阅读说明：本技术 拉曼光谱法与高级光学显微法的集成系统及其应用 (Integrated system of Raman spectroscopy and advanced optical microscopy and application thereof ) 是由 方宁 董斌 陈匡财 赵菲 于 2019-08-16 设计创作，主要内容包括：一种用于样本上的多模态成像的集成光谱显微系统包含反射差分干涉对比度(RDIC)显微镜、与所述RDIC显微镜光学耦合的拉曼光谱仪以及与所述RDIC显微镜光学耦合的全内反射荧光/散射(TIRF/TIRS)显微镜,使得所述集成光谱显微系统能够同时获取同一样本上的RDIC图像、拉曼光谱和TIRF/TIRS图像。(An integrated spectral microscopy system for multimodal imaging on a sample includes a Reflection Differential Interference Contrast (RDIC) microscope, a Raman spectrometer optically coupled to the RDIC microscope, and a total internal reflection fluorescence/scattering (TIRF/TIRS) microscope optically coupled to the RDIC microscope such that the integrated spectral microscopy system is capable of simultaneously acquiring RDIC images, Raman spectra, and TIRF/TIRS images on the same sample.)

1. An integrated spectroscopic microscopy system for multi-modal imaging on a sample, comprising:

a Reflective Differential Interference Contrast (RDIC) microscope;

a Raman spectrometer optically coupled to the RDIC microscope to enable the integrated spectroscopic microscopy system to simultaneously acquire an RDIC image and a Raman spectrum on the same sample.

2. The integrated spectroscopic microscopy system of claim 1, further comprising a total internal reflection fluorescence/scattering (TIRF/TIRS) microscope optically coupled to the RDIC microscope to enable the integrated spectroscopic microscopy system to simultaneously acquire the RDIC image, the raman spectrum, and a TIRF/TIRS image on the same sample.

3. The integrated spectroscopic microscopy system of claim 2, wherein the integrated spectroscopic microscopy system is capable of separately acquiring the RDIC image, the Raman spectrum, and the TIRF/TIRS image on the same sample by removing selected optical components.

4. The integrated spectroscopic microscopy system of claim 2, wherein each of the RDIC microscope, the Raman spectrometer, and the TIRF/TIRS microscope comprises: a delivery member for delivering incident light emitted from a corresponding light source to the sample placed on a substrate to irradiate the sample with the incident light; and a collecting member for collecting light from the illuminated sample in response to illumination by the incident light.

5. The integrated spectroscopic microscopy system of claim 4, wherein the delivery means of the RDIC microscope comprises a linear polarizer, a beam splitter, a Nomarski prism, and an objective lens placed in a first optical path that is an incident light path of the RDIC microscope such that: the beam splitter is placed at 45 ° to the incident light; the Nomarski prism is positioned at a back focal plane of the objective lens and is operably translatable laterally across the optical path to introduce an offset delay to achieve optimal image contrast of the sample; and the objective lens is positioned proximate to the sample and is operable to collimate the shearing orthogonal wavefront onto the sample.

6. The integrated spectroscopic microscopy system of claim 5, wherein the delivery means of the Raman spectrometer comprises the beam splitter, the objective lens, and a operably-removable mirror positioned between the linear polarizer in the first optical path and the beam splitter such that when the operably-removable mirror is removed, incident light of the Raman microscope is not delivered to the sample.

7. The integrated spectroscopic microscopy system of claim 4, wherein the delivery means of the TIRF/TIRS microscope comprises a prism positioned relative to the sample and a plurality of optical components placed in an incident light path of the TIRF/TIRS microscope for delivering incident light to the prism at an angle of incidence greater than a critical angle such that the incident light is fully reflected and an evanescent field is generated to selectively illuminate and excite fluorophores of the sample within the evanescent field, wherein the substrate is between the sample and the prism.

8. The integrated spectroscopic microscopy system of claim 5, wherein the collection means of the RDIC microscope comprises the objective lens, the Nomarski prism, the beam splitter, an analyzer, and a tube lens placed in a second optical path such that light from the sample in response to illumination of the collimated orthogonal wavefront is collected by the objective lens and focused onto an interference plane of the Nomarski prism, passes through the beam splitter and the analyzer, and is focused by the tube lens onto a camera.

9. The integrated spectroscopic microscopy system of claim 8, wherein the collection means of the raman spectrometer comprises the objective lens, the beam splitter, the tube lens, and a mirror placed in the second optical path such that raman signals emitted from the sample in response to illumination by the incident light of the raman spectrometer are collected by the objective lens and pass through the beam splitter and the tube lens and are reflected to a detector of the raman spectrometer through a third optical path.

10. The integrated spectroscopic microscopy system of claim 8, wherein the collection means of the TIRF/TIRS microscope comprises the objective lens, the beam splitter and the tube lens placed in the second optical path such that light emitted from the selectively excited fluorophore of the sample is collected by the objective lens and passes through the beam splitter and is focused on the camera by the tube lens.

11. The integrated spectroscopic microscopy system of claim 4, wherein the beam splitter plate is a dichroic mirror.

12. The integrated spectroscopic microscopy system of claim 4, wherein the substrate comprises a quartz slide coated with a nano-thickness gold film, the sample being placed on the quartz slide.

13. The integrated spectroscopic microscopy system of claim 2, further comprising a sample stage for coupling a prism-type TIRF microscope to the integrated spectroscopic microscopy system.

14. The integrated spectroscopic microscopy system of claim 13, wherein the sample stage has a recess designed to accommodate the prism to enable us to mount a sample plate directly on top of the prism, thereby allowing simultaneous acquisition of a TRIFM image, a RDIC image and a Raman spectrum.

15. The integrated spectroscopic microscopy system of claim 13, wherein the sample stage is an in situ thermal annealing stage and/or an in situ solvent annealing stage.

16. The integrated spectroscopic microscopy system of claim 2, further comprising an autofocus module for providing high stability of the integrated spectroscopic microscopy system.

17. The integrated spectroscopic microscopy system according to claim 2, further comprising a multi-modality imaging collection module for different microscopy/spectroscopy modalities.

18. The integrated spectroscopic microscopy system of claim 17, wherein the multi-modal multi-view imaging collection module is a fully automated multi-modal dual view imaging and spectroscopy module capable of simultaneous single molecule fluorescence imaging and single molecule spectroscopy imaging; 2D/3D super-resolution imaging based on single molecule positioning; two-color imaging; and polarization imaging.

