Microscopic examination method and system

Abstract

To provide a sample holder for use in an optical microscope.SOLUTION: The sample holder includes a plasmonic layer defining a periodic array of sub-micron structures, where the periodic array of sub-micron structures comprises an array of separated plasmonic regions. The regions may be a circle, torus, ellipse, cross, rectangle, square, line, or strip. Methods of performing reflection and fluorescence microscopy using such a sample holder and other sample holders are also disclosed.SELECTED DRAWING: Figure 1a

Description

Technical Field

[0001] Field of the Disclosure The present disclosure relates to the field of optical microscopy. In one form, the present disclosure provides systems and methods that use an optical microscope and an enhanced sample holder.

Background Art

[0002] Background of the Disclosure PCT / Australian Patent Application Publication No. 2018 / 050496 in the name of La Trobe University (the entire content of which is incorporated herein by reference) discloses an optical microscopy system and method that provides enhanced image contrast through the use of a sample holder having a plasmonic layer including a periodic array of submicron structures. In this disclosure, reference to the nanoslides is a reference to the sample holder according to the teachings of PCT / Australian Patent Application Publication No. 2018 / 050496 or Australian Patent Application Publication No. 2018904553, entitled "Microscopy method and system", of the applicant in co-pending litigation as of November 29, 2018, the entire contents of both of which are incorporated herein by reference for all purposes, and an international patent application claiming priority to Australian Patent Application Publication No. 2018904553 filed on the same day as this application. Microscopy using such a sample holder is referred to herein as histoplasmonics or color contrast microscopy (abbreviated as CCM). The sample is placed on the sample holder adjacent to the plasmonic layer. During use, the sample and the sample holder are illuminated and an image of the sample is generated. The inventors have observed that color contrast is presented in the image of interest through the interaction of light with the sample and the plasmonic layer. In particular, regions of the sample having different dielectric constants appear in the image in different colors. An increase in intensity contrast is also achieved. In contrast to CCM, images obtained from conventional optical microscopy using non-specific staining typically exhibit only an intensity contrast of a single color corresponding to the staining used. Even when contrast staining or biomarkers are used, these prior arts only provide an image of a specific color. Summary of the Invention Means for Solving the Problems

[0003] Summary of the Disclosure As is known to those skilled in the art, reflection light microscopy is, in a broad sense, a microscopy technique that uses light reflected from a sample to form an image of the sample. The exemplary embodiments of the nanoslides disclosed in PCT / Australia Patent Application Publication No. 2018 / 050496 can be used in reflection microscopy without modification, but the inventors have determined that such a sample holder can be enhanced to improve its use in reflection light microscopy. As used herein, "forming an image" includes forming a human-perceivable image (e.g., by focusing light) so that a user can perceive an image of the sample (or a part thereof), or generating a digital or photographic image of the sample (or a part thereof) for storage, transmission, display, or other downstream processing.

[0004] Accordingly, a first aspect of the present invention provides a sample holder for use in an optical microscope, the sample holder including a plasmonic layer defining a periodic array of submicron structures, the sample holder being configured to support an object such that the periodic array of submicron structures is adjacent to the object when supporting the object, the periodic array of submicron structures including an array of separated plasmonic regions.

[0005] Accordingly, a second aspect of the present invention provides a sample holder for use in an optical microscope, the sample holder being a plasmonic layer extending over a region of the sample holder and including a plasmonic layer defining a periodic array of submicron structures, the sample holder being configured to support an object such that the periodic array of submicron structures is adjacent to the object when supporting the object, within the region, the plasmonic layer fills 80% or less of the area of the region by the periodic array of submicron structures.

[0006] In some forms of the second aspect, a periodic array of submicron structures fills the plasmonic layer with no more than any one of 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% of the area. The area can cover the entire side surface of the sample holder.

[0007] In some embodiments of either the first aspect or the second aspect of the present invention, the plasmonic region can be islands of plasmonic material. In other embodiments, the plasmonic region can be lines or stripes of plasmonic material separated from adjacent lines or stripes by non-plasmonic strips or lines to form a one-dimensional array of separated plasmonic regions.

