WO2020070489A1

WO2020070489A1 - Microscope slide

Info

Publication number: WO2020070489A1
Application number: PCT/GB2019/052773
Authority: WO
Inventors: Thomas WAIGH; Jian Ren LU; Ruiheng LI
Original assignee: University of Manchester
Current assignee: University of Manchester
Priority date: 2018-10-02
Filing date: 2019-10-02
Publication date: 2020-04-09
Anticipated expiration: 2021-04-02

Abstract

There is described a slide or coverslip for use with a fluorescence microscope. The slide or coverslip comprises a substrate and a graphene oxide layer over at least a portion of the substrate. The graphene oxide layer has an average thickness of ≥2nm. The slide or coverslip can be used to increase the contrast of a fluorescent sample in fluorescence microscopy.

Description

MICROSCOPE SLIDE

FIELD

[01] The present invention relates to slides and coverslips for microscopes. More specifically, the present invention relates to graphene oxide coated slides and coverslips for improving contrast in fluorescence microscopy.

BACKGROUND

[02] Fluorescence microscopy is a widely used tool in modern laboratories. There are a number of forms of fluorescence microscopy including standard, confocal, selective plane illumination, near field and super-resolution. Super-resolution fluorescence microscopy techniques include as stochastic optical reconstruction microscopy (STORM), photoactivated localization microscopy (PALM), stimulated emission- depletion (STED) microscopy and structured illumination microscopy (SIM). Such techniques can enable researchers to perform high quality non-invasive imaging experiments with a resolution approaching 20 nm.

[03] To use fluorescence microscopy effectively, specific regions of the sample must be labelled with fluorescent moieties, also known as fluorophores. However, the labelling of the samples is also often accompanied by a background of non-specifically bound fluorescent moieties attached to the surfaces of the sample holder e.g. the glass slide to which the sample is attached. This can result from excess fluorescent moieties forming on the substrate due to sedimentation and adsorption. These non-specifically bound fluorescent moieties can lead to high background noise during fluorescence imaging, substantially reducing the contrast and thus resolution in fluorescence microscopy experiments, in particular with imaging of biological materials and soft condensed matter. With more fluorescent moieties, there is a stronger image signal, but this comes hand in hand with increased background noise, so it is difficult to balance the amount of fluorescent moieties that are used.

[04] Considerable effort is currently required to remove the contributions from non- specifically bound fluorescent moieties e.g. using complicated, time consuming and often only partially successful methods to wash them off. Such methods have included the introduction of blocking agents to obstruct adsorption such as BSA, casein, gelatin or animal derived serum, or counter stains that can mask weak background fluorescence (e.g. Evans blue). However, such techniques suffer from problems including cost, low throughput, unwanted sample perturbations and sub-optimal contrast, for example if all of the fluorophores are not blocked or there is leakage of light from one colour channel into another.

[05] It is therefore an object of aspects of the present invention to address one or more of the abovementioned or other problems. In particular, it is an object of the present invention to improve contrast between the sample and the background in images obtained using fluorescence microscopy.

SUMMARY

[06] According to a first aspect of the present invention there is provided a slide or coverslip for use with a fluorescence microscope, the slide or coverslip comprising a substrate and a graphene oxide layer over at least a portion of the substrate, wherein the graphene oxide layer has an average thickness of ³2nm, suitably of >2nm.

[07] According to a second aspect of the present invention, there is provided a method for increasing the contrast of a fluorescent sample in fluorescence microscopy, the method comprising the steps of:

a. performing a fluorescence microscopy technique on the sample arranged on a surface of a slide or coverslip to produce an image of the sample,

wherein the sample comprises attached fluorescent moieties, which may be autofluorescence within the sample and/or an attached extrinsic fluorescent moiety;

wherein the surface of the slide or coverslip on which the sample to be imaged is arranged further comprises fluorescent moieties that are not attached to the sample to be imaged;

wherein the slide or coverslip comprises a substrate, a graphene oxide layer over at least a portion of the substrate, and optionally a spacer layer over at least a portion of the graphene oxide layer, wherein the sample is arranged on the surface of the slide or coverslip such that it is over at least a portion of the graphene oxide layer, and spacer layer when present; and wherein the graphene oxide layer quenches the fluorescence of at least a portion of the non-attached fluorescent moieties on the surface of the slide or coverslip to increase the contrast of the sample. [08] According to a third aspect of the present invention, there is provided use of a slide or coverslip according to any aspect of the present invention in a fluorescent microscopic technique.