19. The integrated spectroscopic microscopy system of claim 17, wherein the multi-modal multi-view imaging collection module is a multi-modal multi-color 4-channel imaging module capable of 3D single particle tracking capability through bifocal imaging or Point Spread Function (PSF) engineering including parallax imaging, double helix PSF, and astigmatism.

Technical Field

The present invention relates generally to the field of spectroscopy and microscopy, and more particularly to an integrated system of raman spectroscopy and advanced optical microscopy and applications thereof.

Background

The background description provided herein is for the purpose of generally presenting the context of the disclosure. The subject matter discussed in this background of the invention section should not be assumed to be prior art merely because it was mentioned in this background of the invention section. Similarly, the problems mentioned in the background section or associated with the subject matter of the background section should not be assumed to have been previously identified in the prior art. The subject matter in the background section merely represents different approaches that may also be inventions in their own right. Work of the presently named inventors, to the extent it is described in this background section, as well as aspects of the description that may not otherwise qualify as prior art at the time of filing, are neither expressly nor impliedly admitted as prior art against the present disclosure.

Optical microscopy imaging has played an increasingly important role in the study of nanomaterials and biomaterials and in basic chemical processes (e.g., diffusion, absorption, and reaction) on a molecular and nanoscale scale. However, its widespread chemical use has been hampered by the lack of definitive chemical information of the sample in optical microscopy images. Spectroscopic techniques (raman, IR, etc.) are often required as a companion tool to obtain a structural fingerprint of a sample. Systems that can provide both physical and chemical properties of a sample are essential for a more complete understanding of the physicochemical properties [1-9 ].

To date, performing in situ microscopy and spectroscopic measurements on exactly the same sample (or exactly the same region of the sample), particularly on samples undergoing continuous transformations, has remained challenging [10-16 ]. Existing multi-modal spectroscopic microscopy systems employ microscopy modes for a large part for relatively simple purposes such as sample positioning and fluorescence image acquisition. The higher-order features that have become hallmarks of microscopy development over the past decade, including single-molecule sensitivity, sub-diffraction limited spatial resolution, and fast (millisecond-range) temporal resolution, have rarely been combined with spectroscopic measurements.

On the other hand, new challenges and opportunities arise at intermediate scales (from-10 nm to 10 μm) between nano and traditional macro scale (bulk) materials. Many functional materials begin to exhibit their functional behavior at the mesoscale, where nanoscale building blocks are assembled into more complex functional architectures to achieve more varied interaction and greater functionality with the environment. Even though a great deal of research has progressed significantly on nanoscale structures over the past decades, mesoscale structures that are inherently dynamic and metastable remain relatively unexplored. The lack of mesoscale knowledge has limited our ability to predict and optimize the performance of functional materials and often limited their application to trial and error. There is a great need for an in situ multimodal characterization system that can perform and correlate various physical and chemical measurements on the same sample region that is subjected to continuous transformation under environmental stimuli to monitor and understand the evolution of mesoscale materials.

Accordingly, there exists a heretofore unaddressed need in the art to address the aforementioned deficiencies and inadequacies.

Disclosure of Invention

One of the objects of the present invention is to provide a new high sensitivity multifunctional spectroscopic microscopy system that combines far field optical microscopy imaging (fluorescence, dark field, interferometry, Total Internal Reflection (TIR), quantitative polarization, etc.) with raman-based spectroscopic techniques (confocal raman, Surface Enhanced Raman Spectroscopy (SERS), etc.) for in situ studies and kinetics of nano-and meso-scale structures. Different sample stations have also been developed to image under more challenging experimental conditions, such as thermal stress or solvent vapor, to enable in situ monitoring of the sample.

In one aspect of the invention, an integrated spectroscopic microscopy system for multimodal imaging on a sample comprises: a Reflective Differential Interference Contrast (RDIC) microscope; and a raman spectrometer optically coupled to the RDIC microscope to enable the integrated spectroscopic microscopy system to simultaneously acquire an RDIC image and a raman spectrum on the same sample. The RDIC microscope can also be used to collect quantitative polarization microscopy images by removing the Nomarski prism and setting the polarization direction of the light correctly. The integrated spectral microscopy system may also include a total internal reflection fluorescence/scattering (TIRF/TIRS) microscope optically coupled with the RDIC microscope to enable the integrated spectral microscopy system to simultaneously acquire RDIC and polarization microscopy images, the raman spectra, and TIRF/TIRS images on the same sample.

In one embodiment, the integrated spectroscopic microscopy system is capable of separately acquiring both RDIC and polarization microscopy images, Raman spectra, and TIRF/TIRS images on the same sample by removing selected optical components.

In one embodiment, each of the RDIC and polarization microscope, the Raman spectrometer, and the TIRF/TIRS microscope includes: a delivery means for delivering incident light emitted from a corresponding light source to a sample disposed on a substrate to irradiate the sample with light; and a collection member for collecting light from the illuminated sample in response to illumination by the incident light.

In one embodiment, the substrate comprises a quartz slide coated with a nano-thickness gold film on which the sample is placed.

In one embodiment, the delivery member of the RDIC microscope comprises a linear polarizer, a beam splitter, a Nomarski prism, and an objective lens placed in a first optical path that is the path of the incident light of the RDIC microscope such that the beam splitter is placed at 45 ° to the incident light; a Nomarski prism positioned at a back focal plane of the objective lens and operably translated laterally across the optical path to introduce an offset delay to achieve optimal image contrast of the sample; and the objective lens is positioned proximate to the sample and is operable to collimate the shearing orthogonal wavefront onto the sample.

In one embodiment, the collection member of the RDIC microscope includes an objective lens, a Nomarski prism, a beam splitter, an analyzer, and a tube lens placed in the second optical path such that light from the sample in response to illumination of the collimated orthogonal wavefront is collected by the objective lens and focused onto an interference plane of the Nomarski prism, passes through the beam splitter and the analyzer, and is focused onto the camera through the tube lens.

In one embodiment, the delivery means of the raman spectrometer comprises a beam splitter, an objective lens, and an operably removable mirror positioned between the linear polarizer in the first optical path and the beam splitter such that when the operably removable mirror is removed, incident light of the raman microscope is not delivered to the sample.

In one embodiment, the collection member of the raman spectrometer comprises an objective lens, a beam splitter, a tube lens and a mirror positioned in the second optical path such that raman signals emitted from the sample in response to illumination by incident light of the raman spectrometer are collected by the objective lens and pass through the beam splitter and the tube lens and are reflected to a detector of the raman spectrometer through a third optical path.

