GB2553652A

GB2553652A - Scanning microsphere lens nanoscope

Info

Publication number: GB2553652A
Application number: GB1711268.1A
Authority: GB
Inventors: Stanescu Sorin; Guo Wei; Ll Lin
Original assignee: Lig Tech Ltd
Current assignee: Lig Tech Ltd
Priority date: 2016-07-14
Filing date: 2017-07-13
Publication date: 2018-03-14
Also published as: GB201612255D0; WO2018011583A2; WO2018011583A3; GB201711268D0

Abstract

An apparatus 1 suitable for use in super resolution microscopy comprises a microscope 10, an illumination source to illuminate a sample 20 and at least one microsphere 13 positioned between the sample 20 and the objective lens 11 which can provide resolution beyond the diffraction limit. The apparatus also includes a control beam source which can generate control beams 45 having a different wavelength to the illumination source, which captures the microsphere 13 and controls its position by the optical tweezer effect. The control beam source 45 may be a laser and the control beams generated may have wavelengths in the infrared region of the spectrum. The apparatus may also comprise one or more piezo acoustic actuators which can generate acoustic waves in order to control the position of the microspheres. A method of using the apparatus is also claimed. In another aspect, the invention includes an apparatus as above, and method of using said apparatus, however using piezo acoustic modulators in place of a control beam.

Description

(54) Title ofthe Invention: Scanning microsphere lens nanoscope

Abstract Title: Scanning Nanoscope with Radiative Pressure Control of Microsphere Lens (57) An apparatus 1 suitable for use in super resolution microscopy comprises a microscope 10, an illumination source to illuminate a sample 20 and at least one microsphere 13 positioned between the sample 20 and the objective lens 11 which can provide resolution beyond the diffraction limit. The apparatus also includes a control beam source which can generate control beams 45 having a different wavelength to the illumination source, which captures the microsphere 13 and controls its position by the optical tweezer effect. The control beam source 45 may be a laser and the control beams generated may have wavelengths in the infrared region ofthe spectrum. The apparatus may also comprise one or more piezo acoustic actuators which can generate acoustic waves in order to control the position ofthe microspheres. A method of using the apparatus is also claimed. In another aspect, the invention includes an apparatus as above, and method of using said apparatus, however using piezo acoustic modulators in place of a control beam.

At least one drawing originally filed was informal and the print reproduced here is taken from a later filed formal copy.

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SCANNING MICROSPHERE LENS NANOSCOPE

Technical Field of the Invention

The present invention relates to optical microscopy and in particular to super resolution optical microscopy. In particular, the invention relates to super resolution 5 optical microscopy using a single microsphere lens or multiple microsphere lenses.

Background to the Invention

Conventional optical microscopic imaging resolution has a theoretical resolution limit of approximately 200 nm within the visible light spectrum due to the far-field diffraction limit. As a result, conventional optical microscopic imaging is not suitable for observing subjects having structures smaller than this limit, for example live viruses (typically 5-150 nm, with some up to 300 nm). In order to image such structures beyond the optical diffraction limit, other techniques have been used.

Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) are often used to image specially prepared dead virus structures at very high 15 resolutions (10 nm) in vacuum. These techniques require complex sample preparation and are not suitable for in vivo imaging and measurements (the electron beam affects the living cells, viruses etc.).

Atomic force microscopes (AFMs) offer good imaging of small featured samples by a contacting probe. The sample may be easily damaged by the AFM’s tip.

Moreover, this technique does not offer a real image but a reconstructed imaging.

Stimulated emission depletion (STED) fluorescence optical microscopy is a recently established method for the imaging of cellular structures, bacteria and viruses beyond the optical diffraction limit, down to a resolution of 6 nm. This technique is based on the detection of light emitted by the fluorescing specimen when it is excited by laser light of a specific wavelength and switching off part of the fluorescent zone using another laser light of a different wavelength, i.e. the illuminating light spot can be smaller than the optical diffraction limit. STED fluorescent microscopes offer a better resolution through software processing of the images since the objective lenses collecting the images are still subject to diffraction limit, but the sample also requires a complex preparation (fluorescent labelling), which may not be always suitable for living organisms imaging. The fluorescent imaging technique gives good results mainly for organic samples. However, for high resolution, this technique is confronted with the challenge of photo bleaching which limits the minimum exposure time of light exposure to tens of seconds. In addition, the technique is not suitable for imaging non-organic materials such as metals, semiconductors and ceramics.