[0008] In some embodiments of either the first aspect or the second aspect of the present invention, the submicron structures can be arranged in a periodic array having a separation between the submicron structures in the range of 200 nm to 500 nm. The submicron structures can have a maximum dimension in the range of 50 nm to 300 nm. Most preferably, the submicron structures are regions of plasmonic material spanning from about 100 nm to 200 nm. The plasmonic regions can be shaped as any one or more of a circle, a torus, an ellipse, a cross, a rectangle, a square.

[0009] In some embodiments of either the first aspect or the second aspect of the present invention, the plasmonic layer can be formed from one or more metals selected from any one of Al, Ag, Au, Ni, Pt, and Pd. The plasmonic layer can have a thickness in the range of 20 nm to 300 nm.

[0010] In some embodiments of either the first aspect or the second aspect of the present invention, the array of separated plasmonic regions can be a regular array having an equal spacing between adjacent plasmonic regions in the first and second directions. Preferably, the first and second directions are orthogonal directions. However, the array can have different spacings in the first direction and the second direction.

[0011] In some embodiments of either the first or second aspect of the present invention, the sample holder includes a substrate connected to at least a portion of a first surface of the plasmonic layer to provide mechanical support for the plasmonic layer. In some embodiments, the sample holder includes an optically transparent protective layer adhered on top of the plasmonic layer to separate the plasmonic layer. The optically transparent protective layer may have a thickness of less than 150 nm. In some embodiments, the optically transparent protective layer may have a thickness of less than 80 nm. The optically transparent protective layer may include any one or more of silicon oxide, silicon nitride, transparent metal oxides, and polymers. The sample holder may include a microscope slide.

[0012] In some embodiments of either the first or second aspect of the present invention, the sample holder enables light transmitted through the sample to the plasmonic layer to be reflected from the sample holder for the generation of an image including reflected light.

[0013] In some embodiments of either the first or second aspect of the present invention, in use, the incident light illuminates the sample and the sample holder and interacts with the sample and the plasmonic layer. The reflected light includes a characteristic spectrum in which each color depends on the localized dielectric constant of the sample. In this way, a color image encoding the localized dielectric constant of the sample can be formed from the reflected light.

[0014] The inventors further recognized that the nanoslide can also be advantageously used in fluorescence microscopy, as described herein or in PCT / Australia Patent Application Publication No. 2018 / 050496. Most advantageously, this can be done in a microscopy arrangement adapted for reflected light microscopy.

[0015] Accordingly, the present invention is a method of imaging a sample, comprising To provide a sample holder having an upper surface and a lower surface, wherein the upper surface has an associated plasmonic layer, and the plasmonic layer includes a periodic array of submicron structures, Attaching a biological sample to the upper surface of the sample holder, Illuminating the sample with the light such that the light interacts with the sample and the sample holder, Receiving the light emitted from the sample by fluorescence and forming an image thereof A method including the above is also provided.

[0016] The method further includes receiving light after interaction of the sample and the sample holder and forming at least one image thereof, and at least one localized structural characteristic of the sample is visible in the image based on the color of the received light. Such imaging is described in more detail in PCT / Australia Patent Application Publication No. 2018 / 050496. Herein, an image formed in this way by this method is referred to as a color contrast image. Advantageously, in this way, a sample mounted on a sample holder can be imaged by using fluorescence microscopy in addition to using color contrast imaging.

[0017] An image formed from the light emitted from the sample by fluorescence can be formed in a first period, and a color contrast image can be formed in a second period. In some embodiments, illuminating the sample with the light such that the light interacts with the sample and the sample holder can include using a first illumination spectrum in a first period and a second illumination spectrum in a second period. The first illumination spectrum can be selected based on the fluorescence characteristics of the sample. In some cases, the first and second illumination spectra can be the same.

