of the camera.

[0007] In another publication that described other forms of tackling the back reflection issue, a setup was provided aiming to have an angle offset for the light source, combined with the tilt of the disk, to block the reflected excited light by the beam stop. This method has the inherent case of a possible DOF mismatch with the disk tilt plane. More specifically, two ends of the Nipkow disk, for both the output light path and the input light source path, were used - effectively equivalent to using two Nipkow disks for either path. This reduces the back reflection effect, however, this design is mechanically and optically complex, with synchronization issues. The image capture resulted in scan lines being evident, obscuring the sample details - possibly due to de-synchronization of the two paths.

[0008] Existing solutions may suffer from one or more of the following drawbacks: - • a poor signal to noise ratio due to the existence of strong glare effect and being applicable to samples with high reflectivity, especially in ultra-high resolution confocal microscope where a relatively small pinhole size is used;• using a tilted disk along the optical axis may suppress the glare, however this also leads to degradation of the image quality in terms of reduction of image FOV, contrast, resolution and homogeneity;• requiring use of additional optics components, such as prism pairs, to partially compensate for the image degradation, hence increasing optical loss, while still facing FOV reduction and image deformation,• inducing problems such as chromatic lateral shift, as well as extremely critical and difficult optical alignment.[0009J Thus, there is a need for a method and / or system to address at least the problems mentioned above, thereby overcoming the DOF limitation while preserving the benefits of reflection reduction from the tilted disk.Summary

[0010] According to an embodiment, an apparatus for performing confocal microscopy is provided. The apparatus includes an optical arrangement including one or more optical components configured to direct light reflected from a sample under observation; and an imaging sensor configured to capture an image of the sample based on at least part of the reflected light, wherein the optical arrangement further includes a pinhole element configured to spatially filter the reflected light, and wherein the imaging sensor and the pinhole element are configured to simultaneously tilt away from a plane perpendicular to an optical axis of the optical arrangement in a matched manner such that the captured image has optimal resolution and image quality in terms of glare or reflection suppression and reduced imaging distortion.

[0011] According to an embodiment, a method for performing confocal microscopy is provided. The method includes directing, by an optical arrangement, light reflected from a sample under observation, spatially filtering, by a pinhole element of the optical arrangement, the reflected light; and capturing, by an imaging sensor, an image of the sample based on at least part of the reflected light. The imaging sensor and the pinhole element are simultaneously tilted away from a plane perpendicular to an optical axis of the optical arrangement in a matched manner such that the captured image has optimalresolution and image quality in terms of glare or reflection suppression and reduced imaging distortion.Brief Description of the Drawings

[0012] In the drawings, like reference characters generally refer to like parts throughout the different views. The drawings are not necessarily to scale, emphasis instead generally being placed upon illustrating the principles of the invention. In the following description, various embodiments of the invention are described with reference to the following drawings, in which:

[0013] FIG. 1 shows a schematic view of an apparatus for performing confocal microscopy, according to various embodiments.

[0014] FIG. 2A shows a schematic view illustrating a setup based on Scheimpflug principle by using one photographic lens with a Nipkow disk and an imaging sensor, according to one example in the simplest form.

[0015] FIG. 2B shows a schematic view of an imaging setup based on Scheimpflug principle by using two photographic lens with a Nipkow disk and an imaging sensor, according to one example.

[0016] FIG. 2C shows a schematic view illustrating an imaging optic path based on the imaging setup provided by the apparatus of FIG. 1, according to various embodiments.

[0017] FIG. 3 shows a flow chart illustrating a method for performing confocal microscopy, according to various embodiments.

[0018] FIG. 4A shows an image of the Nipkow disk captured from an existing chromatic confocal microscope, according to an example.

[0019] FIG 4B shows the image of FIG. 4A, with a gradual defocus vertically on the image.

[0020] FIG. 5 shows a non-tilted camera image of still (non-spinning) Nipkow disk, along with three selected zoomed-in segments, according to an example.

[0021] FIG. 6A shows an image of a flat reference sample at an object plane that is located after (downstream from) the Nipkow disk, captured from the existing chromatic confocal microscope, according to an example.

[0022] FIG. 6B shows the image of FIG. 6 A, with a gradual defocus across the axis of tilt.

[0023] FIG. 7 shows a schematic view illustrating a ray diagram for an image from the surface of the Nipkow disk to the imaging lens / camera, with focal plane mismatch to marginal rays, according to an example.

[0024] FIG. 8 shows a photograph of a first (conventional) setup constructed with a Jenoptik colour camera, a lx zoom lens, and an Edmund optics DOF target tilted away from the camera sensor plane, according to an example.

[0025] FIG. 9A shows a photograph of a second setup constructed to compare the effect of tilting the camera, according to an example.

[0026] FIG. 9B shows the photograph of the second setup of FIG. 9A with a clear representation of the different planes in the second setup

[0027] FIG. 10A shows an image capture of the non-tilted camera setup of a DOF target, according to an example.

[0028] FIG. 10B shows an image capture of the tilted camera setup of the DOF target, according to an example.

[0029] FIG. 11 shows a profile across 20 Ip / mm band on the DOF target, according to an example.

[0030] FIG 12A shows an image capture of the non-tilted camera setup of a DOF target, with a profile line drawn across the 20 Ip / mm band of the DOF target, according to an example.

[0031] FIG. 12B shows an image capture of the tilted camera setup of the DOF target, with a profile line, drawn across the 20 Ip / mm band of the DOF target, according to an example.

[0032] FIG. 13A and FIG. 13B show photographs respectively depicting a front view and a side view of a non-tilted setup of a Zeiss Axiocam 305 camera, mounted directly to an imaging lens via c-mount, according to an example.

[0033] FIG. 14A and FIG. 14B show photographs respectively depicting a front view and a side view of a tilted setup of the Zeiss Axiocam 305 camera, mounted via a monitor arm 1401, according to an example.

[0034] FIG. 15 shows a tilted camera image of still (non-spinning) Nipkow disk (on the left), and three selected zoomed-in segments (enlarged views on the right), according to an example.

[0035] FIG. 16A shows a 50pm grid patern captured on a chromatic confocal microscope - non-tilted camera, according to an example.

[0036] FIG. 16B shows a 50pm grid pattern captured on a chromatic confocal microscope - tilted camera, according to an example.

[0037] FIG. 17A shows a 50pm grid patern captured on a chromatic confocal microscope - non-tilted camera, with reference lines for plot profiling of 13 rows and 16 columns, according to an example.

[0038] FIG. 17B shows a 50pm grid pattern captured on a chromatic confocal microscope - tilted camera, with reference lines for plot profiling of 13 rows and 16 columns, according to an example.

[0039] FIG 18A and FIG. 18B show plot profiles of row 8 (x-direction) of the non-tilt capture and the tilt capture of 50pm grid captures, respectively, according to various examples.

