WO2013064843A1

WO2013064843A1 - Support for microscope sample comprising a diffraction grating for forming an illumination pattern thereon

Info

Publication number: WO2013064843A1
Application number: PCT/GB2012/052747
Authority: WO
Inventors: Michael Geoffrey Somekh; Chung Wah See
Original assignee: University of Nottingham
Current assignee: University of Nottingham
Priority date: 2011-11-03
Filing date: 2012-11-05
Publication date: 2013-05-10
Anticipated expiration: 2014-05-03
Also published as: GB201118962D0

Abstract

Illumination support (10) for microscope sample (12) comprising a diffraction grating layer (14) and a refractive layer (18), the sample (12) to be viewed with the microscope being disposed on the latter (18), said grating (14) forming an illumination pattern thereon (18).

Description

SUPPORT FOR MICROSCOPE SAMPLE COMPRISING A DIFFRACTION

GRATING FOR FORMING AN ILLUMINATION PATTERN THEREON

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. For a non-confocal scanning system, 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.

According to an aspect of the invention there is provided 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.

One recent, known development in optical microscopy uses structured illumination light to illuminate the sample surface with a series of periodic patterns. The resolution improvement is determined by the spatial frequency of the pattern that can be projected onto the sample. In previous implementations this is limited because it uses the microscope to project the pattern. Consequently the improvement in lateral resolution of such a known system compared to a conventional widefield microscop e is no greater than a factor of two. The grating structure of this invention overcomes these limitations; moreover, it also allows operation in other imaging modalities.

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. In use, 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 μιη.

According to another aspect of the invention there is provided a microscope comprising the microscope sample support of the above-mentioned aspect.

Optionally the microscope comprises a spatial light modulator arranged to direct illumination light onto the diffraction grating. Advantageously 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. According to another aspect of the invention there is provided a method of creating an illumination pattern comprising illuminating the microscope sample support of the above-mentioned aspect. According to another aspect of the invention there is provided 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. Advantageously, 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.

According to another aspect of the invention there is provided 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.

Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings in which:

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. In other embodiments, the substrate may not b e present. In this embodiment the substrate is in the form of a microscope slide. In some examples either amplitude or phase gratings can be used in the present invention. Typically, 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. In this embodiment of the invention, the refractive portion is a refractive layer 18 made from a refractive material having a refractive index, n, where n > 1. In some embodiments refractive materials having different refractive indices may be used - for example, n > 1.5, n > 2, n > 2.5, n > 3, n > 3.5. As will be described in further detail, it can be useful for the refractive portion to have a high refractive index. Examples of refractive materials and their refractive indices in the visible range include:

In general, improved resolution is achieved with high index materials. Referring to figure 1 , in this embodiment the thickness of the refraction layer, d, is about 1 μιη. In other examples, the thickness of the refraction layer might be between 0.5 and 10 μιη. 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),

defined as T_D = 2n , where n is the index of the refractive material, T is the period λ

of the grating and λ is the wavelength of the light. Using typical examples (T = Ι μιη, λ = 0.532μιη, and n = 2.5), then T_D = 9.4μιη. Depending on the application, 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 . Similar light patterns can also be generated using d = OcT_D + MT_D with M being an integer, which is the desired thickness OcT_D with additional thickness equalling multiple T_D' s. As an example only, the pattern at a distance of (T_D + 0. 1 ) μιη is substantially similar to the pattern at a distance of (2T_D + 0.1 ) μιη, or a distance of (5T_D + 0. 1 ) μιη etc. 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.

It is generally more difficult to fabricate a thicker refractive portion having uniform quality than a corresponding uniform quality thinner refractive portion. Therefore in some examples it is desirable to restrict the thickness of the refractive portion to less than one Talbot distance. For example, in a particular high resolution application, the grating period might be less than 1 μιη and the corresponding Talbot distance might be about 10 μιη.

The refractive layer 1 8 comprises a support surface 20 arranged to support the sample 12. In this example, the support surface 20 is substantially planar.