[0018] In some embodiments, receiving light emitted from a sample by fluorescence involves filtering light received from the sample and / or the sample holder (e.g., based on wavelength or spatial filtering) to minimize light received from sources other than the fluorescence.

[0019] In a preferred form, the method includes illuminating the sample from the side of the sample holder on which the sample is placed and receiving, from the same side as the illumination, the light emitted from the sample by fluorescence and also the light after interaction with the sample and the sample holder.

[0020] It should be noted that the terms upper surface and lower surface are not intended to refer to a particular orientation of any sample holder during sample preparation or use.

[0021] In embodiments of the present invention, the method may include spatially correlating an image formed from light emitted from a sample by fluorescence with a color contrast image. The method may include forming a composite image including an image formed from light emitted from a sample by fluorescence and a color contrast image. A plurality of images formed from light emitted from a sample by fluorescence and / or a plurality of color contrast images may be combined into a single image. The combination may be performed optically (e.g., during optical image formation) or digitally (e.g., by combining data values representing the images).

[0022] The sample is preferably a biological sample.

[0023] The sample holder used in embodiments of this aspect of the present invention may be a sample holder according to the embodiments of PCT / Australian Patent Application Publication No. 2018 / 050496, but most preferably is a sample holder according to the embodiments of the first aspect of the present invention.

[0024] The images formed by the method described above may be used in histology and pathology in a manner that may be apparent to those skilled in the art.

[0025] In another aspect, a system for forming an image using an embodiment of any one of the above-described aspects is provided. The system may include an imaging system, an illumination system, and a sample holder having an upper surface and a lower surface, the upper surface having an associated plasmonic layer, the plasmonic layer including a periodic array of submicron structures, and a reflected light microscope. The system may include an image capture system for generating at least one image of the sample.

[0026] Embodiments of the present invention may be applied to an automated or semi-automated method of identifying a structure as taught in an international patent application claiming priority to Australian Patent Application Publication No. 2018904551, entitled "Automated method of identifying a structure", filed on November 29, 2018, by the applicant of the present application and Australian Patent Application Publication No. 2018904551, filed on the same day as the present application, the entire contents of both of which are incorporated herein by reference for all purposes, and may be used to generate a digital image.

[0027] Embodiments of aspects of the present disclosure may be used in embodiments of the teachings of an international patent application claiming priority to Australian Patent Application Publication No. 2018904550, entitled "Method of identifying a structure", filed on November 29, 2018, by the applicant of the present application and Australian Patent Application Publication No. 2018904550, filed on the same day as the present application and incorporated herein by reference for all purposes.

[0028] Brief Description of the Drawings Exemplary embodiments of the present invention will be described by way of non-limiting examples with reference to the accompanying drawings. The accompanying drawings filed with this international application are color images used in embodiments of the present invention and include color images resulting from their use. The color information forms part of the present disclosure of the embodiments. If black-and-white reproduction or grayscale reproduction of the images occurs, the color disclosure can be obtained from the originally filed document.

Brief Description of the Drawings

[0029]

