EP3245663A1

EP3245663A1 - Sample holder for use in both a light optical microscope and a charged particle microscope

Info

Publication number: EP3245663A1
Application number: EP16710017.1A
Authority: EP
Inventors: Jacob Pieter Hoogenboom; Robert Jan MOERLAND
Original assignee: Delmic BV
Current assignee: Delmic BV
Priority date: 2015-01-12
Filing date: 2016-01-12
Publication date: 2017-11-22
Also published as: WO2016114656A1

Abstract

The invention relates to a sample holder for holding a sample, or to a sample arranged on said sample holder, for inspecting said sample with both a light optical microscope and a charged particle microscope. The sample holder comprises a substrate (51) having a first side which is provided with a conductive layer (53), wherein said substrate and said conductive layer are at least substantially optically transparent. Also the sample is arranged at the first side of the substrate, in particular at a side of the conductive layer facing away from the substrate. Between the conductive layer and the sample a spacing layer (60) of a predetermined thickness is provided. Said spacing layer is substantially electrically insulating and at least substantially optically transparent.

Description

Sample holder for use in both a light optical microscope and a charged particle microscope

BACKGROUND

The invention relates to sample holder for inspecting a sample arranged on said holder with both a light optical microscope and a charged particle microscope. The invention further relates to an apparatus comprising such a sample holder, which apparatus is arranged for inspecting a sample with both a light optical microscope and a charged particle microscope. In addition, the invention relates to a method for preparing a sample for inspecting said sample with both a light optical microscope and a charged particle microscope.

Microscopic analysis of samples increasingly relies on the complimentary capabilities of multiple imaging techniques. A prominent example is correlative light and electron microscope where a sample is analysed (mostly first) with the light microscope and identified regions of interest are subsequently inspected with an electron microscope. This can be done with separate stand-alone microscopes (which can also include multiple different forms of light microscopy, or even other inspection tools such as atomic force microscopy) , but also dedicated integrated microscopes exist. Such integrated microscopes are described, for example in WO2012008836.

In order to be able to study the same sample with both a light optical microscope and a charged particle microscope, such as an electron microscope, the holder or substrate onto which the sample needs to be mounted preferably is arranged to be compatible with both inspection techniques, in particular holder or substrate preferably is arranged to allow inspection with both photons and charged particles .

In light optical microscopy usually optical transparent sample holders or substrates are used.

In charged particle microscopy, such as electron microscopy, the substrate or the combination of sample and substrate needs to be conductive. A typical approach to make sample plus substrate conductive is coating with a conductive material such as gold. However, such a gold layer is not optically transparent.

In many situations, e.g. in the important case when thin sections of slices of biological tissue are examined, it is sufficient if the substrate is conductive. Such tissue sections are typically very thin (20 - 200 nm) and also typically stained with heavy metal substances such as osmium tetroxide or uranyl acetate. This makes that charging of this kind of sample on a conductive substrate is negligible.

Such a conductive substrate may be a silicon wafer. However, a silicon substrate is not transparent at the wavelengths used in light optical microscopy.

An alternative, as for example disclosed in EP 1998206, is to use a glass substrate with a layer of a transparent conductive material, such as Indium-Tin-Oxide (ITO) . When using such a glass substrate, light microscopy can be performed through the optical transparent glass substrate, which increases collection efficiency, resolution, ease of working, and is compatible with most procedures in light microscopy. In addition, an ITO-coated glass substrate can be used for imaging a sample in an electron microscope. Even thin unstained biological samples can be imaged in an electron microscope, using an ITO-coated glass substrate.

Contrary to intuition and common perception, the inventors have found that when studying the fluorescence from a sample on an ITO-coated glass substrate, the intensity of the fluorescent light from the sample is much lower than expected on the basis of the density of fluorescent molecules in the sample. This decrease in the fluorescence intensity, also referred to as quenching, poses a problem for using fluorescence microscopy in a correlative light and electron microscope. Said quenching may result in artefacts in an image obtained by said fluorescence microscopy.

It is an object of the present invention to provide an optimized sample holder to carry out light and charged particle optical microscopy which at least reduces the quenching.

SUMMARY OF THE INVENTION

According to a first aspect, the invention provides a sample holder for inspecting a sample arranged on said sample holder with both a light optical microscope and a charged particle microscope, wherein said sample holder comprises :

a substrate having a first side which is provided with a conductive layer, wherein said substrate and said conductive layer are at least substantially optically transparent, and

a spacing layer, wherein said spacing layer is substantially electrically insulating, at least substantially optically transparent, and is arranged on said conductive layer at a side facing away from said substrate. Preferably the spacing layer comprises a layer of inorganic material .

