WO2020218973A1

WO2020218973A1 - Method and system for super resolution imaging

Info

Publication number: WO2020218973A1
Application number: PCT/SG2020/050245
Authority: WO
Inventors: Lianwei CHEN; Yan Zhou; Minghui Hong
Original assignee: Phaos Technology Pte. Ltd.
Priority date: 2019-04-26
Filing date: 2020-04-20
Publication date: 2020-10-29

Abstract

Methods and systems for nanoscopy are disclosed. The method includes moving a microsphere relative to an objective lens of a microscope to bring the microsphere into a first field of view when a first feature of a sample is observable and a second feature of the sample is not observable through the objective lens. The first feature and the second feature are concurrently disposed under visible light or white light illumination within the first field of view, of which the first field of view is provided by a lens system in a first lens configuration. The first lens configuration includes the objective lens but does not include the microsphere. The microsphere is coupled to the microscope and configured for controllable movement in ambient air without contacting the sample.

Description

METHOD AND SYSTEM FOR SUPER RESOLUTION IMAGING

TECHINCAL FIELD

[0001] Example embodiments in the present disclosure are generally directed to optical imaging techniques, and more particularly towards novel methods and systems that provide for super resolution nanoscopy.

BACKGROUND

[0002] Optical imaging utilizing a conventional optical microscope has been widely used in many fields, including examination of biological samples and optical characterization of integrated circuits for failure analyses. However, achievable image resolution under visible light or white light illumination, with the aid of conventional microscope, is limited by the diffraction limit. Particularly, the conventional optical microscope can only resolve objects with the proximity around a half of the incident wavelength and may not be suitable for used in optical characterization of nano-scale features, such as live viruses. Other non-optical techniques, such as scanning electron microscope (SEM) and transmission electron microscope (TEM) may be used to perform nano-scale observation. However, these techniques require complex sample preparation and working conditions and may also be destructive to the samples.

[0003] Recently, super resolution imaging techniques, including Optical Microsphere Nanoscope (OMN) was realized to observe nano-scale features. However, this technique also has its limitations. For example, it requires the deposition of microsphere directly onto the surface of the sample. Furthermore, the field of view is restricted to the location of the microsphere.

[0004] In view of the foregoing, there is a need to provide a super resolution nano imaging system and method to visualize nano-scale features without being invasive to the samples. It is also desirable to provide a highly flexible, easy-to-use and economical scheme for super resolution nano-imaging. SUMMARY

[0005] In the present disclosure, according to an aspect, a method of nanoscopy is presented. The method includes moving a microsphere relative to an objective lens of a microscope to bring the microsphere into a first field of view when a first feature of a sample is observable and a second feature of the sample is not observable through the objective lens, the first feature and the second feature being concurrently disposed under visible light illumination within the first field of view, wherein the first field of view is provided by a lens system in a first lens configuration, the first lens configuration includes the objective lens and does not include the microsphere, the microsphere being coupled to the microscope and configured for controllable movement in ambient air without contacting the sample.

[0006] The method may further include when the microsphere is in the first field of view, obtaining an image through the lens system, the image includes a first image portion obtained through the lens system in the first lens configuration and a second image portion concurrently obtained through the lens system in a second lens configuration, the second lens configuration includes the objective lens and the microsphere. Additionally, the method may further include moving the microsphere to bring the second feature into focus in the second image portion without changing the objective lens.

[0007] In one example embodiment, the method may include moving the microsphere within the first field of view while keeping the sample substantially stationary relative to the objective lens. The microsphere may be moved relative to an optical axis of the objective lens. In another example embodiment, the method includes moving the microsphere out of alignment with the optical axis of the objective lens and into alignment with the second feature, and bringing the second feature into focus in the second image portion without changing the objective lens.

[0008] In another aspect, the present disclosure provides a method of nanoscopy. The method includes obtaining a first image portion of a target area observable in a first field of view, the first field of view being determined by an objective lens of a microscope configured to operate with a visible light illumination source; obtaining a second image portion of a selected feature, the selected feature being disposed in the target area, the second image portion being obtainable through a second lens configuration of a lens system, the second lens configuration includes the objective lens and a microsphere coupled to the microscope; and controllably moving the microsphere relative to the objective lens to obtain a subsequent image portion of another selected feature through the lens system in the second lens configuration of the lens system, wherein the first image portion is obtainable concurrently with either one of the second image portion and the subsequent image portion.

[0009] The method may further include moving the microsphere relative to the objective lens to bring different parts of the target area of a sample into focus, without relative movement between the sample and the objective lens. Alternatively, the method may include moving the microsphere relative to the objective lens to bring different parts of the target area into focus, without changing either the objective lens or a working distance of the objective lens.

[0010] The method may include providing an adapter, wherein the adapter includes an opening and a receptacle portion, wherein the opening is completely vacant and the receptacle portion is configured to hold the microsphere. In one example embodiment, the opening and the receptacle portion are distinct and are spaced apart from each other. The receptacle is configured to hold a plurality of microspheres.