Figure 1a

Figure 1b

Figure 1c

Figure 1d

Figure 1e

Figure 1f

Figure 1g

Figure 1h

Figure 1i

Figure 1j

Figure 2a

Figure 3A

Figure 3B

Figure 3C

Figure 3D

Figure 4

Figure 5a

Figure 5b

DETAILED DESCRIPTION OF THE INVENTION

[0030] Detailed description of the embodiment Figure 1a shows an embodiment of a sample holder used in an example of the present disclosure. Figure 1a shows a cross-section across the entire sample holder suitable for use in the present invention. The sample holder 100 includes a substrate on which the plasmonic layer 102 is deposited. Figures 1b and 1c show two types of plasmonic layers 102 as exemplified in PCT / Australia Patent Application Publication No. 2018 / 050496, having submicron arrays that are fabricated and can be used in embodiments. The layers are silver salt films each having a thickness of 150 nm, although other suitable materials can be used. Figure 1b has a submicron array in the form of circular nanoapertures with a 450 nm period arranged in a hexagonal pattern. Figure 1c has cross-shaped nanoapertures on a rectangular pattern. The cross-shaped nanoapertures have a 450 nm period in one direction (here defined as the 0° direction) and a 400 nm period in the orthogonal direction (defined as the 90° direction). These arrays have surface plasmon polariton (SPP) resonance modes in the range of 470 - 550 nm (within the visible region of the electromagnetic spectrum). To protect the surface of the plasmonic layer 102, a layer 104 (10 nm ± 1 nm) of hydrogen silsesquioxane (HSQ), a glass-like material, is deposited after the fabrication of the plasmonic layer 102. After capping with HSQ, the sample holder 100 has an upper surface similar to that of a conventional microscope slide on which a sample can be supported. In use, the HSQ layer also presents a polar surface that promotes tissue adhesion. In other embodiments, a metal oxide capping layer (e.g., SiO2) can be used instead of HSQ.

[0031] The imaging target sample is prepared according to the embodiments of the international patent application claiming priority to PCT / Australian Patent Application Publication No. 2018 / 050496 in the name of La Trobe University or Australian Patent Application Publication No. 2018904553 with the name "Microscopy method and system" of the present applicant in the co-pending application filed on November 29, 2018, and Australian Patent Application Publication No. 2018904553 filed on the same day as this application, and is placed on a sample holder. In a preferred embodiment of the present invention, a sample 106 (usually a slice of biological tissue) that does not need to be labeled with a stain or label is placed on a sample holder adjacent to the plasmonic layer as shown in Figure 2a.

[0032] Figure 3A is a schematic diagram of a system 300 in which a sample holder 100 is used in reflected light microscopy. The techniques and equipment used in reflected light microscopy with conventional slides are known to those skilled in the art, and in order to avoid obscuring the details of the present invention, the description of known techniques will be omitted.

[0033] System 300 includes a light source 310 that emits incident light 311 to illuminate the sample 106. The illumination is performed, in this example, from the side of the sample holder where the sample is placed. The reflected light 315 is retroreflected to an imaging system 312 for generating a color contrast image. The sample holder 100 is a nanoslide having a plasmonic layer.

[0034] When system 300 is used in reflected light microscopy, the reflected light forming the image interacts with the plasmonic layer of the sample holder and the sample so as to possess a reflection spectrum that varies according to the local dielectric constant of the sample. Therefore, the reflected light image displays color contrast.

[0035] Figure 4 shows a reflection image of a thin section (70 nm) of the optic nerve of a mouse. The tissue was prepared by a conventional method and embedded in epoxy resin. The 70-nm-thick section was cut on a Leica UC7 ultramicrotome. The image was captured by using a 20× magnification and was taken in reflection mode. In the image, the visible stripes that are within the visible region of the Em spectrum and thus generate visible fringes within the image occur from the fact that the submicron structures within the plasmonic layer of the nanoslides include structures having a periodicity of about 450 nm. By using an array with a smaller periodicity or a different shape (e.g., hexagonal), such artifacts can be avoided.

[0036] The special reflection spectrum becomes somewhat similar to the inverted one of a normalized transmission spectrum (such as that shown in PCT / Australian Patent Application Publication No. 2018 / 050496) having grooves that appear at the peaks within the transmission spectrum. However, various combinations of plasmon resonance modes can exist. Figure 5a shows exemplary simulated received light spectra when the nanoslides were used in reflection mode for the following three samples: A sample having R = 1 (i.e., no sample or the sample is air), A sample having R = 1.33 (e.g., a sample made of water), A sample having R = 1.5 (e.g., a material such as a polymer).

[0037] The simulated spectra are based on a nanoslide having circular islands of plasmonic material with a radius of 90 nm arranged in an array having a separation of 480 nm in a first direction within a hexagonal lattice array.