The present invention thus provides a sample holder with provided at least a reduction of the quenching of the fluorescence, by arranging a spacing layer between the conductive layer on the substrate, and the sample. The term Spacing layer' in this application also encompasses a multilayer stack.

The spacing layer is a substantially electrically insulating layer, preferably the spacing layer is a dielectric layer. The inventors found that, contrary to intuition and common perception, transparent conductors, despite their transparency and near-zero imaginary part of the refractive index, can substantially dissipate the energy of an emitter of light, such as a fluorescent molecule, nanoparticle, or protein, by near-field interaction. This dissipation may be caused by Ohmic (resistive) dissipation of the induced local currents in the conductor.

If one looks at an emitter of light, for example a fluorescent molecule, nanoparticle, or protein, not only is energy radiated into the far field, there is also a near field contribution. This near field contribution is not radiated out, but as the name suggests is only contained in a local (evanescent) field. As the near field decays according to a power law with distance from the emitter, a non-radiative dissipation of the energy by a conductor takes place in close proximity (0 - 20 nanometers) from such a conductor. Therefore, for emitters within this close proximity, a substantial amount of their excited state energy is quenched in this way, leading to strongly reduced fluorescence yield and thus measured intensity. It follows that in case of a thin tissue section or other sample with fluorescent markers, the fluorescence measurement is strongly biased: the fluorescence from molecules close to the surface is very dim or not emitting light at all, while the fluorescence of molecules further away from the substrate appears with normal emission intensity. Thus, any material with a finite conductivity will give resistive near-field quenching of the fluorescence of molecules which are arranged close to said material, even if the material is transparent for far-field radiation. The spacing layer according to the invention, which is an insulating layer, provides a distance between the conductive layer and the sample, and thereby reduces the resistive quenching of the fluorescence of the molecules in the sample which are close to the sample holder.

Furthermore, the substrate, conductive layer and the spacing layer are at least substantially optically transparent to allow to obtain an light optical image from a sample through said sample holder.

It is noted that the EP1160192 publication discloses a work substrate which is made of a glass substrate onto which a thin ITO film (189 nm thick) is evaporated, and which is then dip-coated with a thin polystyrene film (15 nm thick) to increase the adhesion force between the object and the substrate. Such a work substrate has various disadvantages when used in both a light optical microscope and a charged particle microscope: a. A polymer layer, in particular a polystyrene film, degrades in time when exposed to charged particles in general, and electrons in particular. b. Although not explicitly disclosed in EP1160192, dip-coating usually provides a thin film on both the upper and the lower surface of the work substrate, which has a negative influence on an optical image of a sample when the image is obtained through the work substrate.

c. The polymer layer, in particular a polystyrene film, may produce cathodoluminescence light, i.e. light generated by the electron beam falling on the polymer layer, which cathodoluminescence light from the polymer layer may interfere and/or hinder the acquisition of an optical image of a sample on top of said polymer layer.

By using a spacing layer comprises a layer of inorganic material, as in the present invention, one or more of the above disadvantages are at least partially obviated.

As described above, the sample holder of the invention is provided with a substantially electrically insulating spacing layer to provide a distance between the conductive layer on the substrate and the sample to reduce resistive quenching.

Alternatively, a substantially electrically insulating spacing layer may also be provided on a side of the sample which is arranged in contact with the sample holder. In this case, and according to a second aspect, the invention provides a sample holder with a sample arranged on said sample holder, for inspecting said sample with both a light optical microscope and a charged particle microscope, wherein said sample holder comprises:

the sample, wherein said sample is provided with a spacing layer, wherein said spacing layer is substantially electrically insulating and at least substantially optically transparent, and wherein the spacing layer of said sample is arranged on said conductive layer at a side facing away from said substrate.

With respect to this alternative arrangement of the insulating spacing layer, it is noted that the insulating spacing layer is part of the sample, and as such is only present between the sample and the conductive layer. In this alternative arranged, the insulating spacing layer is substantially not directly exposed to the charged particle beam. Accordingly, in an embodiment of this alternative arrangement, the spacing layer may comprise a polymer layer.