In one embodiment, a delivery member of a TIRF/TIRS microscope includes a prism positioned relative to a sample and a plurality of optical components placed in an incident light path of the TIRF/TIRS microscope for delivering incident light to the prism at an incident angle greater than a critical angle such that the incident light is fully reflected and generates an evanescent field to selectively illuminate and excite fluorophores of the sample within the evanescent field, wherein a substrate is between the sample and the prism.

In one embodiment, the collection member of the TIRF/TIRS microscope comprises an objective lens, a beam splitter and a tube lens placed in the second optical path such that light emitted from the selectively excited fluorophores of the sample is collected by the objective lens and passes through the beam splitter and is focused on the camera by the tube lens.

In one embodiment, the beam splitter plate is a dichroic mirror.

In one embodiment, the integrated spectroscopic microscopy system further comprises a sample stage for coupling the prism-type TIRF microscope to the integrated spectroscopic microscopy system.

In one embodiment, the sample stage has a notch designed to accommodate a prism to enable us to mount the sample plate directly on top of the prism, thereby allowing simultaneous acquisition of a TRIFM image, a RDIC image and a Raman spectrum. In one embodiment, the sample stage is an in situ thermal annealing stage and/or an in situ solvent annealing stage.

In one embodiment, the integrated spectroscopic microscopy system further comprises an autofocus module for providing high stability of the integrated spectroscopic microscopy system.

In one embodiment, the integrated spectroscopic microscopy system further comprises a multimode imaging collection module for different microscopy/spectroscopy modalities.

In one embodiment, the multi-modality multi-view imaging collection module is a fully automated multi-modality dual view imaging and spectroscopy module capable of performing single molecule fluorescence imaging and single molecule spectroscopy imaging simultaneously; 2D/3D super-resolution imaging based on single molecule positioning; two-color imaging; and polarization imaging.

In one embodiment, the multi-modality multi-view imaging collection module is a multi-modality multi-color 4-channel imaging module that can achieve 3D single particle tracking capability through bifocal imaging or Point Spread Function (PSF) engineering including parallax imaging, double helix PSF and astigmatism.

These and other aspects of the present invention will become apparent from the following description of the preferred embodiments, when considered in conjunction with the following drawings, although variations and modifications therein may be effected without departing from the spirit and scope of the novel concepts of the disclosure.

Drawings

The accompanying drawings illustrate one or more embodiments of the invention and, together with the written description, serve to explain the principles of the invention. The same reference numbers may be used throughout the drawings to refer to the same or like elements of an embodiment.

FIG. 1A shows a schematic arrangement of an integrated system according to one embodiment of the invention.

Fig. 1B shows an intensity distribution of a confocal raman laser spot according to one embodiment of the present invention.

FIG. 1C shows a laser spot used in Raman spectroscopy according to one embodiment of the present invention.

Fig. 2A-2B show designs of prism holders for prism-type Total Internal Reflection Fluorescence (TIRF) microscopy and multimodal imaging, respectively, in a system according to an embodiment of the invention.

Fig. 3A-3B illustrate a thermal annealing sample stage and a solvent annealing sample stage, respectively, according to embodiments of the invention.

FIG. 4 illustrates a design of an autofocus module for an upright/inverted microscope according to one embodiment of the present invention.

Fig. 5A-5B illustrate a fully automated multi-modality dual view imaging and spectroscopy module and a multi-modality 4-channel imaging module, respectively, according to embodiments of the present invention.

Figures 6A-6B show Reflection Differential Interference Contrast (RDIC) images of a 1:1P3HT: PCBM blend film before and after 30 minutes thermal annealing, respectively, according to one embodiment of the invention.

Fig. 6C shows RDIC strength traces along the dotted lines shown in fig. 6A-6B before and after thermal annealing according to one embodiment of the invention.

FIG. 6D shows portions of the regular P3HT in the blend before and after thermal annealing along the dotted line shown in FIGS. 6A-6B according to one embodiment of the invention.

Fig. 7A-7B show an RDIC image and a bright field image, respectively, of single layer graphene according to one embodiment of the invention.

Fig. 7C shows raman spectra of points 1 and 2 as shown in fig. 7A according to one embodiment of the present invention.

FIG. 8 shows an RDIC image of a living cell according to one embodiment of the invention.

FIG. 9A shows a Total Internal Reflection Scattering (TIRS) image of 40x80nm (SPR650nm) gold nanorods on a 50nm gold-coated slide, according to one embodiment of the present invention.

FIG. 9B shows the scattering spectrum of the nanorod labeled 1 shown in FIG. 9A, according to one embodiment of the invention.

Detailed Description

The present invention now will be described more fully hereinafter with reference to the accompanying drawings, in which exemplary embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Like reference numerals refer to like elements throughout.

The terms used in this specification generally have their ordinary meaning in the art, both within the context of the invention and in the specific context in which each term is used. Certain terms used to describe the invention are discussed below or elsewhere in the specification to provide additional guidance to the practitioner regarding the description of the invention. For convenience, certain terms may be highlighted, for example, using italics and/or quotation marks. The use of highlighting has no effect on the scope and meaning of the term; the scope and meaning of a term is the same in the same context, regardless of whether it is highlighted. It should be understood that the same thing can be stated in more than one way. Thus, alternative language and synonyms may be used for any one or more of the terms discussed herein, without any specific meaning being assigned herein, whether or not a term is detailed or discussed. Synonyms for certain terms are provided. The recitation of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification, including examples of any terms discussed herein, is illustrative only and in no way limits the scope and meaning of the invention or of any exemplified term. As such, the present invention is not limited to the various embodiments presented in this specification.

It is to be understood that, unless the context clearly dictates otherwise, the meaning of "a" and "the" as used in the description herein and throughout the appended claims includes plural referents. Also, it will be understood that when an element is referred to as being "on" another element, it can be directly on the other element or intervening elements may be present therebetween. In contrast, when an element is referred to as being "directly on" another element, there are no intervening elements present. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

It will be understood that, although the terms first, second, third, etc. may be used herein to describe various elements, components, regions, layers and/or sections, these elements, components, regions, layers and/or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer or section from another element, component, region, layer or section. Thus, a first element, component, region, layer or section discussed below could be termed a second element, component, region, layer or section without departing from the teachings of the present disclosure.

Furthermore, relative terms, such as "lower" or "bottom" and "upper" or "top," may be used herein to describe one element's relationship to another element as illustrated in the figures. It will be understood that relative terms are intended to encompass different orientations of the device in addition to the orientation depicted in the figures. For example, if the device in one figure is turned over, elements described as being on the "lower" side of other elements would be oriented on the "upper" side of the other elements. Thus, the exemplary term "lower" can encompass both an orientation of "lower" and "upper," depending on the particular orientation of the figure. Similarly, if the device in one of the figures is turned over, elements described as "below" or "beneath" other elements would then be oriented "above" the other elements. Thus, the exemplary terms "under" or "beneath" can encompass both an orientation of above and below.