[09] The graphene oxide layer of the slide or coverslip of any aspect of the present invention may have an average thickness of ³2.1 nm, suitably ³2.2nm, ³2.5nm, ³3nm, ³3.5nm, ³4nm, or ³4.5nm, preferably ³5nm. The graphene oxide layer of the slide or coverslip of any aspect of the present invention may have an average thickness of £20nm, suitably £15nm, preferably £10nm.

[10] The thickness may be measured by ellipsometry, atomic force microscopy or electron microscopy.

[11] The slide or coverslip according to any aspect of the present invention may comprise a spacer layer arranged at least partially over the graphene oxide layer. The spacer layer may be operable to receive a sample to be imaged such as to space the sample from the graphene oxide layer.

[12] Preferably the spacer layer is transparent. Transparency ensures that the optical signal from the fluorophores can propagate through the polymer layer and be detected in the optical microscope.

[13] The average thickness of the spacer layer may be £30nm, for example £25nm, or £20nm, such as £17nm, or most preferably £15nm. The average thickness of the spacer layer may be ³1 nm, ³1 5nm, ³2nm, such as ³3nm, ³4nm or ³5nm.

[14] The spacer layer may comprise a polymeric layer. Suitably the polymeric layer is formed from a biocompatible (co)polymer. Suitably the spacer layer provides a biocompatible surface for receiving the sample to be imaged.

[15] The polymeric layer may be formed from (co)polymers selected from one or more of polystyrene, polymethyl(meth)acrylate, polyurethane, polycarbonate, chitosan, polyethyleneimine, polyvinyl chloride, polydimethylsiloxane, polyamideimide, polydimethylsiloxane, polyethersulphone, acrylonitrile butadiene styrene and polylysine.

[16] The polymeric layer may be formed from a hydrophobic (co)polymer. Such a polymeric layer may have an average thickness of <20nm, such as <18nm or <16nm. Suitably the hydrophobic (co)polymer is selected from one or more of polystyrene, polyurethane, polycarbonate, chitosan, polyethyleneimine, polyvinyl chloride, polyamideimide, polydimethylsiloxane, polyethersulphone, acrylonitrile butadiene styrene and polymethyl(meth)acrylate. Advantageously, the use of hydrophobic polymers reduces swelling in liquid environments and can form a layer with a smooth surface.

[17] The polymeric layer may be formed from a positively charged (co)polymer, such as polylysine, polyethyleneimine or chitosan. Advantageously, the positive charge encourages the strong binding with biological cells (i.e. prokaryotes, eukaryotes and archaea) to reduce image blur due to their motion.

[18] Preferably, the spacer layer has substantially no fluorescence. By substantially no fluorescence it is meant relative to the florescence of the fluorescent moieties on the sample to be imaged. The spacer layer may comprise <5wt% added fluorescent moieties by weight of the spacer layer, suitably <2wt%, preferably <1wt%, most preferably substantially 0wt%.

[19] The spacer layer may have <5% quantum yield, preferably <3%, most preferably <1 %. Quantum yield may be measured with visible light absorption spectroscopy.

[20] The slide or coverslip of any aspect of the present invention may further comprise a sample to be imaged, suitably a biological sample.

[21] The slide or coverslip of any aspect of the present invention may further comprise fluorescent moieties.

[22] The sample according to any aspect of the present invention may have at least one dimension that is at least 10nm, suitably at least 12nm, or 15nm. The fluorescent moieties according to any aspect of the present invention may be smaller than 10nm, suitably smaller than 7nm, 5nm or 3nm.

[23] The sample may be a biological sample. Such as a peptide, peptide aggregate, self-assembled peptide aggregate, protein, lipids, lipid aggregates, cabrohydrates, nucleic acids and/or cell, for example a cancer cell, such as a human cancer cell, a bacterial cell or neuronal cell.