In an embodiment the thickness of the spacing layer is in a range of 7 - 30 nanometers, preferably 8 - 20 nanometers, most preferably 10 nanometer. In particular, these preferred ranges in the thickness of the spacing layer proved to provide a substantial reduction in the quenching of the fluorescence when the sample is observed with a light optical microscope and in addition to enable to image said sample in a charged particle microscope, in particular a scanning electron microscope. The requirement for an insulating spacing layer is at odds with the requirement for a conductive substrate for electron microscopy. To provide a solution for this problem it is first of all noted that the inventors have found that the magnitude of the quenching phenomenon is in agreement with a power law decaying with distance from the conductive layer. Secondly, it is noted that the amount of charging of a sample in an electron microscope depends on the number of ejected or absorbed electrons as a result of scanning with the primary beam of electrons. For a thinner sample, less collisions of the primary and back-scattered beams of electrons will take place, resulting in less charging. It should be noted that this also depends on electron energy: for higher energy, less collisions occur in a layer of specific thickness than at lower energy with the same current. However, inspection of biological samples with a scanning electron microscope typically takes place at low electron energy (< 2keV) . The inventors found that there is an optimum thickness for an insulating spacing layer on top of a transparent conductive layer, with which quenching of fluorescence can be substantially prevented while still allowing for electron beam inspection without charging. This optimized thickness, as described in the embodiment above, may depend on the amount of fluorescence quenching that one still allows, and the electron energy and current needed for imaging .

In an embodiment, the spacing layer is provided with a conductive grid which extends substantially through said spacing layer and connects to said conductive layer. The conductive grid provides a conductive path from the sample to conductive layer on the substrate. The conductive grid may be advantageous for electron beam inspection of a sample when using a relatively thick spacing layer and/or a high current for imaging. However, when observing the fluorescence in such a sample using in a light optical microscope, quenching of the fluorescence may occur at positions close to the conductive grid. In an embodiment, the spacing layer comprises alignment markers. Said alignment markers can be used to aid alignment of images taken with the light optical microscope and with the charged particle microscope. In an embodiment, the alignment markers are embedded into the spacing layer.

The sample holder according to the invention is preferably optimized for Correlative Light and Electron Microscopy (CLEM) and may suitably be used in both an integrated CLEM microscope, but also when shuttling between a stand-alone light microscope and a stand-alone electron microscope. At least in the latter case, it may be beneficial that thin alignment markers are arranged at, or embedded into the spacing layer. These thin alignment markers are for example of a thickness equal to or smaller than the thickness of the insulating spacing layer. Such markers can be arranged by nano-patterning (for example using lithography, charged particle beam lithography, charged particle beam induced deposition) , or by deposition of nanoparticles from a solution (for example by spin-coating) , preferably prior to the deposition of the insulating spacing layer. The alignment markers can then be used to aid registration between images taking with both the light microscope and the electron microscope.

In addition or alternatively, the thin insulating spacing layer may itself be patterned, providing a supporting grid for the (tissue) sample. On the one hand, the grid may aid above mentioned microscope registration and retrieval of regions of interest. On the other hand, a patterned spacer may provide uncovered areas of the transparent conductive layer onto which a ground potential or voltage supply can be attached. Also with such a spacing grid, charging of the spacer layer can be completely prevented when imaging the sample over the mentioned uncovered areas. A type of patterned spacing layer may also consist of a single island' on top of the transparent conductor, where the sample is then placed on top of this island. It should be noted that the conductive layer, onto which the thin insulating spacing layer is supported, should be connected to a reference potential, at least when the sample is illuminated with a charged particle beam, in particular an electron beam. This reference potential may be ground potential, or, for imaging with a decelerating field, a negative bias potential. It is noted that for very thin insulating spacing layers (1 - 2 nanometers) direct charge tunnelling to the ground conductive layer may advantageously take place. However this thickness is too thin to substantially prevent quenching.

In an embodiment, said substrate is a rigid substrate suitable for transfer between a stand-alone light optical microscope and a charged particle microscope. In an embodiment, the substrate comprises a glass substrate, for example a glass microscope slide provided with an indium- tin-oxide coating at least at a side of said slide which is used for carrying the sample.

In an embodiment, the spacing layer comprises a layer of aluminium oxide or Si0₂. Such spacing layers may be grown by atomic layer deposition, vapour deposition, or by thin-film growth from solution or in a sol-gel process. Also other solid dielectric materials can be used as the insulating spacing layer.

It should be noted that the sample holder or substrate design according to the invention may also be beneficial for use in other areas of nanotechnology where optically active or emissive materials need to be placed close to a transparent conductive surface. For example, if a substrate needs to be patterned with a charged particle technique such as electron beam lithography, the substrate also needs to be conductive. If the patterning is then meant to make patterned structures of emissive material, it would also be beneficial to use the invented substrate design to prevent signal loss due to quenching while still allowing high-resolution lithography without charging.

According to a third aspect, the invention provides an apparatus for inspecting a sample with both a light optical microscope and a charged particle microscope, wherein said apparatus comprises a sample holder as described above.