[0040] FIGS. 19A to 19C show FWHM measurements for various grid line rows (horizontal) of non-tilt capture and tilt capture of 50pm grid capture, according to various embodiments.

[0041] FIGS. 20A to 20C show FWHM measurements for various grid lines columns (vertical) non-tilt capture and tilt capture of 50pm grid capture, according to various embodiments.

[0042] FIG. 21 shows a schematic perspective view of a 4-axis (x, y, z and tilt) translation stage designed and fabricated for the disk, according to an example.

[0043] FIG. 22A shows a schematic perspective view of a 3D printed fixed angle mount for sensor, according to an example.

[0044] FIG. 22B shows a schematic perspective view of a 2-axis translation mount for sensor, according to an example.Detailed Description

[0045] The following detailed description refers to the accompanying drawings that show, by way of illustration, specific details and embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled inthe art to practice the invention. Other embodiments may be utilized and structural, logical, and electrical changes may be made without departing from the scope of the invention. The various embodiments are not necessarily mutually exclusive, as some embodiments can be combined with one or more other embodiments to form new embodiments.

[0046] Embodiments described in the context of one of the methods or devices are analogously valid for the other methods or devices. Similarly, embodiments described in the context of a method are analogously valid for a device, and vice versa.

[0047] Features that are described in the context of an embodiment may correspondingly be applicable to the same or similar features in the other embodiments. Features that are described in the context of an embodiment may correspondingly be applicable to the other embodiments, even if not explicitly described in these other embodiments. Furthermore, additions and / or combinations and / or alternatives as described for a feature in the context of an embodiment may correspondingly be applicable to the same or similar feature in the other embodiments.

[0048] In the context of various embodiments, the articles “a”, “an” and “the” as used with regard to a feature or element include a reference to one or more of the features or elements.

[0049] In the context of various embodiments, the phrase “at least substantially” may include “exactly” and a reasonable variance

[0050] In the context of various embodiments, the term “about” or “approximately” as applied to a numeric value encompasses the exact value and a reasonable variance.

[0051] As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0052] As used herein, the phrase of the form of “at least one of A or B” may include A or B or both A and B. Correspondingly, the phrase of the form of “at least one of A or B or C”, or including further listed items, may include any and all combinations of one or more of the associated listed items.

[0053] As used herein, the expression “configured to” may mean “constructed to” or “arranged to”.

[0054] Various embodiments provide glare reduction and Field-of-View (FOV) enhancement in confocal microscope, utilising tilted spinning disk and sensor.

[0055] FIG. 1 shows a schematic view of an apparatus 100 for performing confocal microscopy, according to various embodiments. The apparatus 100 includes an optical arrangement 102 including one or more optical components 104 configured to direct light reflected from a sample under observation; and an imaging sensor 106 configured to capture an image of the sample based on at least part of the reflected light. The optical arrangement 102 further includes a pinhole element 108 configured to spatially filter the reflected light. The imaging sensor 106 and the pinhole element 108 are configured to simultaneously tilt away from a plane perpendicular to an optical axis of the optical arrangement 102 in a matched manner such that the captured image has optimal resolution and image quality in terms of glare or reflection suppression and reduced imaging distortion. The optical arrangement 100 is in optical communication with the imaging sensor 106, as denoted by a line 110. The one or more optical components 104 is in optical communication with the pinhole element 108, as denoted by a line 112.

[0056] In the context of various examples, the optical axis of the optical arrangement 102 defines the path along which the light propagates through the one or more optical components 104 of the optical arrangement 102.

[0057] Having optimal resolution and image quality in terms of glare or reflection suppression and reduced imaging distortion leads to a larger effective FOV of the captured image.

[0058] In various embodiments, in the matched manner, the imaging sensor 106 may be configured to tilt away at a first angle from the plane perpendicular to the optical axis and the pinhole element 108 may be configured to tilt away at a second angle from the plane perpendicular to the optical axis, the first angle and the second angle satisfying Scheimpflug principle.

[0059] In other embodiments, the imaging sensor 106 may be located at a conjugate plane of the pinhole element 108; and in the matched manner, the imaging sensor 106 may be configured to tilt away at a first angle from the plane perpendicular to the optical axis and the pinhole element 108 may be configured to tilt away at a second angle from the conjugate plane, the first angle and the second angle satisfying Scheimpflug principle. The conjugate plane of the pinhole element may be defined based on the pinhole element 108at a non-tilted position in a conjugated configuration. The conjugated plane may be perpendicular to the optical axis in the conjugated configuration.

[0060] In other words, the apparatus 100 involves the imaging sensor 106 (e.g. a camera) being located at the conjugate plane of the pinhole element 108 (e g. involving a Nipkow Disk), and the pinhole element 108 being located at the conjugate plane of the sample surface. By implementing Scheimpflug principle, involving the tilt of the camera, the DOF may be effectively increased, providing DOF enhancement. This ensures that the entire tilted disk and the complete sample surface within its view are consistently in focus at all areas of the image. This possibly leads to enhanced image FOV for the chromatic confocal microscope.

[0061] It should be appreciated that the proposed technology basically aims to integrate Scheimpflug principle into the image capture process of the sample surface. Based on Scheimpflug principle, the imaging sensor 106 is tilted at the first angle in an opposite direction of the pinhole element 108 tilted at the second angle. This avoids the reflection of the illumination light from pinhole element surface propagating back to the imaging sensor 106. The tilting of the imaging sensor 106 serves as an image conjugate for both the tilted pinhole element 108 and the sample surface, in the opposite direction of the intersection point of these image conjugates.

[0062] FIG. 2A shows a schematic view illustrating a setup 201 based on Scheimpflug principle by using one photographic lens 203 with a Nipkow disk 208 and an imaging sensor 206, according to one example in the simplest form. The lens 203 is positioned along a lens plane 203’. The Nipkow disk 208 is positioned and tilted parallel along a subject plane 208’. The imaging sensor 206 is positioned parallel along an image plane 206’. An optical axis 205 is defined perpendicular to the lens plane 203’. The Nipkow disk 208, the lens 203 and the imaging sensor 206 are arranged or adjusted relative to one another such that the lens plane 203’, the subject plane 208’ and the image plane 206’ intersect at a Scheimpflug intersection 207.

[0063] FIG. 2B shows a schematic view of an imaging setup 211 based on Scheimpflug principle by using two photographic lens 213a, 213b with a Nipkow disk 208 and an imaging sensor 206, according to one example. The imaging setup 211 is a variant of the setup 201 of FIG 2A Specifically, FIG. 2B illustrates a more general and commonly usedimaging setup 211 depicting the Scheimpflug intersection arrangement for two lens infinite imaging scenario. The imaging setup 211 may include the same or like elements or components as those relevant parts of the apparatus 100 of FIG. 1, and as such, the same ending numerals are assigned and the like elements may be as described in the context of those relevant parts of the apparatus of FIG. 1, and therefore the corresponding descriptions may be omitted here. As seen in FIG. 2B, the subject plane 208’ is tilted away from a front focal plane 219 of the photographic lens 213a, while the image plane 206” is tilted away from a back focal plane 221 of the photographic lens 213b. The tilted subject plane 208’ and the tilted image plane 206” converge towards a Scheimpflug intersection 217.