[24] It has been found that graphene oxide can provide a quenching response as an acceptor molecule to donor fluorescent moieties over a wide range of emission spectrum wavelengths. Accordingly, the fluorescent moieties may include fluorescent dyes, quantum dots and/or fluorescent polymers. Preferably, the fluorescent moiety is a fluorescent antibody, or fluorescent lectins. [25] The fluorescence microscopy technique may be selected from near field fluorescence microscopy, selective plane illumination microscopy (SPIM), super resolution fluorescence microscopy, stochastic optical reconstruction microscopy (STORM), diffraction limited fluorescence imaging, stimulated emission depletion microscopy (STED), structured illumination microscopy and confocal microscopy.

[26] The graphene oxide may be washed, suitably with a base, prior to arranging the sample on the slide or coverslip. Such a washing step removes debris absorbed to the graphene oxide surface during production.

[27] The slide or coverslip substrate may be formed of Si0₂.

[28] Advantageously, the slides, coverslips and method of the present invention provide improved sample contrast with fluorescence microscopy. They can be easily adapted to a variety of fluorescence microscopy methods. They are also cheaper and more convenient than previous methods of improving sample contrast.

[29] All of the features contained herein may be combined with any of the above aspects in any combination.

[30] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the following experimental data and figures

EXAMPLES

[31] Coverslips and glass slides containing layers of graphene oxide and synthetic polymer, as well as fluorophore labelled samples were tested with direct Stochastic Optical Reconstruction Microscopy (dSTORM).

[32] The fluorophores used include Alex fluor 647 (excitation/emission wavelength 650 nm/665 nm from Thermo Fisher Scientific) attached to BSA. A MEA image buffer was used for Alex Fluor 647. The second fluorophore was Cy3B NHS ester (excitation/emission wavelength 559 nm/570 nm from GE Healthcare Life Sciences). An OxyFlour image buffer was used for Cy3B.

[33] An aqueous solution of graphene oxide was made by a modified Humers’ method as described in Preparation of Graphitic Oxide, J. AM. Chem. Soc., 1958, 80 (6)] followed by a base wash process with 1M of NaOH at 70 °C to further clean it. The solution was then dialyzed for two weeks to remove all ionic impurities (molecular weight cut-off 14K). Graphene oxide layers were then spin-coated on coverslips which was then followed with a 1 hour of backing at 70°C at standard pressure to allow the evaporation of any water molecules and improve the adhesion of the films. By doing this the stability of the graphene oxide layer increased, especially in liquid environments (water or toluene). A graphene oxide layer with a thickness of 6 nm will maintain its thickness for over an hour of a deionized water flush at 1 ml/min. A dense packed graphene oxide layer of a thickness of up to 6 nm was achieved by using a graphene oxide aqueous solution with a concentration of 1.6 mg/ml. The graphene oxide sheets were also washed to remove sulphated impurities.

[34] Thickness measurements were performed with a spectroscopic elipsometer ( J.A . Woollam Co., Inc. ESM-300).

[35] The quenching effect of graphene oxide was studied using direct Stochastic Optical Reconstruction Microscopy (dSTORM). The method employs photoswitchable fluorophores to separate individual fluorescence signals by making the fluorophores blink stochastically. The position of each localization is determined accurately from each frame by fitting a two-dimensional Gaussian to the point spread function (PSF) of each fluorescent signal. The final image is re-constructed by stacking all the localizations from each frame together. The resolution of the final re-constructed image can go down to 20 nm or even less. A Digital CMOS camera (Hamamatsu C11440-22CU) was used to capture diffraction limited images at 100 frames per second and the rest of the STORM apparatus consists of a standard fluorescence microscope with laser illumination. To re-construct one super-resolution image, 10000 frames of diffraction limited images were collected. An ImageJ plugin, ThunderSTORM, was used to analyse the image series.

[36] Figure 1 shows a STORM reconstruction of l₃K fibrils on the surface of coverslips. The self-assembled l₃K fibrils were prepared in pure water at 10 mM peptide concentration. The fibrils were aged for at least a week and then dyed with Cy3B-NHS Ester dye. In figure 1 , a coverslip containing a graphene oxide layer, produced as described above, was used. Image A shows the peptide on a graphene oxide layer and image B shows the peptide directly on glass substrate without graphene oxide. It can be seen that the graphene oxide layer in the coverslip of image A effectively reduces the background signal due to surface adsorbed free dye and labelled peptide monomers.

