WO2013064843A1 - Support pour échantillon au microscope comportant un réseau de diffraction permettant de former un motif d'éclairage sur celui-ci - Google Patents

Support pour échantillon au microscope comportant un réseau de diffraction permettant de former un motif d'éclairage sur celui-ci Download PDF

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Publication number
WO2013064843A1
WO2013064843A1 PCT/GB2012/052747 GB2012052747W WO2013064843A1 WO 2013064843 A1 WO2013064843 A1 WO 2013064843A1 GB 2012052747 W GB2012052747 W GB 2012052747W WO 2013064843 A1 WO2013064843 A1 WO 2013064843A1
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WO
WIPO (PCT)
Prior art keywords
sample support
microscope
diffraction grating
illumination
refractive
Prior art date
Application number
PCT/GB2012/052747
Other languages
English (en)
Inventor
Michael Geoffrey Somekh
Chung Wah See
Original Assignee
The University Of Nottingham
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The University Of Nottingham filed Critical The University Of Nottingham
Publication of WO2013064843A1 publication Critical patent/WO2013064843A1/fr

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Classifications

    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/34—Microscope slides, e.g. mounting specimens on microscope slides

Definitions

  • the present invention relates to an apparatus and method for making illumination patterns, such as for use in microscopy - particularly, but not exclusively, for use in confocal microscopy, two photon microscopy, fluorescence microscopy, Stimulated Emission Depletion microscopy, 3-D imaging etc.
  • This invention finds application in transmission or reflection, both fluorescence and non- fluorescence microscopy.
  • the resolution of widefield conventional microscopes is determined by the numerical aperture (NA) of the imaging optics, and is limited to half of the wavelength of the light used.
  • NA numerical aperture
  • the effect of the system NA manifests itself as the size of the illumination focal spot, with the resolution limit the same as for the widefield configuration.
  • a microscope sample support comprising a diffraction grating and a refractive portion, the refractive portion having a support surface arranged to support a sample to be viewed via a microscope, and the diffraction grating being arranged to direct illumination light through the refractive portion onto the support surface to form an illumination pattern thereon.
  • the refractive portion may comprise a refractive layer.
  • the refractive portion may have a refractive index, n, greater than 1 , optionally greater than 2, optionally greater than 2.5, optionally greater than 3, optionally greater than or equal to 3.5.
  • the refractive portion may comprise gallium phosphide, or titanium dioxide or a photoresist, such as a high index photoresist.
  • the grating period may be about the same as a wavelength of illumination light within the refractive portion. In some instances, the grating period may be larger than a wavelength of illumination light within the refractive portion. In other examples, the grating period may be less than, perhaps half of, a wavelength of illumination light within the refractive portion - for example, when the angle of incidence of the incoming light is large or extreme. In general, a smaller grating period provides a better resolution.
  • the diffraction grating may be fixed adjacent to the refractive portion.
  • Advantageously alignment of the grating relative to the refractive portion is made easier.
  • the diffraction grating may be embedded in the refractive portion.
  • Advantageously alignment of the grating relative to the refractive portion is made easier.
  • the microscope sample support may be provided as an easy-to-move unit that can be easily withdrawn as a whole form a microscope and repositioned efficiently within the same or another microscope without affecting the alignment of the grating relative to the refractive portion.
  • the refractive portion may have a thickness between 0.5 and 10 ⁇ .
  • a microscope comprising the microscope sample support of the above-mentioned aspect.
  • the microscope comprises a spatial light modulator arranged to direct illumination light onto the diffraction grating.
  • the light pattern directed onto the diffraction grating can be easily manipulated. This is particularly useful in conjunction with the microscope sample support of this invention.
  • a method of creating an illumination pattern comprising illuminating the microscope sample support of the above-mentioned aspect.
  • a method of creating different illumination patterns comprising variably illuminating the microscope sample support of the above-mentioned aspect.
  • the different illumination patterns may comprise patterns suitable for use in, for instance, stimulated emission depletion microscopy, confocal fluorescence microscopy or multi-photon fluorescence microscopy.
  • a first one of said different illumination patterns may comprise a pattern of excitation spots, and a second one of said different illumination patterns comprises a complimentary pattern of de-excitation spots.
  • the different spot patterns can be easily, consistently and repeatably created using this invention by using a single diffraction grating with different wavelengths of incoming light, for example.
  • Variably illuminating the microscope sample support may comprise any one or more of: varying a position of an illumination beam relative to the diffraction grating; or varying an angle of incidence of an illumination beam relative to the diffraction grating.
  • Variably illuminating the microscope sample support may comprise varying the thickness of the refractive portion.
