WO2025226751A1

WO2025226751A1 - Dark-field absorbance circular dichroism of oriented chiral thin films

Info

Publication number: WO2025226751A1
Application number: PCT/US2025/025871
Authority: WO
Inventors: Garth SIMPSON; Gwendylan A. TURNER; Caitlin E. DUNLAP
Original assignee: Purdue Research Foundation
Current assignee: Purdue Research Foundation
Priority date: 2024-04-26
Filing date: 2025-04-22
Publication date: 2025-10-30
Anticipated expiration: 2026-10-26

Abstract

A circular dichroism (CD) spectroscopy system includes a. source of circularly polarized light having a predetermined wavelength shone along a predetermined projection axis, a film which is translucent to the predetermined wavelength with chiral molecules disposed thereon placed in a plane perpendicular to the predetermined projection axis, light exiting the film represents coherent and incoherent components, a focusing lens having a predetermined focal length placed a first distance away from the film such that the coherent component of light focuses at a predetermined focal point based on the predetermined focal length of the focusing length, a beam block disposed at the predetermined focal point to block substantially all of the coherent component of light from the film, and an optical detector configured to receive substantially only the incoherent light from the film and generate a signal representing chirality of the chiral molecules.

Description

PRF-70689-02 DARK-FIELD ABSORBANCE CIRCULAR DICHROISM OF ORIENTED CHIRAL THIN FILMS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present non-provisional patent application is related to and claims the priority benefit of U.S. Provisional Patent Application Serial 63/639,012, filed April 26, 2024, the contents of which are hereby incorporated by reference in its entirety into the present disclosure. STATEMENT REGARDING GOVERNMENT FUNDING [0002] This invention was made with government support under CHE 2305178 awarded by the National Science Foundation. The government has certain rights in the invention. TECHNICAL FIELD [0003] The present disclosure generally relates to circular dichroism (CD), and in particular to a system and method for selective isolation of low-angle scattered CD spectral signals (incoherent light) through implementation of a dark-field detection geometry; and focused coherent and incoherent light through implementation of a confocal detection geometry. BACKGROUND [0004] This section introduces aspects that may help facilitate a better understanding of the disclosure. Accordingly, these statements are to be read in this light and are not to be understood as admissions about what is or is not prior art. [0005] The drug Thalidomide was first introduced to the marketplace in 1957 in at the time West Germany inter alia for various sleep disorders and morning sickness for pregnant women. The PRF-70689-02 drug caused severe birth defects in tens of thousands of newborns, culminating in its removal from the marketplace by 1961. Thalidomide is a chiral molecule which means it cannot be superposed on its mirror image by any method including rotation, translation, or positional changes. While one handedness of Thalidomide was effective, the other handedness was severely toxic. Furthermore, separating the safe and effective handedness of the molecule would still result in vivo chiral transformation of the drug. Thus, chirality became an important aspect of drug safety and efficacy that needs to be understood. [0006] Circular dichroism (CD) spectroscopy is a technique used to study chirality of a molecule. In short, a beam of linearly polarized light is converted to a right-handed circularly polarized light and to a left handed circularly polarized light and each passed through a sample and detected thereafter to determine the way the sample absorbs these two circularly polarized light. The difference can provide information about chirality of the molecule. [0007] Thus, CD spectroscopy is a vital tool extensively employed for the characterization of biomolecules and the precise determination of absolute chiral configurations in small molecules. Additionally, CD spectroscopy furnishes valuable insights of the secondary structures of proteins, nucleic acids, and other macromolecules. Furthermore, CD spectroscopy provides valuable data on the stereochemical arrangements of small chiral molecules. As a non- destructive and expeditious technique, CD spectroscopy has become an integral component of research methodologies across scientific disciplines. While CD spectroscopy of isotropic systems (e.g., a solution with chiral molecules in a cuvette being analyzed by a CD system) is well- established, the theoretical underpinnings for CD measurements of uniaxial surface assemblies are less developed, despite the natural ubiquity of such assemblies and their roles in molecular self assembly, interfacial biochemical