[0011] As another option, the method may include providing an adapter, wherein the adapter includes an opening and a receptacle portion, wherein the opening is configured to be a light transmission window and the receptacle portion is configured to hold the microsphere.

[0012] In yet another aspect, a method of nanoscopy is disclosed. The method includes obtaining a first image of a target area observable in a first field of view, the first field of view being determined solely by an objective lens of an optical microscope configured to operate under visible light illumination, the first image being formed by light received by the objective lens through a first window of an adapter, obtaining a second image of a selected feature, the selected feature being disposed in the target area and observable in a second field of view, the second field of view being determined by a microsphere disposed on the adapter, the second image being formed by light received by the objective lens and the microsphere, and controllably moving the adapter relative to an optical axis of the objective lens to switch between obtaining the first image and the second image, wherein the microsphere is disposed on the adapter. [0013] In one example embodiment, obtaining the first image includes aligning the first window with the optical axis of the objective lens such that the first image is characterised by microscopic resolution. Further, obtaining the second image includes aligning the microsphere between the objective lens and the selected feature, the microsphere being disposed to receive evanescent waves from the selected feature such that the second image is characterised by super resolution. The method may further include disposing the microsphere out of alignment with the optical axis of the objective lens.

[0014] The present disclosure also includes an imaging system for use with the method as recited above. The imaging system includes an optical microscope having a lens system and a control unit coupled to the optical microscope. The control unit is configured to perform a method of capturing a first image at a microscopic level of resolution when the lens system is in a first lens configuration, and capturing a second image at a nano-level of resolution when the lens system is in a second lens configuration, wherein the first lens configuration includes the objective lens and does not include the microsphere, and wherein the second lens configuration includes the objective lens and the microsphere.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Fig. 1A shows a portion of an imaging apparatus having an adapter in accordance with at least some embodiments of the present disclosure;

[0016] Fig. IB shows a top view of an embodiment of the adapter of Fig. 1A;

[0017] Fig. 1C shows a cross-sectional view of the adapter of Fig. IB;

[0018] Fig. ID shows a top view of another embodiment of the adapter of Fig. 1A;

[0019] Fig. IE shows a top view of yet another embodiment of the adapter of Fig. 1A;

[0020] Fig. 2A shows a portion of an imaging apparatus having a lens system in a first lens configuration in accordance with at least some embodiments of the present disclosure;

[0021] Fig. 2B shows an image obtained through the arrangement of the lens system of Fig. 2A; [0022] Fig. 3A shows a portion of an imaging apparatus having the lens system in a second lens configuration in accordance with at least some embodiments of the present disclosure;

[0023] Fig. 3B shows an image obtained through the arrangement of the lens system of Fig. 3A;

[0024] Fig. 3C shows another embodiment of an image obtained through the arrangement of the lens system of Fig. 3A;

[0025] Fig. 4A shows a portion of an imaging apparatus having the lens system in a second lens configuration in accordance with at least some embodiments of the present disclosure;

[0026] Fig. 4B shows an image obtained through the arrangement of the lens system of Fig. 4A;

[0027] Figs. 5A-5C show example imaging results taken during various stages of operating an imaging apparatus in accordance with some embodiments of the present disclosure;

[0028] Fig. 6 illustrates another embodiment of the adapter;

[0029] Fig. 7 shows a chart illustrating a method of nanoscopy in accordance with at least some embodiments of the present disclosure;

[0030] Figs. 8 shows a flow chart illustrating yet another method of nanoscopy in accordance with at least some embodiments of the present disclosure; and

[0031] Fig. 9 shows a block diagram illustrating the configuration of a system for operating an imaging apparatus in accordance with at least some embodiments of the present disclosure.

DETAILED DESCRIPTION

[0032] It will be readily understood that the components of the embodiments, as generally described and illustrated in the figures herein, may be arranged and designed in a wide variety of different configurations in addition to the described example embodiments. Thus, the following more detailed description of the example embodiments, as represented in conjunction with the figures, is not intended to limit the scope of the embodiments, as claimed, but is merely representative of example embodiments. [0033] Reference throughout this specification to “one embodiment”, “another embodiment” or“an embodiment” (or the like) means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearance of the phrases “in one embodiment” or“in an embodiment” or the like in various places throughout this specification are not necessarily all referring to the same embodiment.

[0034] Furthermore, the described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details are provided to give a thorough understanding of embodiments. One skilled in the relevant art will recognize, however, that the various embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, some or all known structures, materials, or operations may not be shown or described in detail to avoid obfuscation.

[0035] As used herein, the singular“a” and“an” may be construed as including the plural“one or more” unless clearly indicated otherwise.