It will be further understood that the terms "comprises" or "comprising" or "has" or "carries" or "contains" or "involves" and the like are open-ended, i.e., mean including but not limited to. When used in this disclosure, they specify the presence of stated features, regions, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and/or groups thereof.

Unless defined otherwise, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present invention and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

As used herein, "about," "about," or "approximately" shall generally mean within 20%, preferably within 10%, and more preferably within 5% of a given value or range. The quantities given herein are approximate, meaning that the terms "about", "about" or "approximately" can be inferred if not expressly stated.

As used herein, the terms "comprising," "including," "carrying," "having," "containing," "involving," and similar terms are to be understood as open-ended, i.e., meaning including but not limited to.

As used herein, at least one of the phrases A, B and C should be construed to mean logic (a or B or C) that uses a non-exclusive logical or. It should be understood that one or more steps within a method may be performed in a different order (or simultaneously) without altering the principles of the present invention.

The following description is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses. The broad teachings of the present disclosure can be implemented in a variety of forms. Therefore, while this disclosure includes particular examples, the true scope of the disclosure should not be so limited since other modifications will become apparent upon a study of the drawings, the specification, and the following claims. For purposes of clarity, the same reference numbers will be used in the drawings to identify similar elements. It should be understood that one or more steps within a method may be performed in a different order (or simultaneously) without altering the principles of the present invention.

The present invention relates to a novel high sensitivity multifunctional spectroscopic microscopy system that combines far field optical microscopy imaging (fluorescence, dark field, interferometry, total internal reflection, polarization, etc.) with raman-based spectroscopic techniques (confocal raman, Surface Enhanced Raman Spectroscopy (SERS), etc.) for in situ studies and dynamics of nanoscale and mesoscale structures. Different sample stations have also been developed to image under more challenging experimental conditions, such as thermal stress or solvent vapor, to enable in situ monitoring of the sample.

The in situ imaging capabilities of the system of the present invention make it a valuable characterization technique complementary to other conventional methods such as electron microscopy and scanning probe microscopy. Which provides a high-throughput optical instrument that operates under ambient or controlled conditions in which the light source is used. It stimulates a new scientific quest that was previously unattainable by providing scientists with a new ability to dynamically acquire and correlate a wide range of information of their samples at sub-micron spatial resolution.

When the transformation is fine or requires spectroscopic measurement of fine sample structures, there is a need for high resolution and high contrast microscopy techniques that can quickly locate and observe fine structures while performing spectroscopic measurements. Subsequently, high resolution and high contrast images of changes in physical form can be correlated with spectral changes indicative of dynamically changing chemical properties. High throughput is an advantage of wide-field optical microscopy imaging techniques over other high resolution and high contrast techniques such as electron microscopy, scanning probe microscopy, atomic force microscopy, and near-field scanning optical microscopy. Near-field scanning optical microscopy has low incident light intensity, hampers excitation of weak fluorescent molecules, and is not suitable for imaging soft materials due to the high spring constant of optical fibers [8]. High resolution and high contrast optical microscopy techniques can be used to rapidly scan and locate regions, structures or domains of interest under physical or chemical transformations. Physical and chemical processes can be related and distinguished by a combination of microscopic and spectroscopic techniques. Optical microscopy imaging techniques are also advantageous as being less invasive; thus, sample integrity is typically maintained throughout the sampling process, enabling the study of dynamic processes over an extended time frame. In general, for samples viewed under optical microscopy imaging techniques, no special sample preparation is required; thus, the sample can be observed and studied under its working conditions. Raman spectroscopy can provide rich information about chemical structure without special sample preparation requirements, enabling it to perform in situ experiments [8, 17]. Also, since the system can operate in an open ambient environment, different conditions such as temperature, pH, and electronic voltage can be applied to the sample through various designs of the sample holder. By varying the submerged target, it is possible to sample in different environments, such as water or air.

In one aspect of the invention, an integrated spectroscopic microscopy system for multimodal imaging of a sample includes a Reflection Differential Interference Contrast (RDIC) microscope, a Raman spectrometer optically coupled to the RDIC microscope, and a total internal reflection fluorescence/scattering (TIRF/TIRS) microscope optically coupled to the RDIC microscope, such that the integrated spectroscopic microscopy system is capable of simultaneously acquiring RDIC images, Raman spectra, and TIRF/TIRS images on the same sample.

In one embodiment, the integrated spectroscopic microscopy system is capable of separately acquiring an RDIC image, a Raman spectrum, and a TIRF/TIRS image on the same sample by removing selected optical components.

In one embodiment, each of the RDIC microscope, the Raman spectrometer, and the TIRF/TIRS microscope includes: a delivery means for delivering incident light emitted from a corresponding light source to a sample disposed on a substrate to irradiate the sample with light; and a collection member for collecting light from the illuminated sample in response to illumination by the incident light.

In one embodiment, the substrate comprises a quartz slide coated with a nano-thickness gold film on which the sample is placed.

In one embodiment, the delivery member of the RDIC microscope comprises a linear polarizer, a beam splitter, a Nomarski prism, and an objective lens placed in a first optical path that is the path of the incident light of the RDIC microscope such that the beam splitter is placed at 45 ° to the incident light; a Nomarski prism positioned at a back focal plane of the objective lens and operably translated laterally across the optical path to introduce an offset delay to achieve optimal image contrast of the sample; and the objective lens is positioned proximate to the sample and is operable to collimate the shearing orthogonal wavefront onto the sample.

In one embodiment, the collection member of the RDIC microscope includes an objective lens, a Nomarski prism, a beam splitter, an analyzer, and a tube lens placed in the second optical path such that light from the sample in response to illumination of the collimated orthogonal wavefront is collected by the objective lens and focused onto an interference plane of the Nomarski prism, passes through the beam splitter and the analyzer, and is focused onto the camera through the tube lens.

In one embodiment, the delivery means of the raman spectrometer comprises a beam splitter, an objective lens, and an operably removable mirror positioned between the linear polarizer in the first optical path and the beam splitter such that when the operably removable mirror is removed, incident light of the raman microscope is not delivered to the sample.

In one embodiment, the collection member of the raman spectrometer comprises an objective lens, a beam splitter, a tube lens and a mirror positioned in the second optical path such that raman signals emitted from the sample in response to illumination by incident light of the raman spectrometer are collected by the objective lens and pass through the beam splitter and the tube lens and are reflected to a detector of the raman spectrometer through a third optical path.