[0064] The tilts shown in FIGS. 2A and 2B extend the DOF of the imaging sensor 206 in the z direction (i.e. along the optical axis 205). This DOF increases at the subject plane 208’ then translates to an increased DOF after magnification at the sample surface (not shown in the figures), leading to imaging resolution, contrast, and homogeneity enhancement at the outer edges of the sample surface. Image quality enhancement and imaging homogeneity enhancement are achieved.

[0065] In various embodiments, the apparatus 100 may further include a fixed mount coupled to the imaging sensor 106. The fixed mount may be designed in a manner to secure the imaging sensor 106 at the first angle. The fixed mount may be fabricated using 3D printing, or any other suitable fabrication techniques.

[0066] In various embodiments, the apparatus 100 may further include an adjustable mount coupled to the imaging sensor 106. The adjustable mount may be designed in a manner to adjust and releasably lock the imaging sensor 106 at the first angle.

[0067] For example, the adjustable mount may include a 2-axis translation mount.

[0068] In various embodiments, the apparatus 100 may further include a tilt stage configured to translate the imaging sensor 106 in a direction along a rotational axis of the pinhole element. In this context, the term “translate” refers to adjust or manipulate.

[0069] The pinhole element 108 may be coupled to a 4-axis translation mount operable to tilt the pinhole element 108 to the second angle, and adjust the pinhole element 108 along three orthogonal axes, one parallel to the optical axis and two orthogonal to the optical axis

[0070] The imaging sensor 106 may be configured to tilt along the three orthogonal axes. In other words, the imaging sensor may be tilted in three dimensions.

[0071] In various embodiments, the apparatus 100 may further include a substrate arranged to hold the sample. The one or more optical components 104 may further include a lens arrangement positioned between the pinhole element 108 and the substrate. The substrate may be configured to tilt at a third angle away from an object plane perpendicular to an optical axis of the lens arrangement in the matched manner, the second angle being proportional to the third angle by a magnification power (or factor), M measured between the pinhole element 108 and the substrate.

[0072] The substrate may be coupled to or may include a sample mount operable to tilt the substrate to the third angle.

[0073] In various embodiments, the apparatus 100 may further include an imaging lens configured to focus the spatially filtered reflected light onto the imaging sensor 106. The imaging sensor 106 may be configured to tilt away from the imaging lens. The imaging lens and the imaging sensor 106 may be parts of a camera optically coupled to the optical arrangement 102. Alternatively, the camera may be a typical camera with the imaging lens interface modified. The imaging lens may have a magnification power (or factor), M.

[0074] The imaging sensor 106 may include a charged-couple device (CCD) array. Other types of imaging sensor may include frame image sensor arrays with flat surface (such as Complementary Metal-Oxide Semiconductor (CMOS) sensors) and image sensor arrays with curved surface. For an image sensor array with curved surface, specially designed lens may be used to match the curvatures of the image plane and the image sensors of the image sensor array.

[0075] The pinhole element 108 may include a pinhole mask coupled to a spinning motor. For example, the pinhole mask may be a Nipkow disk.

[0076] In various embodiments, the apparatus 100 may further include a controller configured to adjust the imaging sensor 106 and the pinhole element 108 tilting away from the plane perpendicular to the optical axis in the matched manner. The controller may further be configured to adjust the substrate tilting away from the object plane in the matched manner. The controller may be operated manually or automatically.

[0077] FIG. 3 shows a flow chart illustrating a method 320 for performing confocal microscopy, according to various embodiments. As shown in FIG. 3, at Step 322, light reflected from a sample under observation is directed by an optical arrangement (e.g. 102 of FIG 1). At Step 328, the reflected light is spatially filtering by a pinhole element (e.g.108 of FIG. 1) of the optical arrangement. At Step 326, an image of the sample is captured, by an imaging sensor (e.g. 106 of FIG. 1), based on at least part of the reflected light. The imaging sensor 106 and the pinhole element 108 are simultaneously tilted away from a plane perpendicular to an optical axis of the optical arrangement 102 in a matched manner such that the captured image has optimal resolution and image quality in terms of glare or reflection suppression and reduced imaging distortion.

[0078] The method 320 may include the same or like elements or components as those of the apparatus 100 of FIG. 1, and as such, the same numerals are assigned and the like elements may be as described in the context of the apparatus 100 of FIG. 1, and therefore the corresponding descriptions are omitted here.

[0079] In various embodiments, in the matched manner, the imaging sensor 106 may be tilted away at a first angle from the plane perpendicular to the optical axis and the pinhole element 108 may be tilted away at a second angle from the plane perpendicular to the optical axis, the first angle and the second angle satisfying Scheimpflug principle.

[0080] In other embodiments, the imaging sensor 106 may be located at a conjugate plane of the pinhole element 108; and in the matched manner, the imaging sensor 106 may be tilted away at a first angle from the plane perpendicular to the optical axis and the pinhole element 108 may be tilted away at a second angle from the conjugate plane, the first angle and the second angle satisfying Scheimpflug principle.

[0081] For example, the first angle may range from 0° to 45°, or preferably 6° to 45°. The second angle may range from 0° to 45°, or preferably 6° to 45°.

[0082] It should be appreciated that for the apparatus 100 and the method 320 described herein, the angle of 6° may be a minimal angle needed to avoid pinhole element (Nipkow disk) reflection back to the imaging sensor 106 (or camera). For other system design with smaller illumination light beam diameter or with lens having a longer focal length, the minimal angle may be smaller (i.e. less than 6°) and approach to but not reach 0°. Themaximal angle is about 45°. The range of 0° to 45° may be dependent on the illumination beam diameter and lens focal length.

[0083] In one embodiment, in the matched manner, the imaging sensor 106 may be secured at the first angle by a fixed mount coupled to the imaging sensor 106.

[0084] In another embodiment, in the matched manner, the imaging sensor 106 may be adjusted and releasably locked at the first angle by an adjustable mount coupled to the imaging sensor 106, and the imaging sensor 106 may be translated in a direction along a rotational axis of the pinhole element 108 by a tilt stage.

[0085] The pinhole element 108 may be tilted to the second angle and may be adjusted along three orthogonal axes, one parallel to the optical axis and two orthogonal to the optical axis and to each other, by a 4-axis translation mount coupled to the pinhole element 108. The imaging sensor 106 may be tilted along the three orthogonal axes.

[0086] In various embodiments, the method 320 may further include providing a substrate to hold the sample. The substrate may be tilted at a third angle away from an object plane perpendicular to an optical axis of a lens arrangement of the one or more optical components (e.g. 104 of FIG. 1) in the matched manner, the lens arrangement being positioned between the pinhole element 108 and the substrate, and the second angle being proportional to the third angle by a magnification power (or factor), M measured between the pinhole element 108 and the substrate.