assays, and applications in chiroptic design. [0008] Typical CD spectrometers are limited in the analysis of thin films, which can contain CD spectral components of both molecular chirality as well as chirality introduced by the nature of the assembly itself. Current CD spectrometers have no intrinsic way to isolate each component, and resulting spectra are a mix of both. In a typical CD spectrometer, the film may be placed in the path of circularly polarized light (light that is modulated between one circular polarization and an orthogonal circular polarization). The assembly chirality has been shown to invert via PRF-70689-02 rotation along the axis perpendicular to the light beam axis from a normal orientation to a flipped orientation. In principle, summation of the two orientations could provide the molecular response. However, the because the signal strength from the assembly chirality is orders of magnitude larger relative to the molecular chirality, the molecular response is often on the order of the noise of the assembly response, resulting in a low signal to noise ratio. [0009] Therefore, there is an unmet need for a novel system that can isolate the assembly response from the molecular response and vice versa. Such a method would provide improved chirality information about a chiral molecule and overcome signal to noise ratio issues in conventional CD spectroscopy. SUMMARY [0010] A circular dichroism (CD) spectroscopy system is disclosed. The system includes a source of circularly polarized light having a predetermined wavelength shone along a predetermined projection axis, a film which is translucent to the predetermined wavelength with chiral molecules disposed thereon placed in a plane perpendicular to the predetermined projection axis, light exiting the film represents coherent and incoherent components, a focusing lens having a predetermined focal length placed a first distance away from the film such that the coherent component of light focuses at a predetermined focal point based on the predetermined focal length of the focusing length, a beam block disposed at the predetermined focal point to block substantially all of the coherent component of light from the film, and an optical detector configured to receive substantially only the incoherent light from the film and generate a signal representing chirality of the chiral molecules. [0011] According to one embodiment, in the CD spectroscopy system of claim 1, the predetermined wavelength is between about 190 nm and about 700 nm. [0012] According to one embodiment, in the CD spectroscopy system the film is made of S- naproxen. [0013] According to one embodiment, in the CD spectroscopy system the source of circularly polarized light is based on a system includes a light source providing light at a plurality of PRF-70689-02 wavelengths, a selective wavelength device adapted to receive light the plurality of wavelengths and output light at a selective wavelength, a linear polarization filter adapted to receive light at the selective wavelength and output a linearly polarized light at the selective wavelength, and a circular polarization filter adapted to receive the linearly polarized light and at the selective wavelength and output a circularly polarized light. [0014] According to one embodiment, in the CD spectroscopy system the selective wavelength device is a diffraction grating. [0015] According to one embodiment, in the CD spectroscopy system light from the light source is provided to the diffraction grating via one or more mirrors, including a parabolic mirror. [0016] According to one embodiment, in the CD spectroscopy system light output from the source of circular polarized light is provided from the diffraction grating via one or more mirrors, including a parabolic mirror. [0017] According to one embodiment, in the CD spectroscopy system the film is rotated about an axis perpendicular to the projection axis from a normal position to a flipped position 180 rotated from the normal position to thereby generate signals associated with normal and flipped orientations. [0018] According to one embodiment, in the CD spectroscopy system the film rotation is based on continuous basis there by providing a plurality of signals associated with normal and flipped orientations, thus allowing an averaging of said signals. [0019] According to one embodiment, in the CD spectroscopy system a difference in signals associated with normal and flipped orientations is about 10× higher than if both coherent and incoherent signals were registered by the optical detector. [0020] A circular dichroism (CD) spectroscopy system is also disclosed. The system includes a source of circularly polarized light having a predetermined wavelength shone along a predetermined projection axis, a film which is translucent to the predetermined wavelength with chiral molecules disposed thereon placed in a plane perpendicular