  • Variably illuminating the microscope sample support may comprise varying properties of a spatial light modulator arranged to direct illumination light onto the diffraction grating.
  • an image processor arranged to process light information gathered using the method of the above- mentioned aspect to form an image of the sample, wherein the image processor is arranged to take into account the illumination pattern formed on the support surface by the diffraction grating.
  • Figure 1 shows a schematic representation of a microscope sample support according to one embodiment of the invention
  • Figure 2a shows a schematic representation of a square diffraction grating for use with the microscope sample support of figure 1 ;
  • Figure 2b shows a schematic representation of a triangular diffraction grating for use with the microscope sample support of figure 1 ;
  • Figure 3 is a flowchart outlining a method of creating an illumination pattern according to an embodiment of the invention.
  • Figure 4 is a flowchart outlining a method of creating different illumination patterns according to an embodiment of the invention.
  • Figures 5a to 5c show intensity distributions of illumination patterns created in one embodiment of this invention
  • Figures 6a to 6f comprise graphical simulations showing different illumination patterns created according to an embodiment of the invention
  • Figure 7 graphically illustrates properties of an illumination pattern created in one embodiment of the invention.
  • Figure 8 shows a series of intensity distributions at different positions within a microscope sample support system according to an aspect of the invention.
  • Figure 1 shows a microscope sample support 10 upon which a sample 12 is located.
  • the sample 12 is to be viewed through a microscope within which the support 10 can be inserted.
  • the sample support 10 comprises a diffraction grating 14 mounted on a substrate 16 in this embodiment.
  • the substrate may not b e present.
  • the substrate is in the form of a microscope slide.
  • amplitude or phase gratings can be used in the present invention.
  • an amplitude grating is formed with a thin (typically about 50nm) layer of metal with repeating patterns with square or triangular symmetry such as those shown in figures 2a and 2b and described below.
  • Phase gratings will be formed with regions of variable optical path length formed typically with areas of different refractive index. This could be achieved by interlacing low index photoresist and high index photoresist.
  • the phase grating has the advantage in that more energy is transmitted to the sample and that, in general, the strength of the higher diffraction orders is greater which results in higher spatial resolution and better lateral resolution.
  • Square and triangular gratings are just two examples of grating patterns - other forms of grating can be used as appropriate, as will be apparent to the skilled person.
  • the support 10 also comprises a refractive portion.
  • the refractive portion is a refractive layer 18 made from a refractive material having a refractive index, n, where n > 1.
  • refractive materials having different refractive indices may be used - for example, n > 1.5, n > 2, n > 2.5, n > 3, n > 3.5.
  • the thickness of the refraction layer, d is about 1 ⁇ . In other examples, the thickness of the refraction layer might be between 0.5 and 10 ⁇ . In yet further examples, the thickness may vary significantly.
  • the illumination pattern is repeated periodically with a change in thickness at a distance called the Talbot distance, as a result of the Talbot effect.
  • the Talbot distance (T D ) is a distance called the Talbot distance.
  • T D 2n , where n is the index of the refractive material, T is the period ⁇
  • the thickness, d, of the refractive layer is OcT D , where a is between a number greater than 0, for example 0.05, and 1 .
  • the light pattern at the sample surface can be shifted by angularly scanning the illumination beam, and apart from this the light pattern formed at the surface should be manipulated as described above in order to provide a pattern suitable for a desired application.
  • the thickness of the refractive portion it is desirable to restrict the thickness of the refractive portion to less than one Talbot distance.
  • the grating period might be less than 1 ⁇ and the corresponding Talbot distance might be about 10 ⁇ .
  • the refractive layer 1 8 comprises a support surface 20 arranged to support the sample 12.
  • the support surface 20 is substantially planar.
  • the diffraction grating 14 is embedded within the refractive layer 1 8.
  • the diffraction grating may be located adj acent, effectively adj acent or near (within a few nm of) the refractive portion. Substantially, there is no air gap between the diffraction grating and the refraction portion.
  • the diffraction grating is fixed (by being embedded) to the refractive portion. This feature offers accurate and repeatable alignment between the grating and the refractive portion, and hence the surface upon which the sample will rest, in use.
  • the diffraction grating is about 50 nm thick. In general, it should be thick enough to block an incident light beam.
  • the microscope sample support 10 is illuminated by illumination light 22, in this embodiment in the form of a beam of coherent light.
  • the illumination light 22 strikes the diffraction grating 14 at an incident angle, ⁇ .
  • the light is then directed via the diffraction grating 14 through the refractive layer 18 onto the support surface 20 where it forms an illumination pattern.