[0087] The substrate may be tilted to the third angle by a sample mount coupled to the substrate. The substrate may include a sample mount tilted to the third angle. The third , , i i i iangle may be calculated b.

[0088] In various embodiments, the method 320 may further include prior to capturing the image at Step 326, focusing, by an imaging lens, the spatially filtered reflected light onto the imaging sensor 106. The imaging sensor 106 may be tilted away from the imaging lens. The imaging lens may have a magnification power (or factor), M.

[0089] The first angle and the second angle may be identical when M is equal to 1. In other words, M=1 from the pinhole element 108 (e.g. involving a Nipkow disk) to the image sensor 106.

[0090] The first angle and the second angle may be different when M is not equal to 1.

[0091] In various embodiments, spatially filtering the reflected light at Step 328 may include spinning the pinhole element 108 by a spinning motor coupled to the pinhole element 108.

[0092] Tn various embodiments, the method 320 may further include adjusting, by a controller, the imaging sensor 106 and the pinhole element 108 to tilt away from the plane perpendicular to the optical axis in the matched manner.

[0093] While the method described above is illustrated and described as a series of steps or events, it will be appreciated that any ordering of such steps or events are not to be interpreted in a limiting sense. For example, some steps may occur in different orders and / or concurrently with other steps or events apart from those illustrated and / or described herein. In addition, not all illustrated steps may be required to implement one or more aspects or embodiments described herein. Also, one or more of the steps depicted herein may be carried out in one or more separate acts and / or phases.

[0094] The apparatus 100 and the method 320 aim at reducing glare and enhancing FOV. Both the imaging sensor 106 (camera) and the pinhole element 108 (Nipkow disk) may be tilted in collective means with matched angles with respect to the optical axis of the apparatus 100. Non-tilted Nipkow disk leads to non-tilted camera.

[0095] With reference made to the apparatus 100 and the method 320, the Nipkow disk is tilted. This objective is different from existing systems where the Nipkow disk is not tilted to enlarge the image depth or compensate the tangential speed difference of objects located at different radius of the sample e.g. a wafer.

[0096] It should also be appreciated that not only the camera and the Nipkow disk are tilted, the sample plane (i.e. the substrate) is also tilted. The sample plane may be tilted away from the object plane. However, the tilt angle of the sample plane may be about 1 / M time of the tilt angle of the Nipkow disk, where M is the magnification power from the sample plane to the Nipkow disk plane.

[0097] In some examples, the camera, the Nipkow disk and the sample plane may be tilted in matched manners. For microscope with large M (magnification) number (or power or factor), the tilt angle of object plane (or in another word, the sample mounting plane) may be neglected. For example, for M>20, the tilt angle is small and has neglectable impact for the application described herein. But for low M or low magnification microscope design,such tilt of the sample plane also needs to be included in the design. Also, if the tilting angles of all three planes (camera, Nipkow disk and sample) are not matched with each other, e.g. one such angle is derived from proper value significantly, it may lead to opposite effects as expected. Hence, the matched manners are not mere routine design changes and the enhanced performance of the apparatus 100 and the method 320 cannot be achieved in any obvious manner.

[0098] The image quality reaches maximum value at matched angles. The optimal angle at which the Nipkow disk is tilted may be determined to maximize the DOF at the sample plane. This angle may be then translated to the camera imaging plane surface to match this angle prior to the imaging plane. This way, the defocus may be improved with no change to the sample or the disk. The sign of the angle deviation from its optimal value only changes the sign of optical aberration. The absolute value of optical aberration continuously increases with the angle derivation.

[0099] Accordingly, the angles of all tilted optics elements require sophistic control with certain accuracy to achieve targeted performance. Random or over-tuned angles degrade the system performance rather than enhance it. In order to guarantee the accuracy of the tuning mechanism, the Nipkow disk may be mounted on a 4-axis (x, y, z and tilt) translation mount to achieve the required disk tilting and the camera / sensor tilt mount may be attached to a tilt stage to allow for translation in the direction along the axis of rotation of the Nipkow disk. To the best of the inventors’ knowledge, no existing systems, where no accurate tilt angle control was applied, require at least these two features.

[0100] FIG. 2C shows a schematic view illustrating an imaging optic path based on the imaging setup provided by the apparatus of FIG. 1, according to various embodiments. As shown in FIG. 2C, 01 is the tilt angle of the sample plane 215’ (i.e. the third angle) with respect to the object plane 215, 02 is the tilt angle of the Nipkow disk (i.e. the second angle), and 03 is the tilt angle of the camera (i.e. the first angle), fl is the focal length of lens LI in relation to the conjugate plane of the Nipkow disk 208b. fl’ is the focal length of lens LI’ in relation to the camera plane 206’ . f2 is the focal length of lens L2 in relation to the sample plane 215’. f2’is the focal length of lens L2’ in relation to the Nipkow disk plane 208a. The various tilt angles may have the following relationships, as seen in Equation (1) and Equation (2):-Tan02 = Mtan01 - Equation (1)Tan03 = M’ tan02 - Equation (2)where M is the magnification power between the Nipkow disk plane 208a and the sample plane 2015’, provided by M =and M’ is the magnification power between the camera plane 206’ and the Nipkow disk plane 208a, provided by M' = ^7.

[0101] Examples of the apparatus 100 (FIG. 1) and the method 320 (FIG. 3) will be described in more details below.|0102| Intention

[0103] Chromatic confocal microscope has similar structure except a chromatic aberration lens is inserted in the optical path to generate required chromatic dispersion, A chromatic confocal microscope was used as the experimental basis in this work. However, the results obtained in this work also can be applied to its traditional counterpart. The Nipkow disk (which acts as the pinhole mask) is mounted on a spinning motor. Although the disk surface usually has anti-reflection coating, the backward reflection of the illumination to the camera still causes very strong glare. This reflection / glare may be ten times higher than the image signal, depending on the pinhole size and reflectivity of the sample. Hence, to suppress the strong glare, the Nipkow disk is tilted at a small angle, about 6-10°, to avoid the back reflection entering the camera or sensor. However, this tilt makes the edge of the Nipkow disk fall outside the DOF of the detection of the camera. Leading to the resultant image being blurry at the outer edges of sample, effectively reducing field of view of the system.[0104| The evident effect of tilting the Nipkow disk leads to a mismatch of DOF to the image plane on the disk. FIG. 4A shows an image of the Nipkow disk captured from an existing chromatic confocal microscope It can be observed that FIG. 4B shows the image of FIG. 4 A, with a gradual defocus vertically on the image - this direction of defocus also relates to the direction across the axis of tilt of the Nipkow disk. FIG. 5 shows a non-tilted camera image of still (non-spinning) Nipkow disk (on the left), and three selected zoomedin segments (enlarged views on the right).