In an embodiment, said apparatus comprises a light optical microscope to observe the sample, wherein the light microscope is arranged to detect luminescence or fluorescence light emitted from the sample, a charged particle microscope to observe or modify the sample with a beam of charged particles, and a sample holder to support the sample in a position in which it can be observed with both microscopes, wherein the sample holder comprises:

a spacing layer, wherein said spacing layer is substantially electrically insulating, at least substantially optically transparent, and is arranged on said conductive layer at a side facing away from said substrate. Preferably, the spacing layer comprises a layer of inorganic material .

In an alternative embodiment, said apparatus comprises a light optical microscope to observe the sample, wherein the light microscope is arranged to detect luminescence or fluorescence light emitted from the sample, a charged particle microscope to observe or modify the sample with a beam of charged particles, and a sample holder to support the sample in a position in which it can be observed with both microscopes, wherein the sample holder comprises :

a sample, wherein said sample is provided with a spacing layer, wherein said spacing layer is substantially electrically insulating, at least substantially optically transparent, and wherein the spacing layer of said sample is arranged on said conductive layer at a side facing away from said substrate.

In an embodiment, the conductive layer is connected to a supply unit which is arranged to keep the conductive layer at a substantially constant voltage, at least during illumination of the sample holder with the beam of charged particles. In an embodiment, the constant voltage is a ground potential. In an alternative embodiment, the constant voltage is a negative bias voltage.

According to a fourth aspect, the invention provides a method for preparing a sample on a sample holder for inspecting said sample with both a light optical microscope and a charged particle microscope, wherein said sample holder comprises a substrate having a first side which is provided with a conductive layer, wherein said substrate and said conductive layer are at least substantially optically transparent, and wherein said method comprises the steps of:

providing one side of the sample with a spacing layer, wherein said spacing layer is substantially electrically insulating, at least substantially optically transparent, and

arranging the sample on the first side of the substrate, wherein the spacing layer is arranged between the sample and the conductive layer.

In an embodiment, the spacing layer comprises one or multiple polymer layers of desired thickness, which are deposited on said one side of the sample. Such a layer may, for example, be arranged on said one side of the sample using a so-called Langmuir-Blodgett technique, which is described in more detail below with reference to figure 4.

The various aspects and features described and shown in the specification can be applied, individually, wherever possible. These individual aspects, in particular the aspects and features described in the attached dependent claims, can be made subject of divisional patent applications .

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be elucidated on the basis of an exemplary embodiment shown in the attached drawings, in which :

Figures 1A and IB schematically depict basic designs of an apparatus for inspecting a sample with both a light optical microscope and a charged particle microscope,

Figure 2 is a schematic representation of a sample on a sample holder and a graph schematically showing the Intensity of the fluorescence signal as a function of the distance to the transparent conductive layer of the sample holder,

Figure 3 is a schematic representation of a sample on a sample holder, wherein an insulating spacing layer is arranged between the sample and the transparent conductive layer of the sample holder, and a graph schematically showing the Intensity of the fluorescence signal as a function of the distance to the conductive layer of the sample holder,

Figure 4 is a schematic representation of a sample on a sample holder, wherein a insulating spacing layer is arranged between the sample and the transparent conductive layer of the sample holder, and the spacing layer is provided with a conductive grid,

Figure 5 is a schematic representation of a sample on a sample holder, wherein a insulating spacing layer is arranged between the sample and the transparent conductive layer of the sample holder, and the spacing layer is provided with alignment markers,

Figure 6 is a schematic representation of a sample on a sample holder, wherein a patterned insulating spacing layer is arranged between the sample and the conductive layer of the sample holder, and Figure 7 is a schematic representation of the steps of a method for preparing a sample on a sample holder for inspecting said sample with both a light optical microscope and a charged particle microscope

DETAILED DESCRIPTION OF THE INVENTION

By way of exemplifying a typical context of the present invention, a principle and relatively simple set up of a so-called optical SEM combination as known from the prior art will be provided. This example however by no means excludes any known or yet unknown variation or alternative thereof .

With reference first to figure 1A, the basic design of a first example of an inspection apparatus 1 of the invention is explained. It comprises in combination at least an optical microscope 2, 3, 4 and a charged particle microscope 7, 8, such as an ion- or electron microscope.