[0038] When the plasmonic layer covers the entire area of the nanoslides having only small voids provided by the submicron structure therein, compared to the example of PCT / AU2018 / 050496, when used in reflection microscopy, the arrangement of the submicron structure fills a smaller area (in terms of area) of the area covered by the plasmonic layer, and it has been found that it may be advantageous to provide a plasmonic layer area. In some cases, the plasmonic layer area may be filled with only one of 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% (in terms of area) of the area or less. The reduction in the filling rate reduces the reflection of incident light (i.e., allows more transmission), and as a result, the spectral peaks in the reflected light can be more easily distinguishable from the grooves, enhancing the color contrast effect in the received image, which can be advantageous in reflection microscopy.

[0039] Figure 5b shows a simulated image of such a sample showing the visible color of the result from such a sample. As can be seen, The RI = 1 sample (left side) appears blue corresponding to the position of the peak in its reflection spectrum, The RI = 1.33 sample (center) appears orange corresponding to the position of the peak in its reflection spectrum, The RI = 1.5 sample (right side) appears red corresponding to the position of the peak in its reflection spectrum, It is demonstrated that the image formed using the nanoslides in reflection microscopy displays color contrast based on the dielectric constant of the sample related to the refractive index of the sample.

[0040] Importantly, the use of nanoslides enables such color contrast to be obtained without staining the sample and when using a nearly transparent sample. Thus, the received reflected light includes light reflected from the plasmonic layer, in contrast to the upper surface of the sample. This also means that the absorption within the sample itself is relatively low.

[0041] The microscope uses a conventional optical microscope with an eyepiece for viewing by the user, but may alternatively or additionally include an image capture system for generating a digital image for display, storage, or other later use. In some forms, the microscope may form part of an automated slide scanner. System 300 may include a user terminal for display of the captured digital image of the sample and a data storage system for storing the captured images.

[0042] When performing reflected light microscopy using a nanoslide, the inventors have demonstrated that the plasmonic layer can be specialized to produce improved results in some cases as to where / when / why the dots are better. In particular, a periodic array of submicron structures may include an array of isolated plasmonic regions. The plasmonic regions may be islands of plasmonic material separated by gaps. The plasmonic regions will typically be arranged in a periodic array having a separation between submicron structures in the range of 200 nm to 500 nm. Each plasmonic region may have a maximum dimension in the range of 50 nm to 300 nm. Most preferably, the submicron structure is a region of plasmonic material spanning from about 100 nm to 200 nm. The plasmonic regions may be shaped as any one or more of a circle, torus, ellipse, cross, rectangle, square.

[0043] As described in PCT / Australia Patent Application Publication No. 2018 / 050496, the plasmonic layer may be formed from one or more metals such as Al, Ag, Au, Ni, Pt or Pd. The plasmonic layer may have a thickness in the range of 20 nm to 300 nm.

[0044] Figures 1d - 1j show examples of such periodic arrays of submicron structures including an array of isolated plasmonic regions.

[0045] Figure 1d shows a plasmonic layer 102 including a regular rectangular array of square plasmonic regions 103. The spacing of the plasmonic regions 103 in the first and second directions is equal.

[0046] Figure 1e shows a plasmonic layer 102 including an array of square plasmonic regions 103 arranged at equal intervals in the first and second directions, where each row is offset from its neighbor to form a hexagonal array of plasmonic regions 103. The separation between regions in the first and second (x and y) directions can be the same (as shown) or different.

[0047] Figure 1f shows a plasmonic layer 102 including an array of square plasmonic regions 103, where the intervals between the plasmonic regions 103 in the first and second directions are not equal. As described in PCT / Australia Patent Application Publication No. 2018 / 050496, such an example can enable the use of polarized illumination to change the spectrum of the received light by switching the relative polarization of the received light with respect to the rows and columns of the array.

[0048] Figure 1g shows an exemplary plasmonic layer 102 including circular plasmonic regions 103. In this example, the intervals between the plasmonic regions 103 in the first and second directions are equal, although other array arrangements may be used.