[0105] FIG. 6 A shows an image of a flat reference sample at an object plane that is located after (downstream from) the Nipkow disk, captured from the existing chromatic confocal microscope. It can be observed that FIG. 6B shows the image of FIG. 6A, with a gradual defocus across the axis of tilt.

[0106] This phenomenon occurs due to the mismatch of the focal plane of the camera / imaging lens and the surface of the Nipkow disk. FIG. 7 shows a schematic view illustrating a ray diagram 731 for an image from the surface of the Nipkow disk 708 to the imaging lens 713 / camera 706, with focal plane 735 mismatch to marginal rays. The Nipkow disk is mounted on an adjustable mount 737 and is tilted at an angle with respect to an axis 739. The focal plane 735 is angled relative to the surface of the Nipkow disk 708, causing the rays 733a, 733c to be focused above and below the focal plane 735, respectively. The rays 733b is focused on the focal plane 735. The thickness of the focal plane 735 may be determined from the DOF of the camera 706 and the imaging lens 713. In some cases, if the DOF is large enough, this tilt effect may be negligible to the final image.

[0107] The following section (Preliminary Studies and Test) further describes the testing of this problem and the proposal of addressing it by utilising Scheimpflug principle at sensor level by tilting the camera irrespective of the imaging lens, such that the focal plane matches the tilt of the Nipkow disk surface as much as possible. This way, the DOF may be effectively increased.

[0108] Preliminary Studies and Test

[0109] FIG. 8 shows a photograph of a first (conventional) setup 801 constructed with a Jenoptik colour camera 806, a lx zoom lens 813, and an Edmund optics DOF target 815 tilted away from the camera sensor plane by 10°. The setup 801 is to replicate the first image conjugate path of a confocal microscope (camera sensor to tilted disk).

[0110] Redesigning conventional camera sensor - imagine lens interface to allow tilt is unconventional when compared to industry solutions and standards. This inherently affects the optical path of the system at the root instead of at the sample surface.

[0111] FIG. 9A shows a photograph of a second setup 901 constructed to compare the effect of tilting the camera 806. The imaging lens 81 in this case is mounted to the camera806 using a 3D printed replacement C-Mount piece 941 which tilts the camera 806 at a 10° angle to the imaging lens 813. The second setup 901 may be described in similar context with the apparatus 100 of FIG. 1 , and as such, the same ending numerals are assigned and the like elements may be as described in the context of the apparatus 100 of FIG 1 , and therefore the corresponding descriptions are omitted here.

[0112] FIG. 9B shows the photograph of the second setup 901 with a clear representation of the different planes (optical axis 905, image plane 906’, imaging lens plane 913, object plane 915) in the second setup 901.

[0113] After capture of the target using both the non-tilted and tilted setup, the images were analysed using ImageJ software. FIG. 10A shows an image capture 1001 of the nontilted camera setup of a DOF target, according to an example. FIG 10B shows an image capture 1003 of the tilted camera setup of the DOF target, according to an example. A difference in illumination homogeneity was observed between the two setups. The tilted setup has a vignette toward the right of the image. This is attributed to a mismatch of the centre of the sensor to the centre of the aperture of the 3D printed mounting piece. This is addressed in the next experimental setup. For this preliminary test setup, the image capture is sufficient to allow analysis of the FOV change between the tilted and non-tilted setup -as the FOV calculation may be a normalised grey scale value of the peaks on the DOF target

[0114] The 20 line pairs per millimeter (Ip / mm) line on the DOF target is selected for comparison between the two setups as the density of lines relative to the resolution of the camera is evident to visual comparison. Using ImageJ software, a plot profile of a line 250 pixels was drawn across the 20 Ip / mm line. FIG. 11 shows a profile across 20 Ip / mm band on the DOF target, according to an example.

[0115] The aim of this test is to measure the plot profile till an acceptable percentage contrast target value of 20%. Then drawn again on the other end of the image till it is met again, then the distance between the two lines measured, effectively measuring the lateral field of view along the image. FIG. 12A shows an image capture 1201 of the non-tilted camera setup of a DOF target, with a profile line (denoted by an arrow 1205) drawn across the 20 Ip / mm band of the DOF target, according to an example. FIG. 12B shows an image capture 1203 of the tilted camera setup of the DOF target, with a profile line (denoted byan arrow 1207), drawn across the 20 Ip / mm band of the DOF target, according to an example.

[0116] The percentage contrast value is calculated using this formula, seen in Equation (3) where Tmaxand Imm are the gray value of the peak and the trough of the middle of the band, respectively:- Equation (3)

[0117] The test resulted in DOF values of 14.75 units (distance left profile to right profile) for the non-tilted setup, and 15.50 units for the tilted setup. The distance units are not specifically defined, as the DOF target ruler is meant to be read while the target is 45° not at 10° as in the setup. Hence, the distance is a component of the actual reading from the DOF target, and for the sake of comparison, the units shall remain undefined (arbitrary).

[0118] It may be observed that there is about a 4 83% increase in FOV from the non-tilted to tilted setup. Even with a non-ideal setup for the tilted setup (uneven vignetting), this improvement in FOV indicates that the imaging from the full confocal microscopy capture may yield better DOF and FOV results. However, this is simply a proof of concept of the workings of Scheimpflug’s rule. The purpose of this implementation is to check if the principle still applies when the tilted surfaces are image conjugates to the actual object plane.

[0119] Implementation to Chromatic Confocal Microscope

[0120] Experimental Setup

[0121] For the testing of this principle, an existing chromatic confocal microscope may be used - in which already exists a Nipkow disk, tilted at 10° to the camera sensor. Thus, the only variable between the comparison is the tilt of the camera relative to the optical axis. Due to the mismatch of the sensor form factor (rectangle) to the image lens aperture (circular), care is put into ensuring the optical axis remain aligned during this tilt. FIG. 13A and FIG. 13B show photographs respectively depicting a front view and a side view of a non-tilted setup of a Zeiss Axiocam 305 camera 1306, mounted directly to an imaging lens via c-mount 1301, according to an example. FIG. 14A and FIG. 14B show photographs respectively depicting a front view and a side view of a tilted setup of the Zeiss Axiocam305 camera 1306, mounted via a monitor arm 1401, tilting the camera at an angle of 10°, according to an example.

[0122] After implementation of both setups (FIGS. 13 A, 13B, 14A and 14B), steps were taken to ensure valid comparison of images before and after - i.e. by ensuring that the stage is not moved in z direction, ensuring that the sample is not moved in x-y direction. To negate the illumination inhomogeneity effect and lopsided vignetting (as previously discussed), after mounting the tilted camera 1306, the camera 1306 was manually moved in the x-y direction to ensure the optical axis of the system was in line with the now tilted camera 1306 (sensor).