Recently, super resolution imaging has been demonstrated using arrays of 15 microspheres positioned between objective lens and sample. The microspheres used in such arrays are typically of the order of 5-10 pm in diameter. Use of microspheres enables the capture of evanescent waves present close to the target and optical imaging lenses. These evanescent waves carry high spatial frequency sub-wavelength information and decay exponentially with distance. Hence microspheres close to a surface are more effective at detection of said evanescent waves than a conventional objective lens.

The previous approach was to spread microspheres on the target surface for the imaging. Such an approach is not practical since microspheres may not be placed at the area of interest and position of microspheres at the correct position is challenging.

It is therefore an object of the present invention to enable super resolution microscopy that at least partially overcomes or alleviates some of the above problems.

Summary of the Invention

According to a first aspect of the present invention, there is provided an 5 apparatus for super resolution microscopy, the apparatus comprising: a microscope; an illumination source operable to illuminate a sample; one or more microspheres provided between the objective lens of the microscope and the sample; and a control beam source operable to generate one or more control beams of a different wavelength from the illumination source, so as to control the position of the one or more microspheres in relation to the sample and objective lens by the optical tweezer effect.

The apparatus above thus provides for simple control of the positioning of one or more microspheres to enable super resolution imaging. The use of a control beam with a different wavelength to the illumination source enables microscopy and in particular imaging to take place without interference from the control beam.

The apparatus may be an apparatus for super resolution optical microscopy. In such cases, the one or more microspheres may be an optically transparent microspheres.

The one or more microspheres may have a diameter of in the range 1-250 pm. In one embodiment, the one or more microspheres may have a diameter in the range 530 pm. In particular, the one or more microspheres may have a diameter of around 10 pm.

The one or more microspheres may have a refractive index in the range of 1.5

- 4. In one embodiment, the one or more microspheres may have a refractive index in the range of 1.5-2.5. In particular, the one or more microspheres may have a refractive index of around 1.59.

The one or more microspheres may be formed from any suitable material, including but not limited to polystyrene (PS), silica, BaTiCb.

The one or more microspheres may be provided in a chamber. The chamber may be provided on or over the sample. The chamber may be filled with fluid within which the one or more microspheres are dispersed. In some embodiments the fluid may be water, preferably distilled water. In other embodiments, the fluid may be an oil. The fluid is preferably a bacteria free transparent fluid.

The chamber may contain multiple microspheres. This can provide multiple opportunities to capturing and controlling the position of any one or more microspheres.

The chamber may be sealed or unsealed. Preferably, a sealed chamber is used with larger microspheres and an unsealed chamber is used with smaller microspheres.

Whilst a sealed chamber has the benefit of being reusable and hence more cost effective, 15 the focal length of the microsphere reduces as its diameter reduces. A sealed chamber can also prevent fluid escaping or evaporating due to heating by the illumination source or the control beam. Accordingly, it is impractical to provide a chamber base between the fluid and the sample without unduly impeding the utility of the apparatus for super resolution microscopy. As an example, an unsealed chamber is more suitable for microspheres with diameters of the order of, say, 30pm or smaller and a sealed chamber is suitable for microspheres with diameters of the order of, say, 30pm or larger, say between 30 and 250 pm.

In one embodiment, an unsealed chamber may comprise sidewalls in the form of a closed loop provided directly open to the surface of the sample. The sidewalls may be formed from an elastomeric material. The elastomeric material may be mounted on a thin transparent glass. The glass may have a thickness in the range 50 μιη - 150 pm.