[0049] Figure 1h shows an exemplary plasmonic layer 102 including cross-shaped plasmonic regions 103. In this example, the intervals between the plasmonic regions 103 in the first and second directions are not equal, although other array arrangements may be used.

[0050] Figure 1i shows an exemplary plasmonic layer 102 including rectangular plasmonic regions 103. However, unlike other embodiments, the intervals between adjacent plasmonic regions are relatively narrow. This results in a plasmonic layer where the plasmonic regions occupy a fraction of the plasmonic layer greater than 50% by area.

[0051] FIG. 1j shows an exemplary plasmonic layer 102 including a plasmonic region 103 shaped as a strip separated by a non-plasmonic strip, the plasmonic layer 102 generating a one-dimensional array of separated plasmonic regions.

[0052] As can be seen, the array of plasmonic regions can be a regular array having equal spacing between adjacent plasmonic regions in the first and / or second directions. Preferably, the first and second directions are orthogonal directions. However, the array can have different spacings in the first and second directions.

[0053] The present invention also provides a method of fluorescence microscopy, the method comprising providing a sample holder having an upper surface and a lower surface, the upper surface having an associated plasmonic layer, the plasmonic layer including a periodic array of submicron structures, attaching a sample to the upper surface of the sample holder, illuminating the sample with the light such that the light interacts with the sample and the sample holder, receiving light emitted from the sample by fluorescence and forming an image thereof and including.

[0054] The method further includes receiving light after interaction of the sample and the sample holder and forming at least one image thereof, wherein at least one localized structural characteristic of the sample is visible in the image based on the color of the received light. Such imaging is described in more detail in PCT / Australia Patent Application Publication No. 2018 / 050496. In this specification, an image formed by such a method is referred to as a color contrast image.

[0055] Advantageously, in this way, a sample mounted on a sample holder can be imaged by using fluorescence microscopy in addition to using color contrast imaging.

[0056] As will be apparent to those skilled in the art, fluorescence microscopy is typically (or preferably) performed with a reflected light microscopy arrangement because the sample needs to be illuminated from the same side as the side on which the fluorescence induced within the sample is imaged. Thus, the schematic arrangement of FIG. 3A can also be used for fluorescence imaging of the same sample, as shown in FIG. 3B. In this example, the light source 310 emits incident light 321 to illuminate the sample 106. The illumination causes the sample 106 to fluoresce. The light 325 emitted from the sample by the fluorescence is captured by an imaging system 312 that enables the generation of a fluorescence image of the sample.

[0057] As is known to those skilled in the art, fluorescence microscopy may require specialized sample preparation and labeling techniques. Wavelength-selective illumination and image capture techniques may also be used. Details of common fluorescent sample preparation, illumination techniques, or image capture techniques used in fluorescence microscopy imaging are not described here so as not to obscure the details of the present invention.

[0058] In some embodiments, fluorescence and color contrast microscopy can be performed continuously or simultaneously on the same sample. When performed continuously, an image formed from the light emitted from the sample by fluorescence can be formed in a first period, and a color contrast image can be formed in a second period.

[0059] It may be necessary or advantageous to use special illumination characteristics in each type of microscopy, in which case the light used in color contrast imaging may have a different illumination spectrum than that used in fluorescence imaging. This may require using two light sources interchangeably or using a common light source that is filtered in different ways within each period. However, in some cases, the same spectrum can be used.

[0060] FIG. 3C schematically shows an example in which fluorescence imaging and color contrast imaging of the same sample are performed by using the systems of FIGS. 3A and 3B. In this example, the light source 310 emits light 321 / 311 (which may have the same spectrum or different spectra) to illuminate the sample 106. The illumination causes fluorescence to be generated in the sample 106 and also interacts with the plasmonic layer 102 of the sample and the sample holder 100. The received light 325 is emitted from the sample by fluorescence, and the received light 315 is reflected by the sample holder 100. The received light 315 / 325 is captured by the imaging system 312 to enable the generation of both a fluorescence image and a color contrast image of the sample. As pointed out above, the emission of the light 311 and 321 may or may not be simultaneous.