[0123] Modes of Capture and Initial Analysis

[0124] Two modes of capture were selected for post capture analysis. The image of the Nipkow disk without spin, and image of a 50pm grid sample at the same location. FIG. 15 shows a tilted camera image of still (non-spinning) Nipkow disk (on the left), and three selected zoomed-in segments (enlarged views on the right), according to an example. Comparison may be made with FIG. 5 showing the non-tilted camera image.

[0125] As noted in FIG. 5, the non-tilted setup exhibits stretching at the edges of the Nipkow disk due to the mismatch of the DOF of the camera to the z component of the surface of the Nipkow disk. This is evident from the difference in roundness of the pinholes between those in the centre of the images (where the camera is focused on) against the pinholes at the corners of the images, where they are out of the DOF. In the non-tilted setup of FIG. 5, the pinholes at the comers stretch into an oval shape, indicating they are out of focus, while in the tilted setup of FIG. 15, the pinholes in the corners exhibit the same roundness of compared to those in the middle.

[0126] FIG. 16A shows a 50pm grid pattern captured on a chromatic confocal microscope - non-tilted camera, according to an example. FIG. 16B shows a 50 pm grid pattern captured on a chromatic confocal microscope - tilted camera, according to an example.

[0100] Initial visual analysis of the 50pm grid sample did not bear any conclusion. Further analysis using plot profiles and evaluating the quality of the image needed to be done. It should be noted that the illumination issue for the tilted setup, although less, is stillevident in this aligned image capture. Vignetting is still seen due to the illumination aperture not being large enough to encompass the entire FOV of the tilted capture. As mentioned above, this is sufficient and the analysis done thereafter (as described in the next section under Measurement and Data), takes this into account and compares using data that the illumination has a reduced impact on.

[0127] Measurement and Data

[0128] ImageJ was used to generate gray scale plot profiles of the 50pm grid samples. This reduces the effect of the colour difference in the chromatic confocal microscope, and thereafter the comparison between values is irrespective of the illumination effects. FIG.17A shows a 50pm grid pattern captured on a chromatic confocal microscope - non-tilted camera, with reference lines for plot profiling of 13 rows - the x direction of the image, and 16 columns - the y direction of the image, according to an example. FIG. 17B shows a 50pm grid pattern captured on a chromatic confocal microscope - tilted camera, with reference lines for plot profiling of 13 rows - the x direction of the image, and 16 columns - the y direction of the image, according to an example. Plot profiles for both image captures of FIGS. 17A and 17B were made, along the rows of the grid, then again along the columns of the image, as seen in Fig. 13 This relates to analysis of the data both along the rotation axis of the tilt as well as across the rotation axis of the tilt. This is crucial for the comparison of the effect of the tilt as well as the conclusion of the results.

[0129] FIG. 18A and FIG. 18B show plot profiles of row 8 (x-direction) of the non-tilt capture and the tilt capture of 50pm grid captures, respectively, according to various examples. The plot profiles of each line were then extracted in ImageJ and exported as a csv file to be analysed further in MATLAB. There, codes were written to extract each individual row and column’s plot profile Then, the codes calculate the percentage contrast (as discussed above), and additionally, the full-width-half-maximum (FWHM) of each line on the grid.

[0130] Initial analysis of percentage contrast and general trend of the plot profile tend to favour the non-tilted setup. Due to the larger homogeneity across the plot profile of the non-tilted setup versus the drop of intensity at the edges of the tilted setup. This comparison is valid for tradition brightfield microscopy where homogeneity and uniform pixel responseplay crucial parts in non-area specific measurement. However, for this use case in confocal microscopy, where the individual pixel response is mapped using prior calibration sample to sample, this drawback is reduced. Hence, contrary to the analysis in Preliminary! Studies and Test section above, further emphasis is placed on the FWHM measurements across each peak, that illustrates the resolution at the corners and centre of the image.

[0131] FIGS. 19A to 19C show FWHM measurements for various grid line rows (horizontal) of non-tilt capture 1903 and tilt capture 1901 of 50pm grid capture, according to various embodiments. For ease of analysis, three bands were selected for analysis of the FWHM on the 50pm grid capture - the topmost row (Row 1) as seen in FIG. 19A, the middle (Row 7) as seen in FIG. 19B, and the last row (Row 13) as seen in FIG. 19C. These rows represent the FWHM performance along the axis of rotation, while indicating performance and the marginal edges / rays of the system and central chief rays.

[0132] FIGS. 20A to 20C show FWHM measurements for various grid lines columns (vertical) non-tilt capture 1903 and tilt capture 1901 of 50pm grid capture, according to various embodiments. More specially, the leftmost column (Column 1) as seen in FIG.20A, the middle column (Column 8) as seen in FIG. 20B, and the rightmost column (Column 16) as seen in FIG. 20C, were selected for analysis across the axis of rotation.

[0133] Across all FWHM graphs, a common trend is a low FWHM value toward the middle of the graphs (~ peaks 6 to 8) - which relates to grid lines toward the middle and centre of the images. Additionally, larger FWHM values are seen toward the earlier and later peaks - which relate to the grid lines toward the corner and edges of the image.

[0134] The graphs in FIGS. 19A to 19C exhibit insignificant variance of FWHM values between the tilt and non-tilted setups. The FWHM values are within 1-2 units with the exception of 3 anomalous values (Row 1 peak 3 and 4, Row 13 peak 8). This result is expected as the measurement of FWHM here is along the axis of rotation. The purpose of the tilted setup is to address the defocusing effect as seen in FIGS. 6A and 6B, that lies along the axis of rotation instead of across the axis of rotation.

[0135] The graphs in FIGS. 20A to 20C demonstrate the intended effect of Scheimpflug principle on the Nipkow disk. The graphs similarly exhibit insignificant variance of FWHM values toward the middle peaks, however there is a reduction of FWHM values by3 to 10 FWHM units at the edge peaks ( 1 to 3 and 10 to 12). This is seen across all 3 graphs, as indicated by the side arrows 1905.

[0136] Table 1 summarizes a comparison of the variances between the conventional setup (non-tilt) and the tilted setup.

[0137] Table 1

[0138] The reduction in FWHM values indicate the grid lines are sharper and more focused at the edges of the image capture, in the tilted setup compared to the non-tilted setup. This is likely a direct result of the increased matching of the DOF plane and Nipkow disk plane in the tilted setup. This conclusively shows that there is an improvement of resolution of the grid lines prior to implementing the tilted setup.

[0139] Sensor and Disk Tilt Mounting Mechanisms

[0140] Instead of solely adjusting variables at the sample end or disk plane end, various embodiments of the apparatus 100 may include the adjustments and implementations of improvements on the camera sensor end.