In this embodiment, the diffraction grating 14 is embedded within the refractive layer 1 8. In other embodiments, 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. In this example, 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. In this example, the diffraction grating is about 50 nm thick. In general, it should be thick enough to block an incident light beam.

Referring to figure 1 , 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, φ. 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, φ, 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. Referring to figures 2a and 2b, a square diffraction grating 14a and a triangular diffraction grating 14b are shown as examples.

In the example shown in figure 1 , the illumination light passes through the sample 12 and is collected at an imaging system, such as a camera and image processor. It will be clear to the skilled person that in other types of microscopy, the light may take a different path after the illumination pattern is formed and after interaction with the sample. As an example, after interaction with the sample, reflected light may be collected at an imaging system of a microscope. As another example, 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.

By virtue of using a refractive material of a known refractive index in conjunction with the diffraction grating being close to (in this case embedded in) the refractive portion, the illumination pattern formed at the support surface 20 can be created in a particularly efficient manner. For example, 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. Also, 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. With the present invention, when the refractive portion has a sufficiently high refractive index, 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.

Referring to figure 3, a method 30 of creating an illumination pattern comprises illuminating 32 a microscope sample support as previously described. 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.

Referring to figure 4, a method 40 of creating different illumination patterns comprises variably illuminating 42 a microscope sample support as previously described.

In one embodiment, 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). For example, the position of an illumination source or beam might be varied in any of three dimensions. Alternatively, or additionally, 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. In some embodiments, 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.

In another embodiment, variably illuminating the microscope sample support comprises varying a thickness of the refractive portion. Alternatively, or additionally, 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.

Referring now to figures 5a to 5c, an example of an illumination pattern that can be created according to this invention is illustrated. Figures 5a and 5b show intensity distributions (using different formats) of an illumination pattern that can be obtained using a triangular grating (grating period = 0.29 μιη (base of equilateral triangle)) of the type shown in figure 2b and using a thin film of refractive material having a refractive index of 3.5. The thin film has a thickness of 1.07 μιη. The illumination light has a wavelength of 495 nm. In other embodiments, different combinations of these parameters (grating period, refractive index etc.) may be used to produce similar results, i.e. multiple focal spots.

Referring to figure 5c, the line profile across one of the bright spots represented in figures 5a and 5b is shown. The FWHM of this spot is 84 nm. Therefore it will b e apparent to the skilled person that an illumination pattern comprising spots having a high resolution can be obtained using this invention.

As will be appreciated, a regular, precise pattern of spots is provided. Since 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). In accordance with the method of figure 4, this invention provides an advantageous way of creating different illumination patterns efficiently. As an example, 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.

Using a triangular diffraction grating (such as shown in figure 2b) with a grating period of 0.96 μιη (the base of the equilateral triangle) and a refractive film layer made of BK7 having a thickness of 5.67 μιη, 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.

In other examples, other forms of grating may be used to produce similar results, i.e. focused spots and doughnuts. Also, in other examples different combinations of these parameters (grating period, refractive index etc.) may be used to produce similar results.

Referring to figures 6a and 6b, the wavelength of illumination light used is 495 nm. At this wavelength, the refractive index of BK7 is 1.5218.

Without changing the microscope sample support, 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. For example, unlike prior microscopy systems where 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.

The peaks shown in figures 6a and 6b match exactly (at the support surface) the troughs shown in figures 6c and 6d. Figure 6f illustrates the resultant STED focal distribution. As can be seen from figure 6e, the resultant STED resolution (dotted line) is better then the resolution obtainable using an illumination wavelength of 495 nm alone (solid line).

Therefore, in real time, two different illumination patterns (' spot' and complementary 'doughnut') useful in STED microscopy can be obtained. As will be appreciated, figures 5a, 5b, and 6a to 6d show only a small proportion of the illumination pattern intensity distribution at the support surface. In actual fact, 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.