[0036] Embodiments of the present disclosure provide an improvement by enabling non-contact nanoscopy that overcomes the diffraction limit of visible light or white light, even with the sample being disposed in ambient air without special sample preparation. This is extremely beneficial in many fields, including high-speed optical characterization of viruses, integrated circuit failure analyses, and the development of nanotechnologies. “Visible light” refers to radiation that is perceptible to the human eye, and has at least one wavelength within the visible spectrum ranging from 3850 to 7600 Angstrom units.“White light” refers to a combination of wavelengths of the visible spectrum. For the sake of brevity,“visible light” refers to light that includes at least one wavelength within the visible spectrum. Where“visible light” is used in this document, it can be understood to refer to “visible light” or“white light”, or both.“Non-contact” (or“non-invasive”) as used in this document refers to the imaging or inspection of a sample without any lens coming into contact with the sample, without the need to submerge the sample in some liquid to aid image capture, and/or without damaging or changing the nature or a property of the object for the sake of imaging.“Imaging”,“observing”,“detection”, and“inspection” are used interchangeably as the context will make clear, for the sake of brevity. Further, super resolution imaging, near-field imaging, and nanoscopy are used interchangeably to refer to the observation, imaging, or inspection at a level of resolution beyond the diffraction limit of visible light or white light.

[0037] Fig. 1A shows a portion of an imaging apparatus 100 in accordance with some embodiments of the present disclosure. The imaging apparatus, in one embodiment, includes an optical microscope. The optical microscope is configured to operate under visible light or white light illumination. While an optical microscope may be provided with more than one objective lens, each having different magnification power, it can be understood from the context that reference to the objective lens refers to the objective lens selected for use.

[0038] The optical microscope includes a lens system having at least one objective lens 170, the lens system being configurable between a first lens configuration and a second lens configuration. In the first lens configuration, the objective lens 170 is able to receive or collect light transmitted through or reflected off a sample such that the light is not transmitted through an intermediate optical device. In the second lens configuration, the objective lens 170 is able to receive or collect light transmitted through or reflected off a sample such that the light is transmitted through an intermediate optical device before being received or collected by the objective lens. The intermediate optical device includes a microsphere, such as one suitable for collecting evanescent waves so as to enable near field imaging. In some embodiments, there is provided an adapter coupled to the optical microscope such that the lens system can be switched between the first lens configuration and the second lens configuration without disturbing the sample. One example of a lens system 110 includes the objective lens 170 and an adapter 120, in which the adapter 120 is configured for motion relative to the objective lens 170. In other words, the adapter 120 is configured to move or to be disposed in different positions relative to the optical axis. For the sake of brevity, reference to an axial direction 172 refers to a direction or axis substantially parallel to an optical axis of the objective lens 170 or an axis of symmetry of the objective lens 170.

[0039] The optical microscope includes a sample stage 180 that is configured to support a sample for imaging by the lens system. The sample stage 180 may be configured to provide a first surface 182 in a plane substantially normal to the axial direction 172, the first surface 182 being suitable for fixing a sample in a stationary position relative to the objective lens. In some embodiments, the sample stage may be replaced by a stabilizing device so that the sample or object under inspection can be held in a stable position relative to the objective lens 170.

[0040] According to some embodiments of the present disclosure, an adapter 120 is provided for use with an optical microscope such that the adapter 120 is positionable between the objective lens 170 and the sample stage 180 as shown in Fig. 1A. In some embodiments, the adapter 120 is coupled to the optical microscope so that the adapter 120 is characterised by at least one degree of freedom with respect to the optical axis 172 of the objective lens 170. In some embodiments, the adapter 120 is coupled to the optical microscope so that the adapter 120 is characterised by at least two degrees of freedom with respect to the optical axis 172 of the objective lens 170. In some embodiments, the adapter 120 is coupled to the optical microscope so that the adapter 120 can move in a plane substantially normal to the optical axis 172 ofthe objective lens. In some embodiments, the adapter 120 is coupled to the optical microscope so that the adapter 120 can move in a plane substantially coplanar with the first surface 182 of the sample stage 180. In some embodiments, the adapter 120 is coupled to the optical microscope so that the adapter 120 is configured for movement in which a reference point on the adapter 120 traces a curvilinear path of substantially constant distance from another reference point on the objective lens 170. The adapter 120 is configured to move or to be displaced relative to the objective lens 170. The adapter 120 is a distinct component and spaced away from the objective lens 170. In some embodiments, the adapter 120 may be provided in a range of configurations (e.g., a set of adapters, each of the adapters having a differently sized microsphere), some of which can be used interchangeably in detachable engagement with the same optical microscope.

[0041] Fig. IB shows a top view of an adapter 120 according to an embodiment of the present disclosure, and Fig. 1C shows a cross-sectional view taken at line A-A of Fig. IB. A body 122 of the adapter 120 defines a receptacle 140 and an opening 150. The receptacle is configured to receive at least one microsphere 40. In some embodiments, the receptacle 140 may be held in a fixed position relative to the adapter 120. In other embodiments, the receptacle 140 may be held in a fixed position relative to the opening 150. In such case, the receptacle 140 and the opening 150 are spaced apart by a predetermined separation distance 152 that is greater than zero. For example, the predetermined separation distance 152 may be in the range of about lpm to 1mm. [0042] The opening 150 forms a first window 155 distinct and different from the receptacle 140. In one embodiment, the first window 155 is essentially vacant, that is, the first window is unblocked. In other words, the first window is configured, for light in the visible spectrum to travel through the window 155 without a change in speed. The opening 150 can be configured in different shapes, such as but not limited to, rectangular, circular, or other geometric or irregular shapes. The opening 150 is preferably sized and shaped to permit a sufficient amount of light to transmit therethrough, with insignificant blockage or distortion, for subsequent capture by the lens system. This may include light that is transmitted through or reflected from a sample under observation, as well as light that is incident on the sample. Thus, the first window 155 serves as a light transmission window.