The elastomeric material may be operable to retain the fluid inside the enclosure formed by the glass, the elastomeric material and the test sample surface. The thickness of the elastomeric material should be greater than the diameter of the microspheres used. For example, if the microsphere diameter is 10 pm, then the thickness of the elastomeric material may be 20 pm. The closed loop formed by elastomeric material can be substantially circular but in alternative embodiments may have alternative forms such as square or rectangular. The closed loop may be mounted on a substrate.

In another embodiment, a sealed chamber comprises sidewalls in the form of a closed loop provided upon the surface of a chamber base. Such a chamber may further comprise a chamber top provided upon the upper edge of the sidewalls. The sidewalls may be formed from an elastomeric material. The closed loop may be substantially circular but in alternative embodiments may have alternative forms. The chamber base may comprise a relatively thin sheet of transparent material. In one embodiment, the chamber base may comprise a sheet of sapphire glass or silica class of the order of 2080 pm thick. The relative thinness of the chamber base enables the chamber to be placed on top of a sample without unduly impeding super resolution achieved using the microspheres within the chamber. The chamber top may comprise a sheet of transparent material. In one embodiment, the chamber top may comprise a sheet of glass of the order of 120 pm thick. The relative thickness of the chamber top can improve the structural stability of the chamber may be within 75 - 200 pm.

The control beam source is preferably a laser. The control beam source may generate a beam or multiple beams with a wavelength in the infrared region of the spectrum. Use of an infrared control beam does not impact on imaging in the visible region of the spectrum.

In one embodiment, the control beam may have a wavelength in the range 8501064nm. In a particular embodiment, the control beam may have a wavelength of

975nm. Other examples may include: 808 ± 20 nm (GaAlAs diode laser, InGaAlAs diode laser), 940 nm ± 20 nm (InGaAs diode laser), 1064 nm (Nd:YAG , Nd:YVO4 lasers), 1047-1053 nm (Nd:YLF laser) 1070-1080 nm (Ytterbium fibre laser).

The control beam may have a focused beam diameter in the range 1-20 pm. In one embodiment, the control beam may have a diameter, in focus, of the order of, say,

1.5 pm.

The control beam may have a power in the range lmW-500mW. The power level selected will depend upon the circumstances. In general, higher power levels are 15 required to trap larger microsphere. Higher power levels are required to scan rather than to hold a microsphere in position; higher power levels can also increase the radiation pressure on the microsphere. The problems with excess power levels include the possibility of trapping more than one microsphere within the control beam, the possibility of boiling the fluid between the microsphere and sample, and the possibility 20 of damaging the sample directly.

The separation between the microsphere and the sample may be in the range of nm and 3 pm. The separation between the microsphere and the sample can be selected according to the circumstances. In circumstances where the control beam is operable to transport a microsphere to a particular location without imaging, the separation can be larger (e.g, above 3 pm). If imaging is needed during the microsphere transportation using the tweezer, a lesser separation, say in the range 10 nm - 1 pm is required. In some embodiments, the microsphere may be placed directly on the sample or on the base or top of a chamber during imaging. In such embodiments, the control beam may be switched off once the microsphere is in position.

The illumination source may be operable to generate monochrome or broad spectrum illumination as required or desired. The illumination source may be operable to illuminate the sample in reflection or transmission modes. The illumination source may comprise one or more LEDs or one or more other forms of lamp. Such other forms of lamp may include but are not limited to halogen lamps xenon lamps or similar.

In embodiments where the illumination source is operable to illuminate the sample in reflection, the apparatus may be provided with a restricted aperture between the illumination source and the objective lens. The restricted aperture may be operable to provide a narrow beam of illumination and light collection, thereby improving resolution. In particular, the aperture may enable the generation of a dark field to illuminate the sample and the rejection the out of focus light that might blur an image. Aperture size represents a trade-off between resolution and field of view: a smaller aperture increases resolution, but also results in a restricted field of view. The restricted aperture may comprise a pinhole or a slit. In one embodiment, the aperture has a width in the range 0.2-2mm.

The apparatus may be provided with an imaging device operable to capture an image of the sample as viewed through the objective lens. Typically, the imaging device may comprise an optical sensing array such as a CCD (charge coupled device) array or CMOS (complementary metal-oxide-semiconductor) image sensor.