The charged particle microscope 7, 8 comprises a source 7 for emitting a primary beam 9 of charged particles to a sample supported by a sample holder 10 according to the invention. The sample holder 10 comprises a substrate having a first side which is provided with a conductive layer. To substantially prevent charging of the sample and sample holder 10, the conductive layer of the sample holder 10 is connected to ground potential 54 to keep the conductive layer at a substantially constant voltage, at least during illumination of the sample holder 10 with the beam of charged particles 9. In addition, the sample holder 10 is at least substantially optically transparent to allow the transmission of light 12 from the sample to the optical microscope 2, 3, 4. Typically the sample on the sample holder 10 is arranged on an assembly of stages 5 which are known form the prior art and are therefor not shown in detail. The stages 5 are arranged for moving the sample supported by the sample holder 10 with respect to the optical microscope 2, 3, 4, and/or the charged particle microscope 7, 8. The charged particle microscope further comprises a detector 8 for detection of secondary charged particles 11 backscattered from the sample 10, or emitted, transmitted, or scattered from the sample 10 and possibly induced by the primary beam 9. The charged particle microscope 7, 8 is substantially arranged inside a vacuum chamber 13.

The optical microscope 2, 3, 4 is equipped with a light collecting device 2 to receive in use fluorescence and/or luminescence light 12 emitted by the sample 10 and induced by the primary beam 9 of radiation, and to focus it on a photon-detector 4. The light collecting device 2 may be an objective lens, a mirror or a glass fiber. It may also consist of a plurality of devices to arrange for collecting and focusing of the concerning luminescence light that is emitted by the sample 10, e.g. using a known per se CCD camera. In the present example the optical microscope 2, 3, 4 is of an confocal type having a pinhole 3 between the light collecting device 2 and the photon detector 4. The optical microscope 2, 3, 4 in this example, is placed entirely inside the vacuum chamber 13 of the charged particle microscope 7, 8.

In figure 1A, a controller 15 is provided and useable as an automation unit, e.g. in the form of a computer, including a personal computer provided with dedicated software, implementing one or more methods of use of the inspection apparatus. The controller 15 may typically be provided with one or more screens, e.g. one screen or screen part for depicting the recorded optical image, and the other or another part of the same screen depicting an image, in particular of the same object, i.e. substrate, recorded via the charged particle part of the inspection apparatus 1.

In figure IB a second example of an integrated system 20 is presented, in particular without a confocal detection system, in which the present invention may be applied. The integrated system 20 of this second example comprises a Scanning Electron Microscope (SEM) 27 comprising a vacuum chamber 23 which is connected to a vacuum pump via a connector 35. Inside said vacuum chamber 23, a sample on a sample holder 30 is arranged, which sample can be irradiated with an electron beam 29. Secondary electrons 31 backscattered from the sample, or emitted, transmitted, or scattered from the sample are detected by a detector 28.

The sample holder 30 comprises a substrate having a first side which is provided with a conductive layer. To substantially prevent charging of the sample and sample holder 30, the conductive layer of the sample holder 30 is connected to a supply unit 55 which is arranged to keep the conductive layer at a substantially constant voltage, at least during illumination of the sample holder 30 with the beam of charged particles 29. As schematically indicated in figure IB, the constant voltage is a negative bias voltage.

In addition, the sample holder 30 is at least substantially optically transparent to allow the transmission of light 38 from the sample to a microscope objective 22.

Typically the sample on the sample holder 30 is arranged on an assembly of stages 5 which are known form the prior art and are therefore not shown in detail. The stages 5 are arranged for moving the sample with respect to the Scanning Electron Microscope 27, and/or the light optical microscope system.

Below the sample on the sample holder 30, a microscope objective 22 is arranged inside the vacuum chamber 23, which is part of the light optical microscope system. The other major parts of the light optical microscope system are arranged outside the vacuum chamber 23 in an illumination and detection box 24. The illumination and detection box 24 comprises a light source 21, for example a LED. The emitted light 36 from het light source 21 is directed out of the illumination and detection box 24 via a half transparent mirror or dichroic mirror 25 and is directed into the vacuum chamber 23 via a window 32. This light 37, 38 is coupled into the microscope objective 22 via a mirror 26, for illuminating the sample through the sample holder 30. Light 37, 38 from the sample on the sample holder 30 is collected by the microscope objective 22 and is directed via the mirror 26 and the window 32 towards the illumination and detection box 24, and is imaged 39 via the half transparent mirror or dichroic mirror 25 onto a camera 33, for example a CCD detector.

As shown in figure IB, the light beams for illuminating and imaging the sample on the sample holder 30 enter into and pass from the vacuum chamber 23 via a window 32 which in this example is arranged in a door 34 of said vacuum chamber 23. The illumination and detection box 24 of the light optical microscope system is arranged outside vacuum chamber 23 and may be attached to the outside of the door 34. However, the illumination and detection part of the light optical microscope system may as well be included fully inside, e.g. attached to a bottom part, of the vacuum chamber 23.