[0043] The receptacle 140 is one suitable for non-contact near-field imaging such that the microsphere 40 disposed at the receptable 140 is sufficiently near the sample to detect evanescent waves from the sample without the microsphere 40 physically contacting the sample. The microsphere 40 is thus one suitable for near-field imaging. The receptacle 140 may define a second window that is at least partially filled by at least one microsphere 40 when the adapter 120 is in assembly or in use. The receptacle 140 is distinct and different from the opening 150. In one example, the receptacle 140 includes a through-hole that extends through the entire thickness (or the axial dimension 124) of the adapter 120, in which the through-hole is situated a microsphere 40. In another example, the receptacle 140 is a concave feature or a depression having a depth less than the axial dimension 124 of the adapter 120, the concave feature or the depression being suitably sized to dispose at least one microsphere therein. The receptacle 140 may be suitably sized and shaped depending on the size of the microsphere 40 and the number of microspheres to be received by the receptacle 140.

[0044] Fig. ID shows a top view of another embodiment of the adapter 120. The opening 150 of the adapter 120, as shown in Fig. ID, may include a transparent material. The transparent material may include any suitable material that is sufficiently transparent to allow light in the visible spectrum to travel through the transparent material, and to enable identification of a point of interest under visible light or white light illumination under ambient air conditions. Thus, the transparent material forms the first window 155 which serves as a light transmission window. [0045] In yet another embodiment, the body 122 of the adapter 120 may include a transparent material. The transparent material may include any suitable material that is sufficiently transparent to allow light in the visible spectrum to travel therethrough, and to enable identification of a point of interest under visible light or white light illumination under ambient air conditions. In such case, a portion of the body 122 of the adapter may be designated as the first window 155 which serves as the light transmission window.

[0046] Fig. IE shows a top view of yet another embodiment of the adapter 120. The opening 150 may be coupled to the receptacle 140 through a connecting portion 145. The connecting portion 145 can be configured in various shapes, such as but not limited to, rectangular, circular, or other geometric or irregular shapes. The separation distance between the opening 150 and the receptacle 140 can be suitably adjusted through the connecting portion 145. For example, the opening 150 and the receptacle 140 can be positioned as close as possible to each other by suitably adjusting the dimension and configuration of the connecting portion 145.

[0047] Embodiments of a method of nanoscopy 700, 800 (Fig. 7, Fig. 8) will now be described with reference to a microscope operable under visible light or white light illumination alone, such as one as described above. The microscope may be configured as schematically illustrated in Fig. 2A with the lens system of the microscope being in a first lens configuration. In the first lens configuration 210, an image captured by, detectable or observable using the microscope is characterised by a first field of view 270. The first field of view is determined essentially by the objective lens 170 selected to form at least part of the first lens configuration. In one preferred embodiment of the method of nanoscopy, counter-intuitively, the objective lens 170 with the lowest magnification power is selected for use (out of the available objective lenses coupled to the microscope). The resulting first image 230 is characterised by the first field of view 270 (schematically shown in Fig. 2B). At this stage, a first focal plane is established in the objective lens. The first image 230 may include one or more first features 262 that are visible or detectable, although a clear and focused image of the first feature may or may not be obtainable based on an order of magnification provided by the selected objective lens 170 alone. There are second features 264, 266 which are too small to be detectable or clearly distinguishable at this first level of magnification. Thus, although second features 264, 266 are shown in Fig. 2B, it will be understood that this is for illustrative purposes only. In some cases, the second feature 264 is part of or disposed in the first feature 262. The second feature may include two or more elements that are spaced so close together that the elements appear indistinguishable from one another. That is to say, each second feature may refer to more than one physical element, although each second feature may also refer to one physical element. The first lens configuration 210 is chosen so that, even if a clear and focused image of the first feature and/or the second feature is not obtainable, a first location of the first feature and a second location of the second feature can be determined with reference to the optical axis 172 of the objective lens 170, and with respect to the first field of view 270.

[0048] After determining the second location of the second feature, a microsphere 40 is provided so that the microsphere 40 is aligned with the second location and the objective lens 170, such that visible light and/or other waves or signals from the second location may be received via the microsphere 40 by the objective lens 170. The provision of the microsphere 40 may be effected by moving the adapter 120 such that the lens system is switched to the second lens configuration 310.