The imaging means may be connected to image processing means operable to process the captured image. The processing may include processing to remove image distortions, stitching of multiple images or scanned images and enhancing the image contrast. Additionally or alternatively, the processing may include other steps such as filtering, shadow removal, edge detection, inversion, imaging stitching, contrast enhancement, distortion correction or the like.

The apparatus may comprise a sample mount upon which sample may be 10 positioned such that it can be viewed through the objective lens. The sample mount may be operable to controllably vary the separation between the objective lens and the sample. The sample mount may be operable to controllably vary the position of the sample relative to the objective lens in a plane perpendicular to the optical axis of the objective lens. In such cases, the sample mount may comprise a scanning stage. This can enable scanning of the sample relative to the objective lens so that an increased area of the sample can be imaged. An x-y translation stage is provided to enable the positioning of the laser tweezer beam or beams and translate the held sphere or spheres to the desired location or performing the scanning of an interested areas for imaging. The x-y translation stage may be manual or motorised.

The microscope may comprise multiple objective lenses. In such cases, the microscope may comprise means for switching between said objective lenses.

In some embodiments, the apparatus may comprise one or more piezo acoustic actuators. The piezo acoustic actuators may be operable to generate acoustic waves to control the position of the microsphere. The piezo electric actuators can thus provide a further level of control of the microsphere position. In one embodiment, the piezo electric actuators may be primarily operable to control the separation between the microsphere and the sample.

According to a second aspect of the present invention there is provided a method of performing super resolution microscopy comprising the steps of: providing one or more microspheres between the objective lens of a microscope and a sample; illuminating the sample using an illumination source; and generating one or more control beams with a control beam source, the one or more control beams operable to 10 control the position of the one or more microspheres by the optical tweezer effect.

The method of the second aspect of the invention can include any or all of the features of the apparatus of the first aspect of the present invention as desired or as appropriate.

The method may include the step of capturing said one or more microspheres 15 with the one or more control beams. The method may involve varying the separation between the sample and the microsphere. This variation may be achieved by varying the power and/or focal position of the control beam.

The method may include varying the separation between the objective lens and the sample. The method may include varying the position of the sample relative to the 20 objective lens in a plane perpendicular to the optical axis of the objective lens. In particular, the method may involve scanning the sample relative to the objective lens.

This enables an increased area of the sample to be imaged.

The method may include processing of the captured image. In particular, the method may include processing to remove image distortions. Additionally or alternatively, the method may include other steps such as filtering, shadow removal, edge detection, inversion, image stitching, contrast enhancement, distortion correction or the like.

The method may include the additional step of generating acoustic waves to control the microsphere position. In particular, the generated acoustic waves may be primarily operable to control the separation of the one or more microspheres and the sample.

According to a third aspect of the present invention, there is provided an apparatus for super resolution microscopy, the apparatus comprising: a microscope; illumination source operable to illuminate a sample; a microsphere provided between the objective lens of the microscope and the sample; and a plurality of piezo acoustic modulators operable to generate acoustic waves to control the position of the microsphere.

The apparatus of the third aspect of the present invention may incorporate any or all features of the first two aspects of the present invention as are desired or appropriate.

The apparatus may be an apparatus for optical super resolution microscopy.

According to a fourth aspect of the present invention there is provided a method of performing super resolution microscopy comprising the steps of: providing one or more microspheres between the objective lens of a microscope and a sample;

illuminating the sample using an illumination source; and generating acoustic waves using a plurality of piezo acoustic actuators, the generated acoustic waves operable to control the position of the microsphere.

The method of the fourth aspect of the present invention may incorporate any or all features of the first three aspects of the present invention as are desired or appropriate.