to the predetermined projection axis, light exiting the film represents coherent and incoherent components, a focusing lens having a predetermined focal length placed a first distance away from the film such that the coherent component of light focuses at a predetermined focal point based on the predetermined PRF-70689-02 focal length of the focusing length, a slit disposed at the predetermined focal point to block substantially all of the incoherent component of light from the film, and an optical detector configured to receive substantially only the coherent light from the film and a portion of the incoherent light exiting the slit and generate a signal representing chirality of the chiral molecules. [0021] According to one embodiment, in the CD spectroscopy system the predetermined wavelength is between about 190 nm and about 700 nm. [0022] According to one embodiment, in the CD spectroscopy system the film is made of S- naproxen. [0023] According to one embodiment, in the CD spectroscopy system the source of circularly polarized light is based on a system which includes a light source providing light at a plurality of wavelengths, a selective wavelength device adapted to receive light the plurality of wavelengths and output light at a selective wavelength, a linear polarization filter adapted to receive light at the selective wavelength and output a linearly polarized light at the selective wavelength, and a circular polarization filter adapted to receive the linearly polarized light and at the selective wavelength and output a circularly polarized light. [0024] According to one embodiment, in the CD spectroscopy system the selective wavelength device is a diffraction grating. [0025] According to one embodiment, in the CD spectroscopy system light from the light source is provided to the diffraction grating via one or more mirrors, including a parabolic mirror. [0026] According to one embodiment, in the CD spectroscopy system light output from the source of circular polarized light is provided from the diffraction grating via one or more mirrors, including a parabolic mirror. [0027] According to one embodiment, in the CD spectroscopy system the film is rotated about an axis perpendicular to the projection axis from a normal position to a flipped position 180 rotated from the normal position to thereby generate signals associated with normal and flipped orientations. PRF-70689-02 [0028] According to one embodiment, in the CD spectroscopy system the film rotation is based on continuous basis there by providing a plurality of signals associated with normal and flipped orientations, thus allowing an averaging of said signals. [0029] According to one embodiment, in the CD spectroscopy system a summation of signals associated with normal and flipped orientations is substantially similar in form as compared to a signal received from an isotropic CD spectroscopy from a sample in a vessel having chiral molecules based solely on coherent light. [0030] According to one embodiment, in the CD spectroscopy system the slit is between about 3.34 mm and about 0.86 mm. BRIEF DESCRIPTION OF FIGURES [0031] FIG.1A is a typical circular dichroism (CD) spectroscopy instrumentation depicting a light source with various wavelengths that is shone onto a monochromator in order to generate a circular polarized light that is shone on a film which is rotated from a normal to a flipped position. [0032] FIG.1B is a schematic of a dark-field CD spectroscopy measurement configuration, according to the present disclosure. [0033] FIG.1C is a schematic for a confocal CD measurement configuration, according to the present disclosure. [0034] FIG.1D is a schematic of a conventional isotropic CD spectroscopy. [0035] FIG.2 is a graph of CD measurements in mdeg vs. wavelength in nm for the configurations shown in FIG.1A (dotted lines indicate coherent and incoherent light) and FIG. 1B (solid lines indicate incoherent light only). [0036] FIG.3A is an integrated peak difference of the CD spectrum from 250 to 350 nm, indicating a loss in low-frequency contributions dominating the nonreciprocal response in FIG. 2. [0037] FIGs.3B and 3C are CD outputs in mdeg vs. wavelength in nm for sum and difference CD results from the confocal configuration shown in FIG.1C for slit sizes 3.34 mm and 0.86 PRF-70689-02 mm, respectively, where in each figure isotropic results based on the configuration shown in FIG.1D is also provided. DETAILED DESCRIPTION [0038] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to the embodiments illustrated in the drawings, and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of this disclosure is thereby intended. [0039] In the present disclosure, the term “about” can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range. [0040] In the present disclosure, the term “substantially” can allow for a degree of variability in a value or range, for example, within 