[0061] Figure 3D schematically shows an example in which fluorescence imaging and color contrast imaging of the same sample are performed by using the systems of FIGS. 3A and 3B. In this example, the light source 310 emits light 321 / 311 (which may have the same spectrum or different spectra) to illuminate the sample 106. The illumination causes fluorescence in the sample 106 and also interacts with the plasmonic layer 102 of the sample and the sample holder 100. The received light 325 is emitted from the sample by fluorescence, and the received light 315 is reflected by the sample holder 100. The received light 315 is captured by the first imaging system 312 to enable the generation of a color contrast reflected image. The received light 325 is captured by the second imaging system 312' to enable the generation of a fluorescence image of the same sample. As pointed out above, the emission of light 311 and 321 may or may not be simultaneous. With respect to FIGS. 3C and 3D, optionally, the sample may generate fluorescence during color contrast microscopy if not desired. This can generally be addressed by filtering out and removing the fluorescence wavelengths during image formation or by not illuminating the sample in the wavelength range that causes fluorescence. Since fluorescence is generated via specific optical transitions within the fluorescent tags, it occurs at very specific wavelengths and thus appears as a line (or lines) within the received spectrum. These characteristic lines can be separated from the smoother continuous background spectrum of the color contrast image. If the fluorescence lines accidentally coincide with strong wavelength peaks within the received color spectrum in the color contrast image, this can be corrected by using nanoslides with different separated submicron structures or, if the nanoslides being used have different separations between submicron structures in different directions, this can be corrected by changing the polarization of the illumination of the color contrast image.As described in the Australian patent applications of the applicant in co-pending, entitled "Automated method of identifying a structure" and "Method of identifying a structure", filed on the same day as this application, this will modify the received light spectrum in the color contrast image rather than the fluorescence image.

[0062] The images captured by this method can be used individually or together to draw conclusions about the sample. In some embodiments of the invention, the method may include spatially correlating an image formed from light emitted from the sample by fluorescence with a color contrast image.

[0063] This can be done by generating a composite image showing both color contrast information and the fluorescence image. Multiple images formed from light emitted from the sample by fluorescence (e.g., one of each fluorescence wavelength) and / or multiple color contrast images (captured at various polarizations) can be combined into a single image.

[0064] The combination can be done optically (e.g., during optical image formation) or digitally (e.g., by combining the data values representing the images). It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features described or apparent from the text or drawings. All of these various combinations constitute various alternative aspects of the invention.

Claims

1. A method for imaging a sample, comprising: providing a sample holder having an upper surface and a lower surface, the upper surface having a plasmonic layer associated therewith, the plasmonic layer comprising a periodic array of submicron structures; Affixing the sample to the upper surface of the sample holder; illuminating the sample with light such that the light interacts with the plasmonic layer of the sample and the sample holder; receiving light emitted from the sample by fluorescence and forming a first image therefrom; receiving light after interaction with the plasmonic layer of the sample and the sample holder and forming a second image therefrom; Including, In the second image, at least one local structural characteristic of the sample is visualized based on the color of the light received.

2. 2. The method of claim 1 , wherein the first image is formed from the light emitted from the sample by fluorescence and the second image is formed from light after interaction with the sample and the plasmonic layer of the sample holder simultaneously.

3. 3. The method of claim 2, wherein the sample holder has a characteristic reflectance spectrum defined by the periodic array of submicron structures, the sample holder being selected such that its characteristic reflectance spectrum is substantially different from an expected fluorescence spectrum associated with light emitted from the sample by fluorescence.

4. 2. The method of claim 1 , wherein the first image formed from the light emitted from the sample by fluorescence is formed in a first time period, and the second image formed from light after interaction with the sample and the plasmonic layer of the sample holder is formed in a second time period.

5. 5. The method of claim 4, comprising illuminating the sample with light having a first illumination spectrum during the first time period and with light having a second illumination spectrum during the second time period.