In one embodiment, a delivery member of a TIRF/TIRS microscope includes a prism positioned relative to a sample and a plurality of optical components placed in an incident light path of the TIRF/TIRS microscope for delivering incident light to the prism at an incident angle greater than a critical angle such that the incident light is fully reflected and generates an evanescent field to selectively illuminate and excite fluorophores of the sample within the evanescent field, wherein a substrate is between the sample and the prism.

In one embodiment, the collection member of the TIRF/TIRS microscope comprises an objective lens, a beam splitter and a tube lens placed in the second optical path such that light emitted from the selectively excited fluorophores of the sample is collected by the objective lens and passes through the beam splitter and is focused on the camera by the tube lens.

In one embodiment, the beam splitter plate is a dichroic mirror.

In one embodiment, the integrated spectroscopic microscopy system further comprises a sample stage for coupling the prism-type TIRF microscope to the integrated spectroscopic microscopy system.

In one embodiment, the sample stage has a notch designed to accommodate a prism to enable us to mount the sample plate directly on top of the prism, thereby allowing simultaneous acquisition of a TRIFM image, a RDIC image and a Raman spectrum. In one embodiment, the sample stage is an in situ thermal annealing stage and/or an in situ solvent annealing stage.

In one embodiment, the integrated spectroscopic microscopy system further comprises an autofocus module for providing high stability of the integrated spectroscopic microscopy system.

In one embodiment, the integrated spectroscopic microscopy system further comprises a multimode imaging collection module for different microscopy/spectroscopy modalities.

In one embodiment, the multi-modality multi-view imaging collection module is a fully automated multi-modality dual view imaging and spectroscopy module capable of performing single molecule fluorescence imaging and single molecule spectroscopy imaging simultaneously; 2D/3D super-resolution imaging based on single molecule positioning; two-color imaging; and polarization imaging.

In one embodiment, the multi-modality multi-view imaging collection module is a multi-modality multi-color 4-channel imaging module that can achieve 3D single particle tracking capability through bifocal imaging or Point Spread Function (PSF) engineering including parallax imaging, double helix PSF and astigmatism.

Without intending to limit the scope of the invention, examples according to embodiments of the invention and their associated results are given below. It should be noted that titles or subtitles may be used in the examples for convenience of a reader, which in no way should limit the scope of the invention. Furthermore, certain theories are proposed and disclosed herein; however, it should in no way be construed as limiting the scope of the invention, whether it is correct or incorrect, so long as the invention is practiced according to the invention without regard to any particular theory of action or scheme.

According to the present invention, the integrated system can be used for the dynamic in situ acquisition of chemical information in raman spectroscopy and simultaneously morphological information in RDIC microscopy and high resolution high sensitivity single molecule and particle imaging in TIRF/TIRS microscopy.

Reflection differential interference contrast microscopy

Differential Interference Contrast (DIC) microscopy is in principle an interferometric detection technique that enables visualization of objects under the diffraction limit of light without the need for fluorescent markers. It has been using decades as a complementary tool to visualize cellular features with higher contrast, better resolution, and shallower depth of field than other far-field optical imaging techniques. We have invested specialized efforts in the development of DIC microscopy in the last decade to transform it into a major research tool for Single Particle Orientation and Rotational Tracking (SPORT) of plasmonic nanoparticles in various chemical and biological systems [18-22 ]. Various imaging probes with multiplexed detection capability have been investigated with different shapes (nanospheres, nanorods, nanowires, nanodumbells, core/shell nanostructures) and compositions (gold, silver mixed nanomaterials) [23-27 ]. We have also demonstrated the ability to modify and extend existing DIC microscopes into multimodal imaging tools [22, 29, 29]. Computer simulations of image formation in DIC microscopy, together with examination of the optical behavior of nano-objects under different microscopic settings and experimental conditions, provide theoretical guidelines and support for future experimental design [30-33 ].

Commercially available Nomarski type DIC microscopes typically employ a two-prism transmitted light configuration; however, many substrates in material science, such as solar cells and Surface Enhanced Raman Scattering (SERS) substrates, are opaque. In the present invention, we extended the applicability of DIC microscopy by constructing a reflex DIC (rdic) microscope. This RDIC microscope is optimized for the simultaneous detection of intermediate features of individual nanoscale building blocks and mesoscale materials on reflective surfaces such as solar cells and gold-coated substrates. Advantages of RDIC microscopy over other conventional methods such as electron microscopy and scanning probe microscopy include non-invasive and non-destructive rapid characterization, relatively simple sample preparation, real-time dynamic measurements, and are relatively less expensive.

FIG. 1A schematically illustrates an RDIC microscope 110 according to one embodiment of the invention. Light 112 emitted from a light source 111, e.g., a Light Emitting Diode (LED), for the reflective DIC microscope 110 is collimated by a set of lenses and filters 113 and passes through a linear polarizer 115 and a beam splitter plate (or dichroic mirror) 118 placed at a 45 degree angle to the incident beam 116. In one embodiment, there may be a mirror 114 placed in the optical path between the set of lenses and filter 113 and the linear polarizer 115, so that the collimated beam of reflected light 112 enters the linear polarizer 115 to obtain an incident beam 116. The light reflected by the beam splitter plate (or dichroic mirror) 118 from the incident beam 116 is then focused onto a Nomarski prism 120 positioned at the back focal plane of an objective lens 121. The Nomarski prism 120 can be translated laterally across the optical path 119 to introduce an offset delay to achieve optimal image contrast for different samples. The objective lens 121 then collimates the shear orthogonal wavefront onto a sample (specimen) 123 placed on a specimen plate/platform 122. Subsequently, the light from the sample (specimen) 123 in response to the illumination of the collimated orthogonal wavefront is collected by the same objective lens 121 and focused onto the interference plane of the Nomarski prism 120 where they recombine to eliminate shear forces. After passing through Nomarski prism 120, the same beam splitter 118, and analyzer (or second linear polarizer) 124, light 128 from sample 123 is focused by tube lens 125 onto camera 129.

In one embodiment, as shown in fig. 2B, the sample plate/platform 122 is a substrate of a quartz slide 122a coated with a nanometer thickness gold film 122B, where the gold film 122B acts as a mirror for RDIC imaging. In one embodiment, the analyzer 124 is used in an RDIC to generate a differential interference image pattern and allow beams having the same polarization direction as the analyzer 124 to interact and form an interference image. In certain embodiments, the analyzer 124 may have an extinction ratio: 510-800nm (>1000:1)，520-740nm(>10000:1) and 530-640nm (>100000: 1); laser damage threshold: about 10W/cm for continuous barrier²And about 25W/cm for continuous passage²(ii) a Clear aperture: about 22.9 mm; optical thickness: 280 +/-50 mu M; and the operating temperature: -20 ℃ to 120 ℃.