Detailed Description of the Invention

In order that the invention may be more clearly understood an embodiment/embodiments thereof will now be described, by way of example only, with reference to the accompanying drawings, of which:

Figure 1 is a schematic illustration of an apparatus according to the first aspect of the present invention;

Figure 2 is a schematic illustration of the optical tweezer effect on a microsphere in the present invention;

Figure 3 is a schematic illustration of an image obtained using an apparatus 15 according to the first aspect of the present invention;

Figure 4 is a schematic illustration of an alternative embodiment of an apparatus according to the first aspect of the present invention;

Figure 5 shows images obtained using the alternative embodiment of the present invention (a) using the additional aperture illustrated in figure 4, and (b) without the additional aperture illustrated in figure 4; and

Figure 6 illustrates (a) a cross-sectional view and (b) a plan view of a sealed chamber containing fluid and microspheres for use in the present invention;

Figure 7 illustrates (a) a cross-sectional view and (b) a plan view of an unsealed 5 chamber containing fluid and microspheres for use in the present invention; and

Figure 8 illustrates (a) a cross-sectional view and (b) a plan view of a sealed chamber containing fluid and microspheres for use in the present invention, where the chamber is additional provided with piezo acoustic modulators;

Turning now to figure 1, a schematic illustrations of an apparatus 1 for super resolution microscopy is shown.

A microscope 10 is provided in position to capture an image of a sample 20.

The microscope has an objective lens 12 mounted on an XY positioner allowing the 15 position of the lens relative to the sample to be varied in the XY plane. A microsphere is provided between the sample 20 and the objective lens 11 to provide super resolution microscopy performance.

In order to capture an image, the microscope 10 is provided with a camera 15.

The camera 15 is provided behind a dichroic mirror 19. To enable control of the 20 imaging performance of the camera 15 it is provided with a lens 16, IR filter 17 and an iris 18.

The sample is illuminated by an illumination source 30, comprising a white light LED illumination unit. Light 35 from the illumination source 30 is incident on the sample by passing through an iris 31 and a pair of dichroic mirrors 19, 39.

To control the position of the microsphere 13, a control beam 45 is generated 5 by control beam source 40. In this example the control beam source 40 is an infrared laser operating at 975nm. To optimise the control beam 45, a collimator 41 and expander lenses 42, 43 are also provided. As the control beam 45 is in the infrared region of the spectrum, it does not impact upon imaging carried out in the visible region of the spectrum. Nevertheless, to guard against any unwanted sensitively of eth camera

15 to infrared light the IR filter 17 is provided.

The control beam source 40 generates a control beam 45 that is directed through the objective lens 11 by dichroic mirrors 19, 39. The control beam 45 captures the microsphere 13 and holds it in position by the optical tweezer effect. The forces on a microsphere 13 due to this effect are illustrated in figure 2. As can be seen adjustment of eth separation between the microsphere 13 and the sample (Z direction) can be seen to be controlled by the gradient force and the radiation pressure. Accordingly, by varying the power of the control beam 45, the radiation pressure may be varied and the thus the separation between the sample and the microsphere may be varied. This can in turn vary the properties of images captured using camera 15. By use of the XY positioner 12, variation of the position of the microsphere 13 in the XY plane can be achieved.

The separation between microsphere and sample is between 100 nm and 3 pm.

Care should be taken not to increase the power level beyond the point at which the control beam 45 may damage the sample 20 or at which the control beam 45 may cause the fluid between the microsphere 13 and the sample 20 to heat excessively or boil.

The sample 20 is provided upon an XYZ scanning stage 21. The scanning stage is operable to be controllably moved with respect to the objective lens 2. In particular, the scanning stage 21 may be moved in the Z direction (aligned with the optical axis of the microscope), towards or away from the objective lens 11. In this way, the separation between the objective lens 11 and the sample 20 can be varied for optimum imaging performance.