[0141] The mounting mechanisms for the camera and the Nipkow disk play a crucial role in determining and efficient implementation of capture. The tilt angle of the camera andthe Nipkow disk are to be adjusted to as close as possible. For the camera, this is determined in this case using a 3D printed camera mount for the preliminary experiment, see FIGS. 8, 9A and 9B, and monitor arm adjustment for the latter experiment, see FIGS. 14A and 14B. The 3D printed mount angle is pre-determined and fixed upon printing, then inserted into the camera and fixed onto the imaging lens. Additionally, the angle using the arm mount, required tilting and measuring for the angle, using the back plane of the system as a reference point. FIG. 21 shows a schematic perspective view of a 4-axis (x, y, z and tilt) translation stage / mount 2101 designed and fabricated for the Nipkow disk 2108, according to an example. Similarly, the Nipkow disk required manual tilting and for measuring for the angle, the back plane of the system as a reference point as well.

[0142] Though the arm mounting is sufficient to introduce the desired tilt angle relative to the Nipkow disk, it requires adjusting every time the system (e.g. the apparatus 100 of FIG.1) needs to be adjusted or moved (e.g. change of disk tilt angle, change of objective). Hence, a tilt mount similar to the disk mount may be used, that may be mounted directly onto the system, with tilt adjustment capability. FIG. 22 A shows a schematic perspective view of a 3D printed fixed angle mount 2201 for sensor, according to an example. FIG.22B shows a schematic perspective view of a 2-axis translation mount 2207 for sensor, according to an example.

[0143] In FIG. 22B, a tilt stage 2205 is designed to mount the Axiocam 305 camera 2206 to the imaging lens 2203. This mount 2207 allows for locking of the tilt at a desired and measured angle, then translation in the direction along the axis of rotation of the Nipkow disk, to further align the mismatch of optical axis - as discussed in the Experimental Setup section above. This setup matches the translation capability of the monitor arm setup, as seen in FIGS. 14A and 14B, without the need for external support of the camera 2206.

[0144] In the various embodiments and examples discussed hereinabove, initial observation of images of the Nipkow disk before and after tilting of the camera showed evident improvement in the resolution of the pinholes at the edges of the Nipkow disk -with the tilted setup showing more focused pinholes and less blurriness. Two tests (percentage contrast and FWHM) were conducted non-tilted and tilted setup experiment on the 50 gm grid sample. The FWHM test bore conclusive results that the tilted setupexhibited higher resolution at the marginal edges of the sample. Notably, this improvement in FWHM of the peaks, is seen across the axis of tilt, as theorized prior to testing.

[0145] Though this data supports the use of tilt to improve resolution of the image for capture. The tests confirm that this 3D reconstruction is far less accurate at the edges of the image due to this effect. With a more homogenous resolution over the whole FOV of the image, from the centre to the edges, the pixel mapping to the z height map is expected to improve as well, as the height map is created from the centre of the image in general.

[0146] Various embodiments of the apparatus 100 (FIG. 1) relate to tilted disk and tilted camera simultaneously configuration to suppress glare but without cost of image homogeneity and useful FOV reduction. This is achieved by the design of camera sensor tilt angle to match the Nipkow disk tilt plane to obtain optimal axial resolution and image quality.

[0147] In various examples, a compact and flexible disk tilted mechanism design with 4 axis alignment capability may be provided to achieve the required disk tilting in confocal or chromatic confocal microscope.

[0148] A tilted camera mounting design matched with the tilted disk may be provided to achieve efficient glare suppression and high-quality confocal imaging simultaneously. Matching the tilt angle of the disk versus the tilt angle of the camera needs to be precise and accurate. Suitable mounting mechanism is to be designed to enable a user to carry out the alignment accordingly.

[0149] There is reduced resource requirement to implement, as there is no need to have separate illumination and imaging pathways, and / or to have a second disk for secondary path.

[0150] Compared to an existing system that focuses on line sensor capture with increased DOF, and aims to minimize disk tilt by adjusting the entire imaging module (i.e. both the imaging lens and the camera are collectively provided as one single system) including the beam splitter, aligning them with a new optical axis to suppress reflections (virtually providing the same effect as simply tilting the disk further), various embodiments of the apparatus 100 (FIG. 1) and the method 320 (FIG. 3) relates to technology applied to area capture, requiring consideration of an additional axis of tilt, and the tilt principle is not usedfor reflection suppression but rather, for maintaining image quality and reducing back reflections.

[0151] In other words, in the existing system, there is no tilt of the camera with respect to the imaging lens or the optical path therebetween. Contrarily, various embodiments of the apparatus 100 and the method 320 involve a tilted camera with respect to the imaging lens or the optical path therebetween. More specifically, various embodiments of the apparatus 100 and the method 320 may involve tilting of all conjugate image or object planes along the whole optical path in a synchronized manner (based on Scheimpflug principle) to avoid image deformation and glare noise simultaneously. For example, the camera, the Nipkow disk, ans the sample may be located at these conjugated planes. In other existing systems, only one or two (not all) of these planes may be tilted in a random manner (without any synchronization between each other / one another).

[0152] The proposed solution, provided by the apparatus 100, is an efficient solution to suppress the glare without degradation of the image quality. A simple and compact optical layout design to enhance FOV of a chromatic confocal system using Scheimpflug principle is provided. It is without need of additional optical pathways design or additional optics component, thereby maximizing the use of the optical components and optical path for area scan imaging, eventually leading to improved FOV. In other words, it is not suffered by any issues mentioned above such as FOV reduction, chromatic shift and complex alignment issue.

[0153] A typical camera with modified imaging lens interface may be utilized. For example, A 3D printed adapter (mount) with pre-defined tilt angle may be employed to ensure the camera (sensor) plane is at a specific angle to the imaging lens plane.

[0154] The proposed method (e.g. 320 of FIG. 3) is a simple and cheap approach to suppress the glare even without the need of extremely high extinction ratio >104~l 06antireflection coating on the disk which may be extremely expensive. With tilt disk imaging, low mechanical complexity may be maintained and inherent performance may be improved.

[0155] Applications of the proposed solution may include chromatic and traditional confocal microscopy using single spinning Nipkow disk, thereby enabling larger DOF capture with each image, and 3D reconstruction of topography either by z-stacking ormapping to chromatic calibration curves. Other applications may be those requiring capture or use of reflective surface for capture, with minimum glare and back reflection.

[0156] While the invention has been particularly shown and described with reference to specific embodiments, it should be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the invention is thus indicated by the appended claims and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced.

Claims

CLAIMS1. An apparatus for performing confocal microscopy, the apparatus comprising: an optical arrangement comprising one or more optical components configured to direct light reflected from a sample under observation; andan imaging sensor configured to capture an image of the sample based on at least part of the reflected light,wherein the optical arrangement further comprises a pinhole element configured to spatially filter the reflected light, andwherein the imaging sensor and the pinhole element are configured to simultaneously tilt away from a plane perpendicular to an optical axis of the optical arrangement in a matched manner such that the captured image has optimal resolution and image quality in terms of glare or reflection suppression and reduced imaging distortion2. The apparatus as claimed in claim 1, wherein in the matched manner, the imaging sensor is configured to tilt away at a first angle from the plane perpendicular to the optical axis and the pinhole element is configured to tilt away at a second angle from the plane perpendicular to the optical axis, the first angle and the second angle satisfying Scheimpflug principle.