[0049] One example of the second lens configuration 310 is schematically illustrated in Fig. 3A. In switching from the first lens configuration 210 to the second lens configuration 310, the focal plane of the lens system is switched from the focal plane of the objective lens 170 to the focal plane of the microsphere 40. In the process of switching, the objective lens 170 is preferably held stationary relative to the sample 290. A working distance 240 of the objective lens refers to a distance between the objective lens 170 and the sample 290 (or the first surface 182 of the sample stage 180). The process of switching the focal plane is carried out without substantially changing the working distance 240. The process of switching may be performed by moving the adapter 120 relative to the objective lens 170. The process of switching may include providing the microsphere 40 between the objective lens 170 and the second location, in which the second location was determined with reference to the first field of view 270. In some alternative embodiments, the process of switching includes realigning the lens system to focus on the second feature 264 after the second location has been determined with reference to the first field of view 270 of the objective lens 170, in which the focusing on the second feature 264 includes adding at least one microsphere 40 to the lens system. In some other alternatives, the process of switching may be described as moving the microsphere 40 into the first field of view 270 of the objective lens 170. [0050] An example of a second image 330 obtainable when the lens system is in the second lens configuration 310 is schematically illustrated in Fig. 3B. In some embodiments, the second image 330 is characterised by a second field of view 272, in which the second field of view 272 is determined by the microsphere 40. The second field of view 272 is smaller or narrower than the first field of view 270. The first image 230 and the second image 330 may be obtained sequentially or concurrently. In other embodiments, as illustrated by Fig. 3C, the second image 330 may be a composite image of a first image portion 232 characterised by a first field of view 270 and a second image portion 332 characterised by a second field of view 272. The first image portion 232 and the second image portion 332 can be obtained sequentially or concurrently.

[0051] In some embodiments, the second image 330 includes a first feature 262 observable using the first lens configuration and includes enlarged view and which is used in determining the second location of the second feature 264. The details of the second feature 264 are now observable and resolved at a nanoscopic level using the second lens configuration 310. In some embodiments, the microsphere 40 can be moved with the aid of a programmable control unit such that the microsphere 40 can be controllably moved in a desired direction 250 or a desired path within a target area 260, the target area 260 referring to an area observed with the first lens configuration 210. In some embodiments, the desired path is generally in a plane substantially normal to the axial direction 172. An image processing unit is configured to receive data on the images captured, and to output a second image 330 as illustrated in Fig. 3C in which the second image portion 332 is superimposed on the first image portion 232. The first field of view 270 provides a user with the ability to identify one or more points of interest in the target area 260. The microsphere 40 may then be steered manually, automatically, or semi-automatically, so that the microsphere 40 travels from one point of interest to another point of interest. The second field of view 272 provides a magnification level that is enhanced over that of the objective lens 170, thereby permitting a user to acquire nanoscopic images while essentially enjoying the advantages of a larger field of view. In some embodiments, the second image 330 is composed by superimposing the second image portion 332 (Fig. 3B) over the first image 230 (Fig. 2B), in which the first image is obtained earlier. For samples that do not include moving features, the user can use the earlier obtained first image 230 to guide the path of the microsphere to the point of interest. In some embodiments, for example, when the sample 290 includes moving features, the user can choose to switch back and forth the first lens configuration 210 and the second lens configuration 310, using the first lens configuration 210 to determine the second location (location of the second feature or the point of interest), and using the second lens configuration 310 to acquire a near-field image of the second feature 264. Alternatively, the imaging system may be configured to simulate a real-time near-field imaging experience by constantly updating the first image and the second image provided to the user. In embodiments where the microsphere 40 is held by the receptacle 140 in a fixed position relative to the adapter 120 or the opening 150, the user can choose to switch back and forth the first lens configuration 210 and the second lens configuration 310 to determine the second location with high accuracy.

[0052] Referring to Fig. 4A, in some embodiments, the microsphere 40 may be moved within the first field of view 270 while keeping the sample 290 substantially stationary relative to the objective lens 170. The microsphere 40, for example, may be moved relative to the optical axis 172 of the objective lens 170. A second image 430 (Fig. 4B) may be captured when the microsphere 40 is out of alignment with the optical axis 172 of the objective lens 170 and in alignment with the feature 266. In some embodiments, the second image 430 is characterised by a second field of view 272’, in which the second field of view 272’ is determined by the microsphere 40 which is out of alignment with the optical axis 172.

[0053] Referring to Fig. 4B, the second image 430 is obtained through the lens system in the second lens configuration 310 which has both the objective lens 170 and the microsphere 40. The lens system may be slightly adjusted to bring the feature 266 into focus without changing the objective lens 170.

[0054] Figs. 5A-5C show example imaging results taken during various stages of operating the microscope 100 in accordance with some embodiments of the present disclosure. Fig. 5 A shows an image 510 taken on a sample which is obtained through the lens system in the first lens configuration. The first lens configuration of the lens system includes the objective lens 170. As shown, some features are observable while some features are not observable or resolved by the lens system in the first lens configuration. Nevertheless, the relatively larger first field of view provided by the first lens configuration enables a second feature 564 or a second location 566 to be identified or selected. As shown, at this stage, the second feature 564 is not aligned with the microsphere 40. [0055] Fig. 5B shows an image 520 which is obtained in the course of moving the microsphere 40 towards the point of interest selected, which in this case is the second feature 564 located at the second location 566. At this stage, the lens system is configured with the highest magnification power achievable using the objective lens 170 of the microscope alone, that is, the lens system is in its first lens configuration.