In use, the microsphere may be one of multiple microspheres provided within a fluid filled chamber (described further in respect of figures 6 & 7). Typically the fluid is distilled water. The control beam is used to capture and position one of the multiple microspheres 13 for use in imaging. The power level of the control beam 45 may need to be varied to ensure capture of a microsphere 13 or to move the microsphere 13. Nevertheless, care should be taken not to use an excessive power setting as that may result in the capture of multiple microspheres 13, thus preventing stable imaging. In particular, the optimum power level may vary with the diameter of the microsphere 13. For instance, the minimum power to hold a lOum PS sphere is: 30mW; minimum power to move the sphere: 45mW; optimal power to move (and scan) the sphere: 90mW; and the minimum power to hold a 30um PS sphere is: 75mW; minimum power to move the sphere: 150mW; optimal power to move (and scan) the sphere: 230mW.

After adjustment of the microsphere 13 position and objective lens 11 focus, an image can be captured. If a wider image of the sample is desired, the scanning stage 21 is moved in the XY plane, perpendicular to the optical axis of the microscope 10. This scanning of the sample 20 past the microsphere 13 allows a wider image to be stitched together. An example of an image obtained using a 30um polystyrene microsphere 13 is shown at figure 3.

In figure 1, the sample 20 is illuminated in reflective mode. The skilled man will however appreciate that it is of course possible to utilise the present invention under transmissive illumination given a suitable sample mount or scanning stage 21. An example of such an imaging set up is shown in figure 4. For simplicity, figure 4a shows only the details of the transmissive illumination arrangement and the sample are shown.

In this example light 3 5t from the light source passes through an iris 36 and a condenser to be incident upon a sample 20. The iris 36 and condenser 37 can have their Z position adjusted using adjustment means 38. Light transmitted through the sample 20 passes through microsphere 13 to objective lens 11 for focusing and capture by the camera 15. The microsphere 13 is once again held in position by the control beam 45.

To further improve resolution of the image, as is shown in figure 4 but may also be applied in other embodiments, an additional aperture 50 is provided typically in the form of a slit. The smaller the size of the aperture 50, the greater the increase in resolution, albeit at the cost of a reduced field of view. This is illustrated in the images of figure 5 whereby figure 5a comprises a captured image utilising an additional aperture 50 and figure 5b illustrates a captured image of the same area captured without the additional aperture 50. The optimal slit width is in the range of 0.2mm-2mm.

Turning to the wider view of figure 4, an apparatus adapted to enable imaging in both reflexion mode and transmission mode is shown. For convenience, like features retain the same reference numerals as previous figures and will not be described further. In order to enable imaging in both modes, figure provides for a reflexion mode light source 3Or to generate illumination 35r for use in reflexion mode in addition to transmission mode light source 30t to generate illumination 3 5t for use in transmission mode. Compared with the transmission mode illumination 35t discussed above, the reflexion mode illumination source is provided with an ND filter 34, a homogeniser 33, apertures 31, 3 lr and a lens 32 to condition the illumination 35r.

As compared with figure 1, the control beam source is provided with an additional mirror 44 for directing the control beam 45 into the illumination 35r.

In addition to the above, a confocal scanning and detection unit 60 may be provided. The confocal scanning and detection unit 60 may be operable to generate confocal beam 65 which is combined with the control beam 45 and reflected light from the sample 20 by dichroic mirror 69. The confocal scanning and detection unit 60 is provided with a filter 61 to block incidence of reflections of control beam 45 from the confocal scanning and detection unit 60.

Whilst the above embodiments are described in relation to capture of a single microsphere 13 by a single control beam 45, the skilled man will appreciate that in 20 alternative embodiments, it may be possible to capture multiple microspheres 13 with one or more control beams 45.

Turning now to figures 6a & 6b, a sealed chamber 60 for retaining fluid 61 with multiple microspheres 13 dispensed therein is illustrated. The chamber 60 is adapted to be provided over the sample 20 (or the substrate upon which the sample 20 is provided). The overall width of the chamber 60 may be of the order of 16mm. The chamber comprises sidewalls 62 of elastomeric material, typically an adhesive pad or similar. The side wall 62 is formed into a closed loop on the sample 20. In the example shown, the closed loop is a substantially circular ring, however, the loop may have other shapes in alternative embodiments. The sidewall 62 may be of any suitable height. In one suitable example, the sidewall 62 is in the region of 75pm high.