Clearly, the illumination and detection box 24 may be configured in other manners and may comprise any kind of microscope, including e.g. cathodoluminescence microscope, laser confocal scanning microscope and wide field microscope. In addition the camera 33 can be replaced by an other type of detector, such as a photodiode or a photomultiplier which measures the light intensity originating from a spot in the image. When using such a spot measuring detector, the light intensity from various spots on the sample is measured by scanning over the sample on the sample holder 30, and the combination of such point to point measurements can provide an image of the sample.

A more detailed view of a sample 40 on a sample holder 50 is shown in figure 2. The sample 40 is provided with fluorescent markers 41. The sample holder 50 comprises a substrate 51 having a first side 52 which is provided with a conductive layer 53. Said substrate 51 and said conductive layer 53 are at least substantially optically transparent. The substrate 51 is, for example, a glass substrate, such as a microscope slide. The conductive layer 53 is, for example, a layer of Indium Tin Oxide (ITO) which is transparent to light. However the conductive layer 53 may also be made from alternative transparent conductors, such as, but not restricted to, Zinc Oxide, Diamond-like Carbon, Graphene or other 2D materials, or stacks of 2D materials like Graphene. To substantially prevent charging of the sample 40 and sample holder 50, the conductive layer 53 of the sample holder 50 is connected to ground potential 54 to keep the conductive layer 53 at a substantially constant voltage, at least during illumination of the sample 40 on the sample holder 50 with a beam of charged particles.

As schematically indicated in the graph on the right-hand-side of figure 2, the inventors found that when fluorescence from fluorescent markers 41 in the sample 40 is observed, the intensity of fluorescence from the fluorescent markers 41 is strongly biased: fluorescent markers 41 close to the conductive layer 53 (population a' ) emit very little fluorescent light, while fluorescent markers 41 further away from the conductive layer 53 (population ' ) emit fluorescent light at substantially the normal emission intensity I_n. The conductive layer 53 quenches the fluorescence from the fluorescent markers 41 in close proximity, which leads to a bias and/or to artefacts in the light microscopy results. The area L in the graph represents the fluorescence signal loss due to the quenching effect.

It is noted that the problem occurs strongest for samples with thickness on the order of or a few times the typical range over which quenching occurs. For a tissue section of approximately 200 nm thickness, only a marginal fraction of the embedded fluorescent materials 41 will be quenched and the bias may not be much of a problem. However, sections of 50 nm thickness are routinely used in biological inspection, and even the use of thinner sections, below 20 nm, is being explored. For these thin samples, a substantial fraction of all embedded emitters will be quenched. The same will hold for samples where total internal reflection microscopy or superresolution fluorescence microscopy is used. It should in this respect also be noted that serial sectioning of biological material is also often used to obtain a 3D view of a sample, by consecutive inspection of the serial sections. Here, the depth (z-, or axial) resolution is determined by how thin the tissue sections can be cut, so it is important to have the sections as thin as possible .

Using a sample holder 50 without a conductive layer 53 in order to circumvent the quenching effect, leads to a charging of the sample 40 when the sample 40 is illuminated by a beam of charged particles, such as electrons. This charging leads to a bias and/or to artefacts in the charged particle microscopy results.

Figure 3 shows a more detailed view of a sample 40 on a sample holder 50 according to the present invention. The sample 40 is provided with fluorescent markers 41. The sample holder 50 comprises a substrate 51 having a first side 52 which is provided with a conductive layer 53 which is connected to ground potential 54. Said substrate 51 and said conductive layer 53 are at least substantially optically transparent.

In addition, an insulating spacing layer 60 with a thickness D is provided between the conductive layer 53 and the sample 40. As indicated in the graph on the right-hand- side of figure 3, the intensity of fluorescence from the fluorescent markers 41 still depends on the distance of the fluorescent markers 41 to the conductive layer 53. However, due to the spacing layer 60, the fluorescent markers 41 and in particular the fluorescent markers 41 at the side of the sample facing the sample holder 50 (population a' ) , are separated from the conductive layer 53 over a distance equal to or larger than the thickness D of the spacing layer 60. In the example shown in figure 3, all fluorescent markers 41 emit fluorescent light at substantially the normal emission intensity I_n. The area L' in the graph representing the fluorescence signal loss due to the quenching effect is very small, and the fluorescence signal loss due to the quenching effect is negligible.

On the other hand, the thickness D of the spacing layer 60 is preferably such that it provides sufficient distance between the fluorescent markers 41 in the sample 40 and the conductive layer 53, and it still allows for charged particle beam inspection substantially without charging.