In another example, consider a set of measurements extracted with different angles of illumination as described above. This data is then processed to form the resultant image. Using different processing approaches one can extract different imaging modalities from this dataset. For example, it is possible to extract of non-confocal, confocal and structured light microscopy information with the same dataset. For instance, envisage scanning an array of spots in two dimensions. The confocal and non confocal images are obtained by measuring the reflected light at the detector; to obtain confocal imaging a small area conjugate with each illumination spot is used, whereas in the conventional imaging a larger area is used. By small and large we mean relative to the size of the diffraction limited spot as projected onto the detector. The structured illumination image is extracted by summing the array of spots to form gratings along different directions as is required for structured illumination. Referring to figure 7, using the same experimental setup as for figures 3a to 3c, there is also provided an illustration of how varying the thickness of the refraction portion changes the intensity distribution at the centre of a light spot. Figure 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 μιη). The peak intensity oscillates axially. Figure 8 shows a series of intensity distribution plots as d is changed from d to d+0.22 μιη. Within this range, the intensity at the centre of the spot changes from a maximum to a minimum. Combining this feature with known confocal imaging techniques, it is possible to obtain an image of a very thin slice of sample. The pattern at different distances from the diffraction grating is known and so imaging using different patterns can be easily carried out at different depths in the sample.

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.

In other embodiments 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. In this way, 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. When 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.

In some embodiments, the grating period may be smaller than a resulting focused spot size at the support surface.

Any material that is optically homogenous can be used as the refractive portion, and in general the higher the refractive index the better as resolution improvement increases as refractive index increases. According to another aspect of the invention 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.

Claims

1. 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.

2. The sample support of claim 1 wherein the refractive portion comprises a refractive layer.

3. The sample support of any preceding claim wherein the refractive portion has a refractive index, n, greater than 1 , optionally greater than 2, optionally greater than 2.5, optionally greater than 3, optionally greater than 3.5.

4. The sample support of any preceding claim wherein the refractive portion comprises gallium phosphide, or titanium dioxide or a photoresist, such as a high index photoresist. 6. The sample support of any of any preceding claim, wherein, in use, the grating period is equal to a wavelength of illumination light within the refractive portion.

7. The sample support of any preceding claim wherein the diffraction grating is fixed adjacent to the refractive portion.

8. The sample support of any preceding claim wherein the diffraction grating is embedded in the refractive portion.

9. The sample support of any preceding claim wherein the refractive portion has a thickness between 0.5 and 10 μιη.

10. The sample support of any preceding claim wherein the refractive portion has a

thickness of less than one Talbot distance, T_D, defined as T_D = 2n— , where n is the index of the refractive material, T is the period of the grating and λ is the wavelength of the light.

1 1. A microscope comprising the microscope sample support of any preceding claim.

12. The microscope of claim 1 1 comprising a spatial light modulator arranged to direct illumination light onto the diffraction grating. 13. A method of creating an illumination pattern comprising illuminating the microscope sample support of any of claims 1 to 10.

14. A method of creating different illumination patterns comprising variably illuminating the microscope sample support of any of claims 1 to 10.

15. The method of claim 14, wherein the different illumination patterns comprise patterns suitable for use in stimulated emission depletion microscopy, confocal fluorescence microscopy or multi-photon fluorescence microscopy. 16. The method of claim 15, wherein a first one of said different illumination patterns comprises a pattern of excitation spots, and a second one of said different illumination patterns comprises a complimentary pattern of de-excitation spots.

17. The method of any of claims 14 to 16, wherein variably illuminating the microscope sample support comprises 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.

18. The method of any of claims 14 to 17, wherein variably illuminating the microscope sample support comprises varying the thickness of the refractive portion.

19. The method of any of claims 14 to 18, wherein variably illuminating the microscope sample support comprises varying properties of a spatial light modulator arranged to direct illumination light onto the diffraction grating. 20. An image processor arranged to process light information gathered using the method of any of claims 13 to 19 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. 21. A microscope sample support, microscope, method of creating an illumination pattern, method of creating different illumination patterns or an image processor substantially as herein described with reference to any one or more of the accompanying drawings.