  • the precise illumination pattern that is formed is dependent upon a number of factors, including the wavelength of the illumination light 22, the angle of incidence, ⁇ , of the illumination light 22, the pattern of the diffraction grating 14, the refractive index of the refractive layer of 18, and the thickness, d, of the refractive layer 18.
  • a square diffraction grating 14a and a triangular diffraction grating 14b are shown as examples.
  • the illumination light passes through the sample 12 and is collected at an imaging system, such as a camera and image processor.
  • an imaging system such as a camera and image processor.
  • the light may take a different path after the illumination pattern is formed and after interaction with the sample.
  • reflected light may be collected at an imaging system of a microscope.
  • the illumination pattern might be arranged to interact with the sample by promoting fluorescence, and the imaging system is arranged to collect light generated via the controlled fluorescence.
  • the illumination pattern formed at the support surface 20 can be created in a particularly efficient manner.
  • the microscope sample support 10 it is an advantage for the microscope sample support 10 to be compact.
  • This invention allows the support 10 to be particularly compact.
  • the high refractive index of the refraction portion directs light of different incident angles in a desired pattern towards the support surface 20.
  • the grating period (which is the minimum distance, along any particular direction, in which the grating structure (diffraction grating) repeats itself) is greater than the wavelength of the illumination light in the refractive portion.
  • the grating period is smaller than a resulting focused spot size at the support surface. Therefore the waves emerging from the diffraction grating and that travel through the refractive portion are propagating waves. This is in contrast to other, known technology such as 'near-field' grating technology where the grating period is less than the wavelength of light in a sample.
  • the wavelength of the light in the refractive portion can be shorter than the wavelength of the light in the sample. This provides further improvement in resolution. This is because the resolution limit is usually taken as half of the optical wavelength. As the wavelength inside the refractive material is shortened by the refractive properties, represented by index n, of the material, the resolution will improve accordingly.
  • a method 30 of creating an illumination pattern comprises illuminating 32 a microscope sample support as previously described.
  • various parameters of the microscope sample support and illumination system can be varied in order to create a desired illumination pattern. Examples of types of illumination patterns can be created are provided below.
  • a method 40 of creating different illumination patterns comprises variably illuminating 42 a microscope sample support as previously described.
  • variably illuminating the microscope sample support comprises varying the position of an illumination beam relative to the diffraction grating (or other illumination pattern creating mechanism in other embodiments).
  • the position of an illumination source or beam might be varied in any of three dimensions.
  • the angle of incidence of the illumination beam relative to the illumination pattern creator might be varied.
  • the effect of this method is to provide a particularly efficient, quick mechanism for accurately creating different desired illumination patterns.
  • Using a single diffraction grating, which is fixed relative to the refractive portion and sample different useful illumination patterns can be created. This is useful in a number of different scenarios. Particularly this might be useful in Stimulated Emission Depletion microscopy as described in more detail below.
  • the illumination beam in order to effect the scanning of the light pattern at the sample, the illumination beam will need to be scanned over a small angle (typically a few degrees, maybe upto 10°) at the grating. Manoeuvring the illumination beam in other manners will usually result in a change in the light pattern at the sample surface, which may be necessary for example for 3D imaging.
  • variably illuminating the microscope sample support comprises varying a thickness of the refractive portion.
  • variably illuminating comprises keeping the grating unit (grating pattern, thickness and index) fixed but changing the illumination pattern. This is achieved in some examples using a spatial light modulator.
  • the thin film has a thickness of 1.07 ⁇ .
  • the illumination light has a wavelength of 495 nm.
  • different combinations of these parameters may be used to produce similar results, i.e. multiple focal spots.
  • the microscope sample support is a single unit where the refraction portion and the diffraction grating are fixed relative to each other, the support 10 can easily be removed and reinserted from a microscope or inserted into a different microscope without the need to substantially realign its components (particularly, the diffraction grating relative to the support surface).
  • this invention provides an advantageous way of creating different illumination patterns efficiently.
  • figures 6a to 6f illustrate how this feature is particularly useful in creating different patterns for use in Stimulated Emission Depletion microscopy.
  • Stimulated Emission Depletion microscopy is a relatively new, known technique which involves creating precise patterns of light around a single point on a sample being interrogated. These precise patterns of light are typically an excitation spot at the single point, followed by a de- excitation 'doughnut' also centred at the single point. The sample's response to the excitation spot and the de-excitation doughnut can be used to obtain a high-resolution image.