[0056] Fig. 5C shows an image 530 which is obtained with the aid of the lens system in the second lens configuration. The second lens configuration includes the objective lens 170 and the microsphere 40. As shown, a second image of the second feature 564 can be captured using the microsphere 40, demonstrating the effectiveness of the present method for identifying one or more points of interest efficiently in a relatively large target area.

[0057] Embodiments of the method disclosed herein can be implemented with various types of adapters. Fig. 6 shows another embodiment of the adapter 120. The adapter 120, as shown in Fig. 6, is configured to hold a plurality of microspheres 40 and to be coupled to the optical microscope 100 as described earlier for non-contact optical microsphere nanoscopy. The adapter 120 includes a receptacle portion 600 and an opening 150. The opening 150, as shown in Fig. 6, is separated and is positioned at a predetermined separation distance 152 away from the receptacle portion 600. The opening 150 is preferably completely vacant to enable light to pass therethrough with insignificant blockage or distortion. Alternatively, the opening 150 may include a transparent material that allows light to pass therethrough.

[0058] In one embodiment, the receptacle portion 600 is configured to hold a plurality of microspheres 40. The plurality of microspheres 40, for example, may be of the same size or may be of different sizes. Further, the plurality of microspheres 40 may be formed of the same or different materials. The receptacle portion 600, in one embodiment, may include a through-hole having a gel-like or hydrogel film or layer filled therein to securely hold the plurality of microspheres 40. The plurality of microspheres 40, for example, may be randomly distributed within the receptacle portion 600 of the adapter 120. In other embodiments, the plurality of microspheres 40 may be uniformly distributed to form a 2-dimensional array of microspheres within the receptacle portion 600. In yet another embodiment, the receptacle portion 600 may also include other suitable forms and shapes to hold the plurality of microspheres. [0059] The adapter 120, as illustrated in Fig.6, may be employed in the method as described with reference to Figs. 2A-4B above to search and examine a target area or other target areas of interest. The adapter 120 as described in Fig. 6, for example, is moveable relative to the sample 290 and the objective lens 170. When the lens system of the imaging apparatus 100 is switched from the first lens configuration to the second lens configuration, the user can freely select one or more of the plurality of microspheres 40 to examine one or more selected features within the target area which are not observable or resolvable through the lens system in the first lens configuration. In another embodiment, the adapter 120 is a membrane. In such case, the receptacle 600 may be a portion of the membrane that is configured to hold multiple microspheres of the same or different sizes. In some embodiments, each of the plurality of microspheres 40 may be held in a fixed position relative to the adapter 120 or the opening 150. Since the position of the microsphere 40 relative to the adapter 120 or the opening 150 is predetermined, the user can choose to switch back and forth the first lens configuration and the second lens configuration with the selected microsphere 40 to determine the location of interest with high accuracy.

[0060] A method of nanoscopy according to one embodiment of the present disclosure is illustrated by Fig. 7. The method 700 includes moving a microsphere 40 relative to an objective lens 170 of the microscope 100 to bring the microsphere 40 into a first field of view 270 when a first feature 262 of a sample 290 is observable and a second feature 264 of the sample 290 is not observable through the objective lens 170. The first feature 262 and the second feature 264 are concurrently disposed under visible light or white light illumination within the first field of view 270, of which the first field of view 270 is provided by a lens system in a first lens configuration 210. The first lens configuration 210 includes the objective lens 170.

[0061] The method of nanoscopy may further include, when the microsphere 40 is in the first field of view 270, obtaining an image through the lens system, the image including a first image portion obtained through the lens system in the first lens configuration 210 and a second image portion concurrently obtained through the lens system in a second lens configuration 310, the second lens configuration 310 including the objective lens and the microsphere. Optionally, the method further includes moving the microsphere 40 to bring the second feature 264 into focus in the second image portion without changing the objective lens 170. The microsphere 40 is coupled to the microscope 100 and configured for controllable movement in ambient air without contacting the sample 290.

[0062] Alternatively, the method of nanoscopy may further include moving the microsphere 40 within the first field of view 270 while keeping the sample 290 substantially stationary relative to the objective lens 170. The method may optionally include moving the microsphere 40 relative to an optical axis 172 of the objective lens 170. The method may include moving the microsphere 40 out of alignment with the optical axis 172 of the objective lens 170 and into alignment with the second feature 264; as well as bringing the second feature 264 into focus in the second image portion without changing the objective lens 170.

[0063] Referring to Fig. 8, according to another embodiment of the present disclosure, a method of nanoscopy 800 includes, at 810, obtaining a first image portion of a target area 260 observable in a first field of view 270. The first field of view 270 is determined by an objective lens 170 of the optical microscope 100 configured to operate with a visible light or white light illumination source. The method also includes, at 820, obtaining a second image portion of a selected feature 264. The selected feature 264 is disposed in the target area 260. The second image portion is obtainable through a second lens configuration 310 of a lens system. The second lens configuration 310 includes the objective lens 170 and a microsphere 40 coupled to the microscope 100. The method further includes, at 830, controllably moving the microsphere 40 relative to the objective lens 170 to obtain a subsequent image portion of another selected feature 266 through the lens system in the second lens configuration 310 of the lens system. The first image portion is obtainable concurrently with either one of the second image portion and the subsequent image portion.