6. The method of claim 5 , wherein the first illumination spectrum is selected based on a fluorescence characteristic of the sample.

7. The method of claim 5 or 6, wherein the first and second illumination spectra are the same.

8. The method of claim 5 or 6, wherein the first and second illumination spectra are different.

9. 9. The method of any one of claims 1 to 8, wherein receiving light emitted from the sample by fluorescence comprises filtering light received from the sample and / or the sample holder to minimize light received from sources other than the fluorescence.

10. 10. The method according to claim 1, comprising illuminating the sample from a side of the sample holder on which the sample is placed and receiving from the same side as the illumination light emitted from the sample by fluorescence and also light after interaction with the sample and the plasmonic layer of the sample holder.

11. 11. The method of claim 1, further comprising spatially correlating the first image formed from light emitted from the sample by fluorescence and the second image formed from light after interaction with the sample and the plasmonic layer of the sample holder.

12. 12. The method of claim 11, further comprising forming a composite image including at least one of the first images formed from the light emitted from the sample by fluorescence and at least one of the second images formed from light after interaction with the sample and the plasmonic layer of the sample holder.

13. The method of claim 12 , wherein the first image and the second image are optically combined before being captured as a digital image or digitally combined after being captured.

14. The method according to any one of claims 1 to 13, wherein the sample is a biological sample.

15. The method of claim 1, wherein at least one of the local structural characteristics includes a localized dielectric constant.

16. 1. A system for forming an image, comprising: a reflected light microscope having an imaging system; A lighting system; a sample holder having an upper surface and a lower surface, the upper surface having a plasmonic layer associated therewith, the plasmonic layer including a periodic array of submicron structures; Including, the illumination system is configured to illuminate the sample disposed on the upper surface of the sample holder with light such that the light interacts with the sample and the plasmonic layer of the sample holder; The reflected light microscope is configured to receive light emitted from the sample by fluorescence and form a first image therefrom, and to receive light after interaction with the plasmonic layer of the sample and the sample holder and form a second image therefrom, wherein at least one local structural characteristic of the sample is visualized in the second image based on the color of the received light.

17. 17. The system of claim 16, wherein the reflected light microscope is configured to form the first image formed from the light emitted from the sample by fluorescence and simultaneously form the second image formed from light after interaction with the sample and the plasmonic layer of the sample holder.

18. 20. The system of claim 17, wherein the sample holder has a characteristic reflectance spectrum defined by the periodic array of submicron structures, the sample holder being selected such that its characteristic reflectance spectrum is substantially different from an expected fluorescence spectrum associated with light emitted from the sample by fluorescence.

19. The reflected light microscope comprises: forming the first image formed from the light emitted from the sample by fluorescence during a first time period; and 17. The system of claim 16, configured to form the second image formed from light after interaction with the plasmonic layer of the sample and the sample holder during a second period of time.

20. The lighting system comprises: illuminating the sample with light having a first illumination spectrum during the first time period; and 20. The system of claim 19, configured to illuminate the sample using light having a second illumination spectrum during the second time period.

21. 21. The system of claim 20, wherein the first illumination spectrum is selected based on a fluorescence characteristic of the sample.

22. 22. The system of claim 20 or 21, wherein the first and second illumination spectra are the same.

23. 22. The system of claim 20 or 21, wherein the first and second illumination spectra are different.

24. The system according to any one of claims 16 to 23, wherein the sample is a biological sample.

25. A system according to any one of claims 16 to 24, comprising an image capture system for generating at least one digital image of the sample.

26. The system of claim 25, wherein the system is configured to form a composite image including at least one of the first images formed from the light emitted from the sample by fluorescence and at least one of the second images formed from the light after interaction with the sample and the plasmonic layer of the sample holder.

27. ​​The system described in claim 26, wherein the first image and the second image are optically combined before being captured as a digital image or digitally combined after being captured.

28. A system described in any one of claims 16 to 27, wherein at least one of the local structural characteristics includes a localized dielectric constant.

Images