  • the illumination patterns having intensity distributions as shown in figures 6a and 6c can be created at the support surface.
  • Figures 6b and 6d illustrate the same intensity distributions as figures 6a and 6c respectively, but in an alternative ('mesh') format.
  • grating period may be used to produce similar results.
  • refractive index may be used to produce similar results.
  • the wavelength of illumination light used is 495 nm.
  • the refractive index of BK7 is 1.5218.
  • this invention allows the distributions shown in figures 6c and 6d to be obtained by changing the wavelength of illumination light to 600 nm. At this wavelength, the refractive index of BK7 is 1.5163.
  • This invention provides the opportunity to create different illumination patterns in real-time.
  • Prior microscopy systems are relatively bulky and cumbersome.
  • the microscope sample support unit of this invention allows a microscope user to transition from the illumination pattern of figure 6a to that of the 6c in real-time.
  • the focused spot and the 'doughnut' are generated with different optical components, they are generated using the same sample support with the present invention. This makes the alignment of the system optics much simpler.
  • FIG. 5a, 5b, and 6a to 6d show only a small proportion of the illumination pattern intensity distribution at the support surface.
  • the microscope sample support will produce more than 100 x 100 light spots/troughs.
  • This invention is therefore particularly useful in gathering high resolution data very quickly compared to prior solutions.
  • the resolution of the data obtained is comparable to known, high resolution single-point microscopy systems. In such systems, for an image containing 1000 x 1000 pixels it would be necessary in a standard high resolution scanning system to obtain 1 million scanning points. In contrast, using the present invention, a scan of only 10 x 10 is necessary since each scan itself covers 100 x 100 high resolution (sub- 100 nm) light spots. For example, this situation would apply relative to conventional confocal microscopy.
  • the structured illumination image is extracted by summing the array of spots to form gratings along different directions as is required for structured illumination.
  • FIG 7 plots the relative magnitude (light intensity) at the centre of the light spot against the distance from the diffraction grating (from 0 to 6 ⁇ ). The peak intensity oscillates axially.
  • Figure 8 shows a series of intensity distribution plots as d is changed from d to d+0.22 ⁇ . Within this range, the intensity at the centre of the spot changes from a maximum to a minimum.
  • This invention is particularly suitable for high-resolution microscopy. It is also particularly suitable for microscopy which requires a plurality, especially a large number, of data points, such as various types of scanning microscopy. This is because clearly the invention has the ability to provide very high resolution data (usually associated with single-point microscopy) in conjunction with the ability to provide simultaneous (often thousands of) point measurements.
  • This invention is also particularly suitable for use in Stimulated Emission Depletion microscopy (a known technology). This is because the invention offers a particularly efficient and accurate way of creating different illumination patterns centred at the same points upon a microscope sample.
  • the invention is also particularly useful when viewing biological or living samples since the increase in resolution allows the use of non-damaging (by virtue of its wavelength) light (i.e. not ultraviolet), particularly optical light. Wavelengths such as ultraviolet are prohibited in these types of applications since there is a possibility or likelihood of damage to cells of the sample under consideration.
  • the microscope sample support of this invention achieves high lateral resolution whilst also providing the benefits of high density parallel scanning. Also, the sample support unit is very convenient to use since it can be inserted directly into a microscope and used as a sample holder (in the same way as a microscope slide in conventional systems).
  • the diffraction grating may be a phase modulating grating instead of, or in addition to, the intensity modulating grating described herein.
  • Different grating shapes may b e used as appropriate.
  • a spatial light modulator may be used to create a desired light pattern incident upon the diffraction grating such that the amplitude and/or phase distribution of light hitting the grating is controllable.
  • the function of the diffraction grating can be changed in real-time so that effectively different diffraction gratings are provided in real-time.
  • Any other illumination pattern creator may be used as a substitute for the spatial light modulator.
  • the light beam passes through the SLM, its intensity distribution is modified. This modified intensity pattern is imaged onto the grating or held in close proximity to the grating. With this arrangement the light pattern at the sample can be altered by using appropriate input to the SLM. This is particularly useful for 3D imaging as the light spots can be focused at different locations along the axial direction.
  • the grating period may be smaller than a resulting focused spot size at the support surface.
  • an image processor in the form of hardware, software or a combination thereof, takes into account the illumination pattern created at the support surface in order to interpret data gathered after the illumination light has interacted with the sample. Taking this illumination pattern information into account allows the image processor to efficiently produce a high resolution image of the sample being viewed.