The chamber 60 comprises a transparent base 63, and a transparent top 64, which together with sidewalls 62 act to trap the fluid 61 and microspheres 13 within the chamber. The sealed chamber 60 has the benefit of being reusable, making it relatively cost effective. In the event the fluid 61 is water, the water may include antimicrobial additives to prevent microbe growth impeding optical performance.

The top 64 is relatively thick, say 120pm, which provides structural stability. The base 63 is relatively thin, for example the base 63 may be formed from 20-80pm thick sapphire glass. This minimises the separation between the captured microsphere 13 and the sample 20 to enable effective imaging. Given that the practical range of separation for imaging decreases with the diameter of the microspheres 13, such a sealed chamber 60 is only suitable for use with microspheres above a threshold diameter. In the present example, considering polystyrene microspheres 13, such a sealed chamber 60 is only suitable for use with microspheres 13 of diameter greater than, say, 30pm.

Turning now to figures 7a & 7b, an unsealed chamber 70 for retaining fluid 71 with multiple microspheres 13 dispensed therein is illustrated. The chamber 70 is open directly upon the sample 20 (or the substrate upon which the sample 20 is provided). The overall width of the chamber 70 may be of the order of 16mm. The chamber comprises sidewalls 72 of elastomeric material, typically an adhesive pad or similar. The side wall 72 is formed into a closed loop on a transparent substrate 74. In the example shown, the closed loop is a substantially circular ring, however, the loop may have other shapes in alternative embodiments. The sidewall 72 may be of any suitable height. In one suitable example, the sidewall 72 is in the region of 75pm high.

In use, the sample 20 is placed directly over the open face 73 of the chamber 70 is placed against. As the chamber is placed directly upon the sample 20, there is no base 63 between the microsphere 13 and the sample. Consequently, it is possible for much smaller separations between the sample 20 and the captured microsphere 13 to be achieved. As such, the unsealed chamber 70 is suitable for use with microspheres smaller than the threshold for use in a sealed chamber 60. In the present example, considering polystyrene microspheres 13, such an unsealed chamber 70 is suitable for use with microspheres 13 of diameter less than, say, 30pm. As the chamber 70 is unsealed, it cannot be readily reused.

Turning now to figure 8, a further embodiment of a sealed chamber 80 containing fluid 81 and microspheres 13 is shown. The chamber 80 comprises a side wall 82, base 83 and top 84 in the same manner as the sealed chamber 60. In contrast to the chamber 60, the side wall 82 forms a substantially square loop. In addition, the chamber 80 further comprises piezo acoustic actuators 85 located at each side of the square loop. The skilled man will of course appreciate that other shapes of loop and other arrangements of actuators 85 are possible. The piezo acoustic actuators 85 are operable to generate acoustic waves which are transmitted through the fluid 81. The acoustic waves can be focussed in different locations throughout the chamber 80 in order to trap and/or move one or more microspheres 13. Control of the operation of the piezo acoustic actuators 85 is achieved by use of a control unit 86. The control unit 86 may be operable in response to commands generated by an external computer or the like. Additionally or alternatively, the control unit may be operable in response to a user actuable interface, such as a joystick or the like, to allow direct user control of microsphere 13 position.

The piezo acoustic actuators 85 can thus be used to provide control of microsphere position in additional to the control beam 45. In particular, the use of the actuators 85 may help stabilise microsphere position in the XY plane and may help vary microsphere position in the Z plane. This can be of particular use in moving microspheres 13 closer to the sample 20 in circumstances where an increase in control beam 45 power is not feasible or desirable.

Whilst the piezo acoustic actuators are described above in connection with use 15 in addition to control beam 45, it is also possible that the piezo acoustic actuators 85 may be used in place of the control beam 45 in alternative embodiments of the invention. This would enable the apparatus 1 to omit the control beam source and to consequently simply the arrangement of optical components. This would be of particular importance if the control beam may have an impact on the sample 20 or on the imaging of the sample 20.