A minimal thickness Z₀,_q of the spacing layer can be defined based on an amount of fluorescence quenching that one still allows. A maximum thickness Z_charglng of the spacing layer can be defined based on the charged particle energy and current needed for imaging. Preferably, the thickness D of the spacing layer should be chosen to a value in between the minimal thickness Z₀,_q and the maximum thickness Z_charglng.

In a first example, the spacing layer 60 is part of the sample holder 50. In this case the sample holder 50 comprises a substrate 51, a conductive layer 53 and a spacing layer 60 which preferably comprises a layer of inorganic material, such as aluminium oxide or Silicon dioxide, with a thickness D of approximately 10 nm. The sample 40 is arranged on top of sample holder 50, in particular on top of the spacing layer 60 thereof. The insulating spacing layer 60 is preferably arranged on the conductive layer 53, using atomic layer deposition of aluminium oxide, for example. Alternatively, the spacing layer 60 can be made of Si0₂, which is also known as Silicon dioxide or Silica, other solid, transparent, dielectric materials .

In a second example, the spacing layer 60 is part of the sample 40. In this case the sample holder 50 comprises a substrate 51 and a conductive layer 53. The sample 40 is arranged on top of the sample holder 50, wherein the spacing layer 60 of the sample 40 is arranged in contact with the conductive layer 53 of the sample holder 50. An example of a method to provide a spacing layer 60 on a side of the sample 40 is described in more detail below, with reference to figure 7.

In a third example, as schematically shown in figure 4, the spacing layer 60 is provided with conductive channels 61 to provide a conductive path from the sample 40 to the conductive layer 53. Preferably the conductive channels 61 are arranged to form a conductive grid in the spacing layer 60. Providing the spacing layer 60 with such conductive channels 61 may be advantageous for thick spacing layers, for example when the minimal thickness Z₀,_q is larger than the maximum thickness Z_charglng, and/or for imaging using high charge particle currents.

In a fourth example, as schematically shown in figure 5, the spacing layer 60 is provided with alignment markers 62. Preferably, said alignment markers 62 can be observed in a light optical microscope and in a charged particle microscope. As shown in the example of figure 5, the alignment markers 62 are imbedded in the insulating spacing layer 60 and are thin, that is smaller than the thickness of the insulating spacing layer 60. However, the alignment markers 62 may also be substantially equal to the thickness of the spacing layer 60. These alignment markers 62 can be used to aid registration and/or correlation between images taken by the light optical microscope and images taken by the charged particle microscope, and/or to find and retrieve regions of interest.

In a fifth example, as schematically shown in figure 6, the insulating spacing layer 60' is patterned. That is, the spacing layer 60' provides a supporting grid for the sample 40. This grid may, just as the alignment markers 62, be used to aid registration and/or correlation between images taken by the light optical microscope and images taken by the charged particle microscope, and/or to find and retrieve regions of interest. In addition, the patterned spacing layer 60' provides uncovered areas 63 of the conductive layer 53, which can make it easier to connect the conductive layer 53 to a supply unit 55 or to ground potential 54, which supply unit 55 or ground potential 54 is arranged to keep the conductive layer 53 at a substantially constant voltage, at least during illumination of the sample 40 on the sample holder 50 with the beam of charged particles .

As indicated above, the present invention also provides a method for preparing a sample, and in particular for provide a spacing layer 60 on a side of the sample 40. In figure 7, a schematic representation of the steps of an example of a method for providing a thin insulating spacing layer onto a sample.

In this example the sample 40 comprises a thin tissue section or slice. Such sections are usually cut with a diamond knife 70 from an epon-embedded biological tissue block 42. Typically, the diamond knife 70 cuts of a thin section or slice, which then falls (arrow A) in a water bath 80. The thin section or slice will be the sample 40 which will be arranged on a sample holder 50 in order to enable inspecting the sample 40 arranged on said sample holder 50 with both a light optical microscope and a charged particle microscope.

As shown the sample 40 floats on the surface of the water 81 and can be picked up or transferred to a sample holder 50. In the example shown in figure 7, the sample 40 is coated with a thin insulating spacer, using a so-called Langmuir-Blodgett technique:

Using a first surfactant 82 in a first water bath 80, a first thin polymer coating 83 is deposited on one side of the sample 40. After coating the sample 40 with one monolayer of the first polymer surfactant 83 in the initial water bath 80, the sample 40 is removed from the water bath

80 (arrow B) .

Subsequently the sample 40 is arranged in a second water bath 80' (arrow C) comprising water 81' and a second surfactant 82', wherein the thin polymer coating 83 is arranged to contact the second surfactant 82' . In this step a second thin polymer coating 84 is deposited at a side of the first thin polymer coating 83 facing away from the sample 40. After coating the sample 40 with a second monolayer of the second polymer surfactant 84 in the second water bath 80' , the sample 40 is removed from the water bath 80' (arrow D) .