3. The apparatus as claimed in claim 1, wherein the imaging sensor is located at a conjugate plane of the pinhole element; and wherein in the matched manner, the imaging sensor is configured to tilt away at a first angle from the plane perpendicular to the optical axis and the pinhole element is configured to tilt away at a second angle from the conjugate plane, the first angle and the second angle satisfying Scheimpflug principle.

4. The apparatus as claimed in claim 2 or 3 further comprising a fixed mount coupled to the imaging sensor, wherein the fixed mount is designed in a manner to secure the imaging sensor at the first angle.

5. The apparatus as claimed in claim 4, wherein the fixed mount is fabricated using 3D printing.

6. The apparatus as claimed in claim 2 or 3 further comprising an adjustable mount coupled to the imaging sensor, wherein the adjustable mount is designed in a manner to adjust and releasably lock the imaging sensor at the first angle.

7. The apparatus as claimed in claim 6, wherein the adjustable mount comprises a 2- axis translation mount.

8. The apparatus as claimed in any one of claims 2 to 7 further comprising a tilt stage configured to translate the imaging sensor in a direction along a rotational axis of the pinhole element.

9. The apparatus as claimed in any one of claims 2 to 8, wherein the pinhole element is coupled to a 4-axis translation mount operable to tilt the pinhole element to the second angle, and adjust the pinhole element along three orthogonal axes, one parallel to the optical axis and two orthogonal to the optical axis and to each other.

10. The apparatus as claimed in claim 9, wherein the imaging sensor is configured to tilt along the three orthogonal axes.

11. The apparatus as claimed in any one of claims 2 to 10 further comprising a substrate arranged to hold the sample, wherein the one or more optical components further comprise a lens arrangement positioned between the pinhole element and the substrate, the substrate is configured to tilt at a third angle away from an object plane perpendicular to an optical axis of the lens arrangement in the matched manner, the second angle being proportional to the third angle by a magnification power, M measured between the pinhole element and the substrate.

12. The apparatus as claimed in claim 11, wherein the substrate is coupled to a sample mount operable to tilt the substrate to the third angle.13 The apparatus as claimed in claim 11, wherein the substrate comprises a sample mount operable to be tilted to the third angle.

14. The apparatus as claimed in any one of claims 1 to 10 further comprising an imaging lens configured to focus the spatially filtered reflected light onto the imaging sensor, wherein the imaging sensor is configured to tilt away from the imaging lens.

15. The apparatus as claimed in claim 14, wherein the imaging lens has a magnification power, M.

16. The apparatus as claimed in any one of claims 1 to 15, wherein the imaging sensor comprises a charged-couple device array or a Complementary Metal-Oxide Semiconductor sensor.

17. The apparatus as claimed in any one of claims 1 to 16, wherein the pinhole element comprises a pinhole mask coupled to a spinning motor.

18. The apparatus as claimed in any one of claims 1 to 17 further comprising a controller configured to adjust the imaging sensor and the pinhole element tilting away from the plane perpendicular to the optical axis in the matched manner.19 A method for performing confocal microscopy, the method comprising:directing, by an optical arrangement, light reflected from a sample under observation;spatially filtering, by a pinhole element of the optical arrangement, the reflected light; andcapturing, by an imaging sensor, an image of the sample based on at least part of the reflected light,wherein the imaging sensor and the pinhole element are simultaneously tilted away from a plane perpendicular to an optical axis of the optical arrangement in a matched manner such that the captured image has optimal resolution and image quality in terms of glare or reflection suppression and reduced imaging distortion.

20. The method as claimed in claim 19, wherein in the matched manner, the imaging sensor is tilted away at a first angle from the plane perpendicular to the optical axis and the pinhole element is tilted away at a second angle from the plane perpendicular to the optical axis, the first angle and the second angle satisfying Scheimpflug principle.

21. The method as claimed in claim 19, wherein the imaging sensor is located at a conjugate plane of the pinhole element; and wherein in the matched manner, the imaging sensor is tilted away at a first angle from the plane perpendicular to the optical axis and the pinhole element is tilted away at a second angle from the conjugate plane, the first angle and the second angle satisfying Scheimpflug principle.

22. The method as claimed in claim 20 or 21, wherein the first angle ranges from 0° to 45°, or preferably 6° to 45°.

23. The method as claimed in any one of claims 20 to 22, wherein the second angle ranges from 0° to 45°, or preferably 6° to 45°.

24. The method as claimed in any one of claims 20 to 23, wherein in the matched manner, the imaging sensor is secured at the first angle by a fixed mount coupled to the imaging sensor.

25. The method as claimed in any one of claims 20 to 23, wherein in the matched manner, the imaging sensor is adjusted and releasably locked at the first angle by an adjustable mount coupled to the imaging sensor, and the imaging sensor is translated in a direction along a rotational axis of the pinhole element by a tilt stage.

26. The method as claimed in any one of claims 20 to 25, wherein the pinhole element is tilted to the second angle and is adjusted along three orthogonal axes, one parallel to the optical axis and two orthogonal to the optical axis and to each other, by a 4-axis translation mount coupled to the pinhole element.

27. The method as claimed in claim 26, wherein the imaging sensor is tilted along the three orthogonal axes.

28. The method as claimed in any one of claims 20 to 27, further comprising providing a substrate to hold the sample, wherein the substrate is tilted at a third angle away from an object plane perpendicular to an optical axis of a lens arrangement of the one or more optical components in the matched manner, the lens arrangement being positioned between the pinhole element and the substrate, and the second angle being proportional to the third angle by a magnification power, M measured between the pinhole element and the substrate.

29. The method as claimed in claim 28, wherein the substrate is tilted to the third angle by a sample mount coupled to the substrate.

30. The method as claimed in claim 28, wherein the substrate comprises a sample mount tilted to the third angle.

31. The method as claimed in any one of claims 28 to 30, wherein the third angle is.

32. The method as claimed in any one of claims 20 to 27, further comprising prior to capturing the image, focusing, by an imaging lens, the spatially filtered reflected light onto the imaging sensor, wherein the imaging sensor is tilted away from the imaging lens.

33. The method as claimed in claim 32, wherein the imaging lens has a magnification power, M.

34. The method as claimed in claim 33, wherein the first angle and the second angle are identical when M is equal to 1.

35. The method as claimed in claim 33, wherein the first angle and the second angle are different when M is not equal to 1.

36. The method as claimed in any one of claims 19 to 35, wherein spatially filtering the reflected light comprises spinning the pinhole element by a spinning motor coupled to the pinhole element.

37. The method as claimed in any one of claims 19 to 36, wherein the pinhole element comprises a pinhole mask, preferably a Nipkow disk.

38. The method as claimed in any one of claims 19 to 37, further comprising adjusting, by a controller, the imaging sensor and the pinhole element to tilt away from the plane perpendicular to the optical axis in the matched manner.

Images