Most of the RDIC optics are shared with built-in confocal raman and TIR microscopy modalities. In contrast to conventional transmitted light DIC configurations, RDIC uses only one Nomarski prism in the optical path to shear linearly polarized incident light into two orthogonally polarized wavefronts and recombine the distorted wavefronts reflected from the sample surface. The objective lens acts as a condenser to impinge incident light onto the sample and focus the wavefront returning from the sample surface for recombination at the same Nomarski prism interference plane. The RDIC image may be viewed as a true 3D representation of the specimen surface geometry, rather than a pseudo 3D appearance of the specimen in the transmitted light DIC.

RDIC configurations were demonstrated as early as 1979 by Lessor et al and were directed to surface topography research applications [34]. Traditionally, RDIC has been used to report surface roughness qualitatively and quantitatively while studying polishing techniques, and also has many uses in visualizing details and defects in semiconductor chips fabricated on silicon dioxide wafers. However, all previously developed RDIC microscopes are limited to stand-alone systems with relatively low magnification and low resolution due to design and instrument challenges. Oil immersion objectives with high numerical aperture and high magnification are essential to achieve high resolution and high sensitivity in RDICs. This also requires complex instrumentation due to limited space and short working distances.

However, according to the present invention, the new instrument is a combined spectral microscopy system that utilizes our expertise in advanced instrumentation capabilities, deep understanding (both theoretically and experimentally) and continuous effort of innovation in relation to the development of DIC based technology [19, 23, 24, 28, 32, 33, 35-38 ]. The combined spectral microscopy system provides in-situ optical characterization with sub-diffraction limited spatial resolution, millisecond temporal resolution for single molecule and single nanoparticle imaging, while enabling coexistence of other imaging modalities.

Integration with confocal Raman spectroscopy

The combination of raman and DIC spectroscopy allows us to correlate surface enhancement and inter-particle distance information. The main design challenge is to achieve coexistence of two very different sets of optics for RDIC and raman while achieving optimal image and spectral quality, respectively.

Raman spectroscopy has been considered as a powerful technique for structural analysis [39-40]. By using lasers of different frequencies from near ultraviolet to near infrared in the raman module, optimal excitation conditions for different samples can be achieved. By selecting a laser with an appropriate frequency, certain electronic transitions can be excited and resonance raman studies of parts of certain components or molecules of a sample can be performed [18, 41]. Comparable to RDIC microscopy [41] compared to infrared absorption spectroscopy]Raman measurements are non-invasive under ambient conditions and do not require special sample preparation techniques [8, 17]. The spatial and temporal resolution of raman scattering is determined by the excitation laser spot size and pulse length. In confocal Raman technology, measurements from a volume of femtoliters (about 1 μm) can be made³) Makes spatially resolved measurements in chromosomes and cells possible [41-42 ]]. For temporal resolution, Raman spectra can be taken on a picosecond time scale, providing information about short lived species such as excited states and reaction intermediates [41]. By using different data analysis techniques based on multivariate analysis, more information about the chemical structure and chemical composition of samples of complex systems can be obtained [43]。

In one exemplary embodiment of the present invention as shown in fig. 1A, the combined spectral microscope system comprises a confocal raman system built into the RDIC microscope 110 that allows simultaneous acquisition of raman spectra and optical microscopy images. In this embodiment, a confocal raman system (referred to as a raman spectroscopy module) includes sample illumination 130a and raman spectral detection 130b, both of which are optically coupled to the RDIC microscope 110.

The sample illumination 130a comprises a light source, e.g. a laser 131 for emitting light 132, and collimating and delivering means 135 for collimating the light 132 as incident light for illuminating/exciting the sample 123 and delivering the incident light 138 through a beam splitter plate (or dichroic mirror) 118. Subsequently, light reflected by the beam splitter plate (or dichroic mirror) 118 from the incident light 138 is focused by the objective lens 121 onto the sample (specimen) 123 placed on the sample plate/stage 122 to illuminate the sample 123 with light. In this exemplary embodiment as shown in fig. 1A, the collimating and delivery member 135 includes, but is not limited to, five mirrors M1-M5, a flip mirror FM 137, two lenses L, and a pinhole/slit, all of which are placed in the optical path of the laser light 135. Specifically, an inverting mirror FM 137 is positioned between the linear polarizer 115 and the beam splitter plate (or dichroic mirror) 118 in the same optical path of the incident beam 116 of the RDIC microscope 110 to reflect light 136 obtained from the laser light 132 into the same optical path of the incident beam 116 of the RDIC microscope 110 as incident light 138 of the confocal raman system. Thus, the collimation and delivery member 135 also contains the beam splitter plate (or dichroic mirror) 118 and the objective lens 121 of the RDIC microscope 110.

The raman spectrum detection 130b includes a collecting means for collecting light (raman signal) from the sample (specimen) 123 in response to irradiation of incident light 138 of the confocal raman system, and a detector 149 for processing data of the collected raman signal into a raman spectrum. In this exemplary embodiment as shown in fig. 1A, the collection means 145 includes, but is not limited to, the objective lens 121, the beam splitter 118, and the tube lens 125 of the RDIC microscope 110, which collects and processes the raman signal from the sample 123 in response to the incident light 138 of the confocal raman system in the collected light 138, as well as mirrors M6, M7, M8, relay lenses, lenses L1-L4, and a pinhole. In particular, mirror M6 is positioned between tube lens 125 and camera 129 in the same optical path of light 128 of the RDIC microscope 110 to reflect the collected light 138 to mirror M7, which in turn reflects the light to a relay lens. The reflected light from the collected light 139, after passing through the relay lens, is reflected by mirror M8 to a series of lenses L1-L4 and pinholes/slits and received by detector 149. The detector 149 is a raman spectrometer or detector array such as a Charge Coupled Device (CCD). Various types of CCDs can be used for optimization over different wavelength ranges. In some embodiments, the pinhole is used to suppress the sample signal due to the out-of-focus position while delivering enough sample signal to the detector. The size of the pinhole depends on the objective lens (the numerical aperture of the objective lens and the magnification factor of the objective lens), the wavelength of the light and the intensity of the signal in use.