Whilst the piezo acoustic actuators 85 have been described in relation to a sealed chamber 80, it is also possible that piezo acoustic actuators 85 can be fitted to an unsealed chamber along the lines of that shown in figures 7a & 7b. In such circumstances, care would need to be taken that the magnitude of the acoustic waves and the fluid volume does not result in fluid and/or microspheres being lost from the chamber.

The above embodiments are described by way of example only. Many 5 variations are possible without departing from the scope of the invention as defined in the appended claims.

Claims

1. An apparatus for super resolution microscopy, the apparatus comprising: a microscope; illumination source operable to illuminate a sample; one or more microspheres provided between the objective lens of the microscope and the

5 sample; and a control beam source operable to generate one or more control beams of a different wavelength from the illumination source, so as to control the position of the one or more microspheres in relation to the sample and objective lens by the optical tweezer effect.

2. An apparatus as claimed in claim 1 wherein the one or more microspheres are

10 provided in a fluid chamber.

3. An apparatus as claimed in claim 2 wherein the fluid chamber contains multiple microspheres.

4. An apparatus as claimed in any preceding claim wherein the control beam source is a laser.

15 5. An apparatus as claimed in claim 4 wherein the control beam source generates one or more control beams with a wavelength in the infrared region of the spectrum.

6. An apparatus as claimed in any preceding claim wherein the apparatus is provided with a restricted aperture between the illumination source and the

20 objective lens.

7. An apparatus as claimed in any preceding claim wherein the apparatus comprises a sample mount upon which the sample is positioned such that it can be viewed through the objective lens and wherein the sample mount is operable to controllably vary the position of the sample relative to the objective lens in a plane perpendicular to the optical axis of the objective lens.

8. An apparatus as claimed in any preceding claim wherein the apparatus comprises one or more piezo acoustic actuators operable to generate acoustic

5 waves to control the position of the microsphere.

9. A method of performing super resolution microscopy comprising the steps of: providing one or more microspheres between the objective lens of a microscope and a sample; illuminating the sample using an illumination source; and generating one or more control beams with a control beam source, the control

10 beam operable to control the position of the one or more microspheres by the optical tweezer effect.

10. A method as claimed in claim 9 wherein the method includes the step of capturing said one or more microspheres with the control beam.

11. A method as claimed in claim 4 or claim 10 wherein the method involves

15 varying the separation between the sample and the microsphere.

12. A method as claimed in claim 11 wherein the variation is achieved by varying the power of the one or more control beams.

13. A method as claimed in any one of claims 9 to 12 wherein the method involves scanning the sample relative to the objective lens.

20

14. A method as claimed in any one of claims 9 to 13 wherein the method includes the additional step of generating acoustic waves to control the microsphere position.

15. A method as claimed in claim 14 wherein the generated acoustic waves are primarily operable to control the separation of the microsphere and the sample.

16. An apparatus for super resolution microscopy, the apparatus comprising: a microscope; an illumination source operable to illuminate a sample; one or more microspheres provided between the objective lens of the microscope and the sample; and a plurality of piezo acoustic modulators operable to generate

5 acoustic waves to control the position of the microsphere.

17. An apparatus as claimed in claim 16 wherein the microsphere is provided in a fluid chamber.

18. An apparatus as claimed in claim 17 wherein the fluid chamber contains multiple microspheres.

10

19. An apparatus as claimed in claim 17 or claim 18 wherein the apparatus comprises a sample mount upon which the sample is positioned such that it can be viewed through the objective lens and wherein the sample mount is operable to controllably vary the position of the sample relative to the objective lens in a plane perpendicular to the optical axis of the objective lens.

15 20. A method of performing super resolution microscopy comprising the steps of:

providing a microsphere between the objective lens of a microscope and a sample; illuminating the sample using an illumination source; and generating acoustic waves using a plurality of piezo acoustic actuators, the generated acoustic waves operable to control the position of the microsphere.

20

21. A method as claimed in claim 20 wherein the method includes the step of capturing said microsphere with the acoustic waves.

22. A method as claimed in claim 20 or claim 21 wherein the method involves varying the separation between the sample and the microsphere.

Intellectual

Property

Office

GB1711268.1

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