[0064] In one embodiment, the method further includes moving the microsphere 40 relative to the objective lens 170 to bring different parts of the target area 260 of a sample 290 into focus, without relative movement between the sample 290 and the objective lens 170. In another embodiment, the method further includes moving the microsphere 40 relative to the objective lens 170 to bring different parts of the target area 260 into focus, without changing either the objective lens 170 or a working distance 240 of the objective lens 170. Optionally, the opening 150 and the receptacle 140 are distinct and are spaced apart from each other. Optionally, the receptacle 140 is configured to hold a plurality of microspheres 40.

[0065] Fig. 9 is a block diagram illustrating the configuration of a system 900 for operating the imaging apparatus or microscope 100. The system 900 includes a control unit 910. The control unit 910 may be a central processing unit that performs various control and processing based on a control program or instructions. An input unit 930 is coupled to the control unit 910. The input unit 930 receives various types of information according to a user input. A storage unit 940 is coupled to the control unit 910 and the storage unit 940 stores the control program or instructions for causing the control unit 910 to perform operations of the various components of the imaging apparatus or microscope 100. An image processing unit 960 is coupled to the control unit 910. The image processing unit 960 is configured to acquire and process images at various stages of the operation.

[0066] The control unit 910, in one embodiment, may be configured to control movement of the sample stage 180. For example, the control unit 910 may output a control signal to a drive unit 970 to move the sample stage 180. Thus, the sample stage 180 is moveable in various directions so that the sample 290 is positioned below and aligned with the objective lens 170.

[0067] The control unit 910, in one embodiment, may be configured to control movement of the adapter 120. For example, the control unit 910 may output a control signal to the drive unit 970 to move the adapter 120 to a position such that the opening 150 is in alignment with the optical axis 172 of the objective lens 170. In such arrangement, the lens system of the imaging apparatus 100 is in the first lens configuration which includes the objective lens 170.

[0068] A visible light or white light is directed through the objective lens 170 and passes through the opening 150. The control unit 910 detects and maintains a first field of view 270 which is provided by the first lens configuration 210. The first lens configuration 210 includes the objective lens. A user can search and identify a target area 260 on the sample 290 through an eye piece of the microscope or through a display unit 950 coupled to the control unit 910. The image processing unit 960 captures and processes an image 230 that is obtained through the first lens configuration 210.

[0069] The control unit 910 outputs another control signal to the drive unit 970 to switch the first lens configuration 210 to a second lens configuration 310 without changing the objective lens 170. For example, the drive unit moves the adapter 120 to a position relative to the objective lens 170 so as to bring the microsphere 40 into the first field of view 270. In such arrangement, the lens system of the imaging apparatus 100 is in the second lens configuration 310 which includes the objective lens 170 and the microsphere 40.

[0070] The visible light or white light is directed through the objective lens 170 and passes through the microsphere 40 in the second lens configuration 310. The control unit 910 detects and maintains a second field of view 272 which is provided by the second lens configuration 310. The image processing unit 960 acquires an image 330 that is obtained through the second lens configuration 310.

[0071] The control unit 910 may output a further control signal to the drive unit 970 so as to move the microsphere 40 relative to the objective lens 170 to obtain a subsequent image 430. Alternatively, or additionally, the control unit 910 may be configured to output further control signal to move the microsphere 40 relative to the objective lens 170 to bring different parts of the target area 260 of the sample 290 into focus so as to produce an optimal high-resolution image.

[0072] The present disclosure describes various methods and systems to achieve enhanced nano-imaging. The methods and systems as discussed involves a scheme that switches between a first lens configuration 210 to a second lens configuration 310. The first field of view 270 offered by the first lens configuration 210 having the objective lens 170 allows a user with the ability to identify one or more points of interest in the target area 260. The second field of view 272 offered by the second lens configuration 310 having both the objective lens 170 and the microsphere 40 allows for high quality and high- resolution nano-scale observation. The methods and systems enable dual field of views to be achieved simultaneously within a single setup. Thus, the systems and the methods as described include benefits of both the optical microscope and the microsphere. As described, the adapter 120 includes a light transmission window and a receptacle within a unitary body. In embodiments where the receptacle is fixed relative to the adapter or the light transmission window, switching of the first lens configuration to the second lens configuration or vice versa can be achieved with high accuracy.

[0073] The methods and systems as discussed in the present disclosure are also highly flexible. The methods and systems can be applied to microsphere 40 with different diameters, various illumination wavelengths, and nearly to all optical designs. Furthermore, the microsphere 40 coupled to the microscope 100 is configured for controllable movement in ambient air without contacting the sample surface. Such non-destructive and non-contact feature enables imaging of various samples while preventing contamination on the surface of the sample.