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  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Microscoopes, Condenser (AREA)

Abstract

La présente invention concerne un support d'éclairage (10) destiné à un échantillon au microscope (12) comportant une couche de réseau de diffraction (14) et une couche de réfraction (18). L'échantillon (12) devant être observé au microscope est disposé sur ladite couche (18). Ledit réseau (14) forme un motif d'éclairage sur ladite couche (18).
PCT/GB2012/052747 2011-11-03 2012-11-05 Support pour échantillon au microscope comportant un réseau de diffraction permettant de former un motif d'éclairage sur celui-ci WO2013064843A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB1118962.8 2011-11-03
GB201118962A GB201118962D0 (en) 2011-11-03 2011-11-03 Apparatus and method for making illumination patterns for microscopy

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WO2013064843A1 true WO2013064843A1 (fr) 2013-05-10

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104459971A (zh) * 2014-12-24 2015-03-25 中国科学院半导体研究所 一种基于集成光子芯片的结构光照明显微成像系统
US10261223B2 (en) 2014-01-31 2019-04-16 Canon Usa, Inc. System and method for fabrication of miniature endoscope using nanoimprint lithography
NL2020623B1 (en) * 2018-01-24 2019-07-30 Illumina Inc Structured illumination microscopy with line scanning
US10966597B2 (en) 2015-08-05 2021-04-06 Canon U.S.A., Inc. Forward and angle view endoscope

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Publication number Priority date Publication date Assignee Title
JP2003015048A (ja) * 2001-06-27 2003-01-15 Nikon Corp 格子照明顕微鏡
US20090225407A1 (en) * 2006-12-12 2009-09-10 Nikon Corporation Microscope device and image processing method

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003015048A (ja) * 2001-06-27 2003-01-15 Nikon Corp 格子照明顕微鏡
US20090225407A1 (en) * 2006-12-12 2009-09-10 Nikon Corporation Microscope device and image processing method

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10261223B2 (en) 2014-01-31 2019-04-16 Canon Usa, Inc. System and method for fabrication of miniature endoscope using nanoimprint lithography
CN104459971A (zh) * 2014-12-24 2015-03-25 中国科学院半导体研究所 一种基于集成光子芯片的结构光照明显微成像系统
US10966597B2 (en) 2015-08-05 2021-04-06 Canon U.S.A., Inc. Forward and angle view endoscope
NL2020623B1 (en) * 2018-01-24 2019-07-30 Illumina Inc Structured illumination microscopy with line scanning
WO2019147584A1 (fr) 2018-01-24 2019-08-01 Illumina, Inc. Microscopie à éclairage structuré à balayage de ligne
KR20200021472A (ko) * 2018-01-24 2020-02-28 일루미나, 인코포레이티드 라인 스캐닝을 이용한 구조화 조명 현미경법
EP3628066A4 (fr) * 2018-01-24 2020-07-15 Illumina Inc. Microscopie à éclairage structuré à balayage de ligne
US10928322B2 (en) 2018-01-24 2021-02-23 Illumina, Inc. Structured illumination microscopy with line scanning
AU2019211234B2 (en) * 2018-01-24 2021-04-29 Illumina, Inc. Structured illumination microscopy with line scanning
KR102306769B1 (ko) * 2018-01-24 2021-09-30 일루미나, 인코포레이티드 라인 스캐닝을 이용한 구조화 조명 현미경법

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