In accordance with the present invention, in a combined spectroscopic microscopy system as shown in FIG. 1A, the intrinsic sensitivity of Raman spectroscopy with respect to the chemistry of the sample 123 and the intrinsic sensitivity of RDIC microscopy with respect to the surface shape geometry of the sample 123 are combined to provide an accurate, high throughput, non-destructive, label-free and in situ useful analytical method under ambient conditions. Raman spectra of the same region of the sample in the RDIC image can be taken by using raman spectroscopy modules (systems) 130a-130 b. In one embodiment, the laser spot size used in the Raman spectroscopy module is about 0.4 μm, as shown in FIG. 1C. In one embodiment, 488nm laser light is used as excitation light incident on the graphene sample through a dichroic mirror and objective lens. The raman signal is collected by the same objective lens 121 and focused onto a spectrometer 149.

In addition, in accordance with the present invention, flip mirror FM 137, mirror M6, Nomarski prism 120, and analyzer 124 shown in FIG. 1A are operably removable. The addition/removal of these optical components changes the optical path and optical interaction to achieve different imaging modalities, and thus multi-modal imaging on the same specimen. For example, if the flip mirror FM 137 and mirror M6 are removed, then the Raman spectroscopy modules 130a-130b are disconnected and the integrated system is only able to acquire RDIC images on the sample. Otherwise, the integrated system may acquire both the RDIC image and the Raman spectrum on the same sample. On the other hand, if the Nomarski prism 120 and analyzer 124 are removed, the integrated system can only acquire the raman spectrum on the sample. At the same time, the integrated system may also acquire TIRF as disclosed below.

Integration with total internal reflection fluorescence microscopy

Total Internal Reflection Fluorescence Microscopy (TIRFM) may be considered the most successful mode of fluorescence microscopy applied in molecular dynamics studies at the liquid/solid interface, involving diffusion [ 60-62%]And absorption [63-64]. Under Total Internal Reflection (TIR) illumination, the angle of incidence of light is at a high refractive index (n)₁) May be used. At angles beyond the critical angle, the incident light is totally reflected and must have a ratio n₁Low refractive index adjacent medium (n)₂) Generating an evanescent wave. The depth of penetration of the evanescent wave varies with the angle of incidence, the wavelength of the light, and the refractive indices of the two media. The TIR geometry provides excellent background rejection for interface measurements. Sequential acquisitions from TIRFM with fixed angle and wide field of view microscopy [65-66]Or from two angles TIRFM 67-70]Can give a good estimate of the axial distance, while using a prism or objective based variable angle TIRFM [62, 7-74 ]]More depth-resolved information can be obtained, where a stack of multi-angle images contains integrated fluorescence intensities over various thicknesses of the sample. One of the common inventors of the present invention has also inventedAuto-collimating scanning angle prismatic total internal reflection fluorescence microscopy and optional variable illumination depth pseudo total internal reflection microscopy for nanometer precision axial position determination is now U.S. Pat. No. 9,012,872 [81]]Which is incorporated herein by reference in its entirety.

In one embodiment of the present invention as shown in fig. 1A, TIRFM is integrated into a combined spectral microscopy system as disclosed above, thus the benefits of both the attached surface imaging with high signal-to-noise ratio from TIRFM and the critical information from RDIC and confocal raman can be fully exploited. In this exemplary embodiment, TIRFM 150 includes a light source, such as a laser 151, for emitting light 152, and a delivery means 155 having, but not limited to, a prism 157 positioned relative to sample 123 on plate 122, and a plurality of optical components placed in the incident light path for delivering light 152 to prism 157 at an angle of incidence greater than a critical angle, such that incident light 156 is fully reflected and an evanescent field is generated that exponentially decays with distance, and only fluorophores of sample 123 within this evanescent field are selectively illuminated and excited. An image of light 158 emitted from fluorophores of the sample 123 selectively excited by the evanescent field may be acquired by the camera 129 of the RDIC microscope 110. Accordingly, TIRFM 150 enables selective visualization of the surface area of sample/specimen 123. As shown in fig. 1A, delivery member 155 may include, but is not limited to, mirrors M1-M5 and lens L placed in the optical path of light 152 and incident light 156, which is the light of the light 152 that passes through delivery member 155 to prism 157. In one embodiment, mirror M2 may be a periscope to redirect and change the height of light beam 152. In addition, the TIRFM 150 may include a collection means for collecting light 158 emitted from fluorophores of the selectively excited sample 123, including the objective lens 121 and the tube lens 125 of the RDIC microscope 110. In a certain embodiment, the two lasers 131 and 151 have different wavelengths, which may be used based on experimental and sample requirements. In certain embodiments, the two lasers 131 and 151 are put together to give the system more flexibility in selecting the excitation wavelengths used in the experiment, and they can be easily used interchangeably with other modules in the system.

As shown in fig. 2A, the present invention also relates in one aspect to a sample stage 170 configured for coupling a prism-type TIRFM into an integrated system of raman spectroscopy and advanced optical microscopy as shown in fig. 1A. The sample stage 170 has a recess 172 designed to receive the prism 157 to enable us to mount the sample slide 122 directly on the top 157a of the prism 157, thereby allowing simultaneous acquisition of the TRIFM image 158, the RDIC image 128, and the Raman spectrum 138, as shown in FIG. 2B. Other designs of sample stage may also be used to practice the invention. In certain embodiments, the substrate 122[75-76 ] of the quartz slide 122a, for example, coated with a nano-thickness gold film 122b]To support the sample 123. The gold film 122b is at the interface of the glass substrate 122a and the sample 123 (e.g., cells or aqueous medium). The incident light 156 is converted into Surface Plasmon Resonance (SPR) at the electron conductive gold film 122 b. The plasma then generates an evanescent wave field that extends into the medium on either side of the gold film 122 b. Penetration depth d into the sample medium_SPRIs that

Wherein epsilon_mIs the dielectric constant of gold. As theoretical prediction [77-79 ]]And also verified experimentally [80]]The penetration depth at SPR varies from about 100nm up to 5 μm as the wavelength increases from about 600nm to 4 μm. The evanescent wave field at the interface with the adjacent medium can be used to excite the probe for TIRFM imaging, while the gold film 122b also acts as a mirror for RDIC imaging.

In some embodiments, the substrate need not always be a gold coated substrate, and this depends on which module of the system is in use. Au was chosen as the coating metal due to its good surface plasmon performance in surface enhanced raman experiments.

In-situ thermal annealing station and solvent annealing station

To perform in-situ microscopy and spectroscopic measurements of exactly the same sample (or exactly the same region of the sample), particularly under different thermal and solvent vapor conditions, we developed an in-situ thermal annealing station and solvent annealing station for the integrated system of the present invention, as shown in fig. 3A and 3B.