[37] Figure 1 shows that STORM can be used to study the self-assembly of aggregates of the surfactant-like peptide l₃K. Due to the dynamic nature of self- assembled peptide structures all unnecessary sample disturbance needs to be minimised. As such, removal of unconjugated dye molecules is not suitable when imaging l₃K fibrils. The large concentration of the unconjugated dye and the labelled peptide monomers can readily adsorb to all surfaces, generating significant noise in STORM images. Fibril identification in STORM images using image analysis algorithms can be challenging and by reducing the background noise to a very low level the graphene oxide layer offers the prospect of more accurate analysis. Furthermore, analysis of localisation distributions in STORM images can provide a method for investigating mixtures of monomers within self-assembled structures. These methods rely upon the accurate counting of the number of monomers within a structure, which is based upon the counting of localisations. If there are a significant number of localisations present in the background around the fibre that provide a large background noise, this will introduce an ambiguity into the measurement. The graphene oxide layer has removed almost all the background localisations due to a small amount of surface adsorbed fluorescent dye or labelled peptide monomer showing on the image; this enhancement in raw data quality has the potential to greatly improve results from these kinds of samples.

[38] Figure 2 shows a STORM reconstruction of encapsulated Escherichia coli bacteria (strain EV36), grown at 37 °C in Luria-Bertani (LB) broth grown until the mid log growth phase, with an optical density at 600nm (OD₆oo) of 0.5. Their capsule thickness was around 200-400 nm and it was labelled with a primary antibody (mouse IgG anti-K1 antibody) at 1 :400 concentration in 1 % BSA and a secondary antibody (anti-mouse F(ab') Alexa Flour 647) at 1 :1000 concentration in 1 % BSA. The length of each bacteria was about 2 pm and their width was 1 pm.

[39] Image A shows the live bacteria immobilized on 2nm poly-l-lysine coated on a slide with no graphene oxide layer. Image B shows the bacteria immobilised on 2nm poly-l-lysine coated over a graphene oxide layer-containing slide, prepared as described above. As can be seen in figure 2, with a 5 nm layer of graphene oxide the non-specifically bound fluorophores are significantly quenched. An image with a much lower background was achieved, because the background due to non-specifically bound antibodies and any other autofluorescence molecules (e.g. proteins form the bacteria) has been eliminated.

[40] A big improvement of signal to noise ratio has been achieved by using a graphene oxide layer placed underneath both live cells and peptide samples to quench background noise. A variety of polymer layers have also been demonstrated to separate graphene oxide layers from the sample to provide optimal surface chemistry for its support, while still quenching the fluorescent background.

[41] Attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

[42] All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

[43] Each feature disclosed in this specification (including any accompanying claims, abstract and drawings) may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[44] The invention is not restricted to the details of the foregoing embodiment(s). The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

Claims

1. A slide or coverslip for use with a fluorescence microscope, the slide or coverslip comprising a substrate and a graphene oxide layer over at least a portion of the substrate, wherein the graphene oxide layer has an average thickness of ³2nm, suitably of >2nm.

2. A method for increasing the contrast of a fluorescent sample in fluorescence microscopy, the method comprising the steps of:

wherein the sample comprises attached fluorescent moieties, which may be autofluorescence within the sample and/or an attached extrinsic fluorophore;

wherein the slide or coverslip comprises a substrate, a graphene oxide layer over at least a portion of the substrate, and optionally a spacer layer over at least a portion of the graphene oxide layer, wherein the sample is arranged on the surface of the slide or coverslip such that it is over at least a portion of the graphene oxide layer, and spacer layer when present;

and wherein the graphene oxide layer quenches the fluorescence of at least a portion of the non-attached fluorescent moieties on the surface of the slide or coverslip to increase the contrast of the sample.

3. A slide or coverslip according to claim 1 or 2, wherein the average thickness of the graphene oxide layer is ³2.1 nm, suitably ³2.2nm, ³2.5nm, ³3nm, ³3.5nm, ³4nm, or ³4.5nm, preferably ³5nm.