[0074] This disclosure has been presented for purposes of illustration and description but is not intended to be exhaustive or limiting. Many modifications and variations will be apparent to those of ordinary skill in the art. The example embodiments were chosen and described in order to explain principles and practical application, and to enable others of ordinary skill in the art to understand the disclosure for various embodiments with various modifications as are suited to the particular use contemplated.

[0075] Thus, although illustrative example embodiments have been described herein with reference to the accompanying figures, it is to be understood that this description is not limiting and that various other changes and modifications may be effected therein by one skilled in the art without departing from the scope or spirit of the disclosure.

Claims

1. A method of nanoscopy comprising:

moving a microsphere relative to an objective lens of a microscope to bring the microsphere into a first field of view when a first feature of a sample is observable and a second feature of the sample is not observable through the objective lens, the first feature and the second feature being concurrently disposed under visible light illumination within the first field of view, wherein the first field of view is provided by a lens system in a first lens configuration, the first lens configuration including the objective lens and not including the microsphere, the microsphere being coupled to the microscope and configured for controllable movement in ambient air without contacting the sample.

2. The method as recited in claim 1, the method further comprising:

when the microsphere is in the first field of view, obtaining an image through the lens system, the image including a first image portion obtained through the lens system in the first lens configuration and a second image portion concurrently obtained through the lens system in a second lens configuration, the second lens configuration including the objective lens and the microsphere.

3. The method as recited in claim 2, wherein the method comprises moving the microsphere to bring the second feature into focus in the second image portion without changing the objective lens.

4. The method as recited in claim 1, wherein the method comprises moving the microsphere within the first field of view while keeping the sample substantially stationary relative to the objective lens.

5. The method as recited in claim 4, wherein the method comprises moving the microsphere relative to an optical axis of the objective lens.

6 The method as recited in claim 5, the method comprising: moving the microsphere out of alignment with the optical axis of the objective lens and into alignment with the second feature; and

bringing the second feature into focus in the second image portion without changing the objective lens.

7. A method of nanoscopy, the method comprising:

obtaining a first image portion of a target area observable in a first field of view, the first field of view being determined by an objective lens of a microscope configured to operate with a visible light illumination source;

obtaining a second image portion of a selected feature, the selected feature being disposed in the target area, the second image portion being obtainable through a second lens configuration of a lens system, the second lens configuration including the objective lens and a microsphere coupled to the microscope; and

controllably moving the microsphere relative to the objective lens to obtain a subsequent image portion of another selected feature through the lens system in the second lens configuration of the lens system, wherein the first image portion is obtainable concurrently with either one of the second image portion and the subsequent image portion.

8. The method as recited in claim 7, the method comprising moving the microsphere relative to the objective lens to bring different parts of the target area of a sample into focus, without relative movement between the sample and the objective lens.

9. The method as recited in claim 7, wherein the method comprises moving the microsphere relative to the objective lens to bring different parts of the target area into focus, without changing eitherthe objective lens ora working distance ofthe objective lens.

10. The method as recited in claim 7, the method comprising providing an adapter, wherein the adapter comprises an opening and a receptacle portion, wherein the opening is completely vacant and the receptacle portion is configured to hold the microsphere.

11. The method as recited in claim 10, wherein the opening and the receptacle portion are distinct and are spaced apart from each other.

12. The method as recited in claim 10, wherein the receptacle is configured to hold a plurality of microspheres.

13. The method as recited in claim 7, the method comprising providing an adapter, wherein the adapter comprises an opening and a receptacle portion, wherein the opening is configured to be a light transmission window and the receptacle portion is configured to hold the microsphere.

14. A method of nanoscopy, the method comprising:

obtaining a first image of a target area observable in a first field of view, the first field of view being determined solely by an objective lens of an optical microscope configured to operate under visible light illumination, the first image being formed by light received by the objective lens through a first window of an adapter;

obtaining a second image of a selected feature, the selected feature being disposed in the target area and observable in a second field of view, the second field of view being determined by a microsphere disposed on the adapter, the second image being formed by light received by the objective lens and the microsphere; and

controllably moving the adapter relative to an optical axis of the objective lens to switch between obtaining the first image and the second image, wherein the microsphere is disposed on the adapter.

15. The method as recited in claim 14, wherein obtaining the first image comprises: aligning the first window with the optical axis of the objective lens such that the first image is characterised by microscopic resolution.

16. The method as recited in claim 15, wherein obtaining the second image comprises: aligning the microsphere between the objective lens and the selected feature, the microsphere being disposed to receive evanescent waves from the selected feature such that the second image is characterised by super resolution.

17. The method as recited in claim 16, further comprising disposing the microsphere out of alignment with the optical axis of the objective lens.

18. An imaging system configured for use with the method as recited in any of claims 1 to 17.

19. An imaging system as recited in claim 18, the imaging system comprising:

an optical microscope having a lens system; and

a control unit coupled to the optical microscope, the control unit being configured to perform a method of:

capturing a first image at a microscopic level of resolution when the lens system is in a first lens configuration; and

capturing a second image at a nano-level of resolution when the lens system is in a second lens configuration, wherein the first lens configuration includes the objective lens and does not include the microsphere, and wherein the second lens configuration includes the objective lens and the microsphere.