For the thermal annealing station, thermoelectric cooler modules, which can also be used as heaters by reversing the current, are inserted in the slots (labeled 1). The specimen on the microscope slide is held by two jaws (labeled 2). The entire thermal annealing station can be easily mounted to the optical microscope stage by using 1/4-20 screws in holes 3.

For the solvent annealing station, the solvent system selected for the annealing process fills the cylinder holder (labeled 4). The entire solvent annealing station can be easily mounted to the optical microscope stage by using 1/4-20 screws in holes 5.

Automatic focusing module

The stability of a microscopy imaging system is critical to the accurate recovery of biological, chemical events, especially when long-term data recording is required. For example, during a thermal annealing process, the focal point may change with temperature. In one embodiment, the autofocus module design 400 as shown in fig. 4 is an easily attachable module for providing high stability of the imaging system. This add-on module is suitable for upright microscopes and inverted microscopes. The autofocus module design 400 uses reflected light generated at the interface between the sample and the cover slip to monitor the axial movement of the sample stage with respect to the objective lens. A separate IR (850nm LED) light source is coupled into the optical path using a short pass dichroic mirror, reflected at the sample/coverslip interface, and then imaged on a CCD camera. The axial displacement of the sample relative to the objective lens is conjugate to the lateral displacement of the reflected beam on the CCD camera. The calibration curve is generated, stored and then used to lock the imaging plane each time the autofocus function is activated.

Multi-modality imaging collection module

Due to the nature of collecting photons at multiple wavelengths for different microscopy/spectroscopy modalities, a multi-modal multi-view imaging collection module is essential to the design performance of the integrated system implementing the present invention. Fig. 5A shows a design of a fully automated multi-modality dual view imaging and spectroscopy module, which is capable of (but not limited to): 1) simultaneously carrying out single-molecule fluorescence imaging and single-molecule spectroscopy imaging; 2) 2D/3D super-resolution imaging based on single molecule positioning; 3) two-color imaging; 3) polarization imaging, etc. The signal (128, shown in fig. 1A) collected from the microscope body is relayed to the camera through a pair of clear lenses and re-imaged on the camera port side, as shown in fig. 5A. A cylindrical lens with a focal length of 1000mm can be inserted in the optical path before the first relay lens and introduce an astigmatism effect for 3D super-resolution imaging. For dual view imaging, the optical signal is split into two separate optical paths (channel 1 and channel 2) using optical components such as dichroic mirrors, polarizers, beam splitters, etc. In channel 1, an equilateral prism is mounted on an electric precision slide, can be coupled to the optical path by two mirrors, and disperses the optical signal laterally by wavelength, thus allowing spectroscopic imaging. Single view and dual imaging modes can be easily switched from one to the other by removing the optical single-splitting components (i.e., dichroic mirror, polarizer, beam splitter). Fig. 5B shows a design of a multi-modality multi-color 4-channel imaging module that can achieve 3D single particle tracking capability through bifocal imaging such as parallax imaging, double helix PSF, astigmatism, or Point Spread Function (PSF) engineering. The 4-channel imaging module operates in a similar manner to the dual view imaging module, fig. 5A, but has more channels to split the signal collected from the microscope body. Each channel has an individual focusing lens (part of the relay lens pair) in front of the camera, thus allowing the depth of focus in each channel to be adjusted individually for bifocal imaging on the same camera (i.e., camera 1 and/or camera 2). Optical components such as wedge prisms, phase masks, cylindrical lenses, etc. may be inserted into the optical path in each channel, thus enabling PSF engineering simultaneously or separately.

Examples of the applications

Polymer active layer in Bulk Heterojunction (BHJ) organic photovoltaic devices: poly (3-hexylthiophene) (P3HT) and [6, 6] -phenyl-C61-methyl butyrate (PCBM) blends are one of the most widely studied polymer active layers in BHJ organic photovoltaic devices. By combining the RDIC and confocal raman modules, the molecular arrangement and molecular changes of the blend under different post-deposition treatments, such as thermal annealing and solvent annealing. Figures 6A-6B show Reflection Differential Interference Contrast (RDIC) images of 1:1P3HT: PCBM blend films before and after 30 minutes thermal annealing, respectively. Fig. 6C shows the RDIC strength traces before and after thermal annealing along the dotted lines shown in fig. 6A-6B. FIG. 6D shows the portion of the regular P3HT in the blend before and after thermal annealing along the dotted line shown in FIGS. 6A-6B.

Two-dimensional material: graphene: by combining the RDIC and confocal raman modules, wrinkles and multi-layer regions on single and double layer graphene can be identified. Figures 7A-7C show raman spectra and comparisons of R-DIC and bright field images of the same single layer graphene sample. The RDIC enhances the contrast of the multi-layer and wrinkled areas of the sample. By focusing the excitation laser beam to the diffraction-limited spot shown in fig. 1C, a raman spectrum can be taken from a sub-micron region of the sample. Thus, a very high resolution correlation between the RDIC image and the Raman spectrum can be obtained.

As can be observed from the RDIC image in fig. 7A, the Chemical Vapor Deposition (CVD) synthesized graphene sample was spatially non-uniform. In samples containing an indefinite number of layers, there are multilayer regions and wrinkles, which can be identified from the corresponding raman spectra. These regions are "defects" created as layers during growth, which have a significant impact on graphene applications [56-59 ].

Imaging of cells on a reflective surface: cultured cells with gold nanoparticles on the reflective surface of, for example, a silicon wafer, gold-coated slide are imaged with high contrast by using an RDIC microscope, as shown in FIG. 8.

Gold nanoparticles on Au-coated slides: the interaction between gold nanoparticles and gold films was studied by combining Total Internal Reflection Scattering (TIRS) and spectroscopy modules. FIG. 9A shows a TIRS image of 40x80nm (SPR650nm) gold nanorods on a 50nm gold-coated slide, and FIG. 9B shows the scattering spectrum of the nanorods labeled 1 shown in FIG. 9A, according to one embodiment of the present invention.

The foregoing description of the exemplary embodiments of the present invention has been presented for the purposes of illustration and description only and is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations are possible in light of the above teaching.

The embodiments were chosen and described in order to explain the principles of the invention and its practical application to thereby enable others skilled in the art to utilize the invention and various embodiments with various modifications as are suited to the particular use contemplated. Alternative embodiments will become apparent to those skilled in the art to which the present invention relates without departing from its spirit and scope. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description and the exemplary embodiments described therein.

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