This procedure can be repeated to coat the side of the sample 40 with a fixed number of alternating polymer monolayers 83, 84 to reach a desired thickness.

Finally, the section is placed with the coated layer (s) onto the sample holder 50 (arrow E) so that the desired geometry of the sample 40 on the sample holder 50, as shown in figure 7, is obtained:

Sample 40, in particular a thin tissue sample

Spacing layer 60, comprising a number of alternating polymer monolayers 83, 84 of a desired thickness

Conductive layer 53, in particular a transparent conductive layer (for example ITO)

Substrate 51 (for example glass)

It is to be understood that the above description is included to illustrate the operation of the preferred embodiments and is not meant to limit the scope of the invention. From the above discussion, many variations will be apparent to one skilled in the art that would yet be encompassed by the spirit and scope of the present invention.

For example, although the description above is mainly describing fluorescent markers 41, the invention is not limited to particular types of fluorescent markers but comprises also fluorescent or luminescent light emitters, such as fluorescent molecules, semiconductor nanoparticles, fluorescent proteins, doped nanoparticles, etc...

In summary, the invention relates to a sample holder for holding a sample, or to a sample arranged on said sample holder, for inspecting said sample with both a light optical microscope and a charged particle microscope. The sample holder comprises a substrate having a first side which is provided with a conductive layer, wherein said substrate and said conductive layer are at least substantially optically transparent. Also the sample is arranged at the first side of the substrate, in particular at a side of the conductive layer facing away from the substrate. Between the conductive layer and the sample a spacing layer of a predetermined thickness is provided. Said spacing layer is substantially electrically insulating and at least substantially optically transparent.

Claims

C L A I M S

1. Sample holder for inspecting a sample arranged on said sample holder with both a light optical microscope and a charged particle microscope, wherein said sample holder comprises:

a spacing layer, wherein said spacing layer comprises a layer of inorganic material, is substantially electrically insulating, at least substantially optically transparent, and is arranged on said conductive layer at a side facing away from said substrate.

2. Sample holder provided with a sample arranged on said sample holder, for inspecting said sample with both a light optical microscope and a charged particle microscope, wherein said sample holder comprises:

3. Sample holder according to claim 1 or 2, wherein the thickness of the spacing layer is in the range of 7 - 30 nanometers, preferably 8 - 20 nanometers, most preferably 10 nanometer.

4. Sample holder according to claim 1, 2 or 3, wherein the spacing layer is provided with a conductive grid which extends substantially through said spacing layer and connects to said conductive layer.

5. Sample holder according to any one of the claims 1 - 4, wherein the spacing layer comprises alignment markers .

6. Sample holder according to claim 5, wherein the alignment markers are embedded into the spacing layer.

7. Sample holder according to any one of the previous claims, wherein said substrate is a rigid substrate suitable for transfer between a stand-alone light optical microscope and a charged particle microscope.

8. Sample holder according to any one of the previous claims, wherein spacing layer comprises a layer of aluminium oxide or Si0₂.

9. Apparatus comprising a sample holder according to any one of the previous claims, which apparatus is arranged for inspecting a sample with both a light optical microscope and a charged particle microscope.

10. Apparatus for inspecting a sample, said apparatus comprises:

a light optical microscope to observe the sample, wherein the light microscope is arranged to detect luminescence or fluorescence light emitted from the sample, a charged particle microscope to observe or modify the sample with a beam of charged particles, and

a sample holder to support the sample in a position in which it can be observed with both microscopes, characterized in that the sample holder comprises:

a spacing layer, wherein said spacing layer is substantially electrically insulating, at least substantially optically transparent, and is arranged on said conductive layer at a side facing away from said substrate.

11. Apparatus according to the preamble of claim 10, characterized in that the sample holder comprises:

12. Apparatus according to claim 10 or 11, wherein the conductive layer is connected to a supply unit which is arranged to keep the conductive layer at a substantially constant voltage, at least during illumination of the sample holder with the beam of charged particles.

13. Apparatus according to claim 12, wherein the constant voltage is a ground potential, or wherein the constant voltage is a negative bias voltage.

14. Method for preparing a sample on a sample holder for inspecting said sample with both a light optical microscope and a charged particle microscope, wherein said sample holder comprises a substrate having a first side which is provided with a conductive layer, wherein said substrate and said conductive layer are at least substantially optically transparent, and wherein said method comprises the steps of:

15. Method according to claim 14, the spacing layer comprises one or multiple polymer layers of desired thickness, which are deposited on said one side of the sample .

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BP/HZ