90%, within 95%, or within 99% of a stated value or of a stated limit of a range. [0041] A novel system and method are provided in the present disclosure that can provide improved chirality information about a chiral molecule and overcome signal to noise ratio issues in conventional circular dichroism (CD) spectroscopy. Towards this end, a novel CD system and method are described that allow for selective isolation of low-angle scattered CD spectral signals (incoherent light) through implementation of a dark-field detection geometry. In addition a novel CD system and method are provided that allow for focusing of coherent and incoherent light through implementation of a confocal detection geometry. The approach described herein provides a modular add-on to available CD systems that allows more effective interrogation of these exiting system. [0042] As discussed above, CD spectroscopy has been in focus for many decades for study of chirality in chiral molecules. In a notable example in 1994, Hecht and Barron developed a theoretical foundation predicting the possibility of fully electric dipole-allowed chiral-specific CD in uniaxial molecular assemblies that exhibit optical scattering. In brief, they noted that a scattered signal lacks an existing co-propagating field for interference, resulting in a self- PRF-70689-02 heterodyned signal scaling with the fourth order in the molecular orientation distribution. This behavior contrasts with conventional isotropic CD, which is based on the attenuation of a pre- existing incident wave and therefore scales with the second moments of the molecular orientation distribution. It also contrasts with models for describing CD scattering from isotropic suspensions of particulates, including structured chiral nanomaterials. Finally, it also contrasts with reciprocal and nonreciprocal CD responses arising from plasmonic surface-assemblies and other nanostructured materials. Notably, the theory from Hecht and Barron is explicitly molecular in nature, expressing the chiral-specific scattering in terms of orientational averages over the molecular polarizability tensors. However, no definitive study has been performed confirming the original predictions based on the molecular orientational model serving as the foundation for the early theoretical work. [0043] One possible explanation for the lack of experimental confirmation of the Hecht and Barron molecular model is the absence of prior experimental means for selectively isolating the scattered signal in the measured CD response of uniaxial surface assemblies. CD spectroscopy of thin surface films is typically performed at normal incidence, with the transmitted beam integrating over both coherent transmission and incoherent low-angle scattered signals. In systems exhibiting both polar and in-plane orientational orders, the CD response can arise coherently from a coupling of linear birefringence and linear dichroism (LB/LD), commonly referred to as apparent CD and generally viewed as a parasitic interference in CD spectroscopy. As one notable example, this coherent contribution in biaxial surface assemblies has been described recently by Salij, Tempelaar, and coworkers, and leveraged in the design of novel Fabry-Pérot cavities for chiral-specific polariton coupling. Recently, some of the authors of this work extended the theoretical foundation to include incoherent contributions to nonreciprocal CD from uniaxial assemblies, building on prior theoretical descriptions of incoherent emission in fluorescence optical rotary dispersion and other 4-wave mixing spectroscopies in uniaxial molecular assemblies. In practice, discrimination between coherent LB/LD in biaxial assemblies as described by Salij et al. versus incoherent nonreciprocal CD in uniaxial assemblies can be nontrivial to differentiate based on classical CD spectroscopy alone. The transmitted intensity of circularly polarized incident light is inherently independent of in-plane orientation, and both the PRF-70689-02 coherent and incoherent mechanisms produce nonreciprocal CD responses, in which the CD inverts upon rotation of the sample surface normal co-parallel vs. antiparallel with the optical axis of the spectrometer. [0044] Based on this observation, the present disclosure describe instrumentations for selective isolation of the low-angle scattered CD spectral signals through implementation of a dark-field detection geometry. Additionally, a separate CD instrumentation is described useful for studying focused coherent and incoherent light coming from a thin film sample. According to the present disclosure, instrumentation for dark-field spectroscopy was designed explicitly to selectively optimize detection of the incoherent CD response predicted in uniaxial assemblies based on electric-dipole allowed surface-specific and chiral-specific nonreciprocal CD spectra accessible in ultraviolet to visible (UV-V) in CD spectroscopy. [0045] Referring to FIG.1A, a typical CD spectroscopy instrumentation is shown depicting a light source with various wavelengths that is shone onto a monochromator. The monochromator includes a pair of normal and parabolic mirror configured to reflect the incoming light onto a diffraction grating which allows for light with only one selective wavelength to exit the diffraction grating. Thus, the diffraction grating receives light with multiple wavelengths, and outputs light at one selective wavelength. The output of the diffraction grating is reflected and output out of the monochromator via a second pair of parabolic and normal mirrors. Thus, the monochromator receives light with multiple wavelengths and outputs light at one selective wavelength. It should be noted that other devices, such as a color-wheel, known to a person having ordinary skill in the art, can also be used to generate the output light with the selective wavelength. The output of the monochromator is then provided to a linear polarization filter, which linearly polarizes the incoming light (i.e., instead of the electric field of the electromagnetic wave oscillating randomly, the linearly polarized light’s electric field only oscillates along a single plane). Thus, the linear polarization filter receives light with a selective wavelength, and outputs a linearly polarized light at the same wavelength. Next, the linearly polarized light at the selective wavelength is input to a circular polarization modulator (e.g., a photoelastic 45º modulator) that is configured to modulate the incoming linearly polarized light to a righthanded and lefthanded circular polarization (or more generally, light with a circular PRF-70689-02 polarization and light with an orthogonal circular polarization). Circular polarization refers to a polarization whereby the electric field vector rotates about a plane that is perpendicular to an axis defining direction of light. The output of the circular polarization modulator is then provided to a thin film that is placed in the path of the circularly polarized light. According to one example, the thin film is made of S-naproxen. The film can be rotated from a normal orientation to a flipped orientation. As the circularly polarized light leaves the film, absorption of the light is measured in the two normal and flipped orientations. The light that leaves the film includes both a coherent element and an incoherent element. Both elements are shone onto an optical detector, e.g., a photomultiplier tube, with signals from both orientations collected for data analysis. Example of the results from a conventional CD spectroscopy is shown in FIG.2 (see the dotted line) for both orientations. The signal strength, a parameter critical to signal to noise ratio, is shown as “A,” which in conventional CD spectroscopy of a film has been traditionally small and thus posing a challenge in data analysis. [0046] To overcome this challenge, the present disclosure provides a novel system and method for dark-field measurements whereby the coherent light component of the detected light is blocked, thus allowing only incoherent light to be shone on the optical detector. This configuration is referred to as the dark-field measurement configuration, shown in FIG.1B, which is a schematic of the dark-field measurement configuration, according to the present disclosure. The dark-field measurements configuration differs from the traditional CD spectroscopy for a thin film by adding a focusing lens, e.g., a 35 mm calcium fluoride lens (numerical aperture of 0.36), in the path of light a distance l₁ away from the film. According to one embodiment, l1 was 25 mm. Both incoherent and coherent light components of light exiting the film begin to focus toward a focal point a distance l2 away from the focusing lens. Distances l₁ and l₂ are dependent on the focusing lens. For the 35 mm focusing lens, these distances are about 25mm and 35mm, respectively. The coherent component of the light focuses onto a focal point at which point is absorbed by an optical sink (i.e., a beam block), e.g., a needle. The incoherent component of the light is also focused but begins to diverge beyond the focal point. Some distance away l3 from the focal point is a collimating lens, e.g., a 75 mm calcium fluoride PRF-70689-02 lens, configured to collimate the incoherent light to be provided to the optical detector. According to one embodiment, l2 + l3 is about 110 mm. [0047] Based on the configuration shown in FIG.1B, Dark-field measurements were performed by passing the beam through the sample, then projecting light onto a thin needle beam-block as shown in FIGs.1B to selectively reject the coherent response and isolate the incoherent scattered response. Denser light areas refer to coherent light and lighter light areas refer to incoherent light. The CD measurements are shown in FIG.2 (see the