4. A slide or coverslip according to any previous claim, wherein the average thickness of the graphene oxide layer is £20nm, suitably £15nm, preferably £10nm.

5. A slide or coverslip according to any previous claim, wherein the slide or coverslip further comprises a spacer layer arranged at least partially over the graphene oxide layer, wherein the spacer layer is operable to receive a sample to be imaged such as to space the sample from the graphene oxide layer, preferably the spacer layer is transparent.

6. A slide or coverslip according to any previous claim, wherein the average thickness of the spacer layer is £30nm, for example £25nm, or £20nm, such as £17nm, or most preferably £15nm.

7. A slide or coverslip according to any previous claim, wherein the average thickness of the spacer layer is at least ³1 nm, ³1 5nm, ³2nm, such as ³3nm, ³4nm or ³5nm.

8. A slide or coverslip according to any previous claim, wherein the spacer layer comprises a polymeric layer, suitably the polymeric layer is formed from a biocompatible (co)polymer, suitably a polymer that substantially does not alter the growth, such as numbers or size or the morphology, of the target sample.

9. A slide or coverslip according to any previous claim, wherein the spacer layer comprises a polymeric layer and wherein the polymeric layer is formed from (co)polymers selected from one or more of polystyrene, polymethyl(meth)acrylate, polyurethane, polycarbonate, chitosan, polyethyleneimine, polyvinyl chloride, polydimethylsiloxane, polyamideimide, polydimethylsiloxane, polyethersulphone, acrylonitrile butadiene styrene and polylysine.

10. A slide or coverslip according to any previous claim, wherein the polymeric layer is formed from a hydrophobic (co)polymer, and preferably the polymeric layer has an average thickness of <20nm, suitably the hydrophobic polymer is selected from one or more of polystyrene, polyurethane, polycarbonate, chitosan, polyethyleneimine, polyvinyl chloride, polyamideimide, polydimethylsiloxane, polyethersulphone, acrylonitrile butadiene styrene and polymethyl(meth)acrylate.

11. A slide or coverslip according to any previous claim, wherein the polymeric layer is formed from a positively charged (co)polymer, such as polylysine, polyethyleneimine or chitosan.

12. A slide or coverslip according to any previous claim, wherein the spacer layer comprises <5wt% added fluorescent moieties by weight of the spacer layer, suitably <2wt%, preferably <1wt%, most preferably substantially 0wt%.

13. A slide or coverslip according to any previous claim, wherein the spacer layer has <5% quantum yield, preferably <3%, most preferably <1 %.

14. A slide or coverslip according to any previous claim, wherein the slide or coverslip further comprises a sample to be imaged, suitably a biological sample.

15. A slide or coverslip according to any previous claim, wherein the slide or coverslip further comprises fluorescent moieties.

16. A slide, coverslip or method according to any previous claim, wherein the sample has at least one dimension that is at least 10nm, suitably at least 12nm, or 15nm.

17. A slide, coverslip or method according to any previous claim, wherein the fluorescent moieties are smaller than 10nm, suitably smaller than 7nm, 5nm or 3nm.

18. A method according to any preceding claim, wherein the sample is a biological sample, suitably a such as a peptide, peptide aggregate, self-assembled peptide aggregate, protein, lipids, lipid aggregates, cabrohydrates, nucleic acids and/or cell, such as a cancer cell, for example a human cancer cell, a bacterial cell or neuronal cell.

19. A method according to any preceding claim, wherein the fluorescent moieties are fluorescent dyes, quantum dots or fluorescent polymers, such as fluorescent antibodies or fluorescent lectins.

20. A method according to any preceding claim, wherein fluorescence microscopy technique is selected from near-field fluorescence microscopy, selective plane illumination microscopy (SPIM), super-resolution fluorescence microscopy, stochastic optical reconstruction microscopy (STORM), diffraction limited fluorescence imaging, stimulated emission depletion microscopy (STED), structured illumination microscopy and confocal microscopy.

21. Use of a slide or coverslip according to any preceding claim in a fluorescent microscopic technique, suitably a microscopic technique according to claim 20.