solid lines for normal and the flipped orientations). As seen the signal strength of the incoherent light is about 10× that of the conventional measurements including both coherent and incoherent light components from the measurements based on FIG.1A. [0048] With reference to FIG.1C, which is a schematic for a confocal measurement configuration, complementary measurements were also performed with an inverted confocal configuration, in which the needle beam-block was replaced by a slit aperture (e.g., a Thorlabs VA100CP) paired to the monochromator output to selectively suppress the incoherent response. While most of the incoherent light is suppressed some incoherent light also exits the slit and onto the optical detector via the collimating lens. These collective measurements were evaluated for quantitative assessment of the degree to which the incoherence component contributes to the nonreciprocal “apparent” CD response. [0049] It should be noted that in FIGs.1B and 1C, the film may be rotated from a normal orientation to a flipped orientation one time or many times whereby for each rotation data is collected and averaged. [0050] Finally, referring to FIG.1D, a schematic of a conventional isotropic CD spectroscopy is provided, in which the sample which includes suspended chiral molecules is provided in a vessel, e.g., a cuvette. As before, light is shone onto the sample and exits from the sample and onto the optical detector. In this configuration, little to no incoherent light is produced out of the cuvette. [0051] Results from measurement from systems shown in FIG.1C and 1D are shown in FIGs. 3A, 3B, and 3C for different slit sizes (see FIG.1C). In FIGs.3B and 3C, the sum and difference of the CD spectroscopy output is shown for the normal and flipped configurations. Interestingly, the shape of the sum signal is relatively similar to the shape of the isotropic CD measurements PRF-70689-02 (see the configuration shown in FIG.1D), even though one curve is based on chiral molecules affixed to a film rotated between normal and flipped orientations, and one curve is associated with chiral molecules suspended in a solution in cuvette. [0052] The theoretical framework for the system of the present disclosure is provided below. The incoherent scattered, non-reciprocal CD response can be cast in terms of the chiral-specific, surface-specific factor f51 =c^YX*c^XX, in which c refers to the linear susceptibility of the local sample is expressed according to Eq. (1), provided below: Y_{X * XX χ YX * χ XX} _≅ −₂ i_{χ χ 2 Im} _{= ( )} CD _{XX 2 YX 2 XX 2 YX 2} (1) wherein χ = linear i, Im = imaginary X and Y describe the X and Y axes in which the surface plane lies, and CD = circular dichroism response. In Eq. Error! Reference source not found., a reference frame is selected in which the Z-axis refers to the surface normal with X and Y being the two equivalent axes lying within the interfacial plane. It is further assumed that the incident beam is aligned with the surface normal and that only the low-angle scattered light is collected, such that Z-polarized components of the optical field can be neglected. Local field correction factors can also be neglected in the ratio, as the factors for the X and Y fields will be identical for a uniaxial assembly. The c-tensor element combinations themselves can be cast in terms of the linear molecular polarizability, which is given by orientational averages in the limit of minimal intermolecular electronic perturbations. Importantly, for the incoherent scattered component of the signal, the averages are performed over the combinations of a^ij*a^kl rather than the molecular polarizability a^ij as would be performed for coherent propagation and absorption, as provided by Eq. (2), below. Y_{X* χ XX =} N_{b ^ R ij * k} _{Yi R R Xk R Xl α α l (2)} wherein N_b = ε0 = electric constant, PRF-70689-02 R_Aa = orientational average for combinations of lab and molecular frame coordinates, i, j, k, and l = molecular frame coordinate system, and α^gh = molecular polarizability. The averaging over four rotation matrices rather than just two accesses fully electric dipole allowed chiral-specific terms not present in coherent absorbance spectroscopy. These terms are arguably easiest to interpret by considering a pair of two uncoupled oscillators with different resonant transition energies within the molecular frame. The two vectors describing the transition moments necessarily define a plane. If the internal angle between the two vectors is given to be d, a positive and negative doublet will result in the CD response predicted by Eq. (1), each peak of which is proportional to the following orientational averages, as provided in Eq. (3), provided below. ^_sin 3 _{θ sin ψ sin δ cos δ ^} CD _{∝ ± ^ ^ μ 2 2} _{n μ m (3)} wherein θ = polar tilt ψ = twist angle of plane in which dipoles lie in, δ = angle between transition dipoles, and μ_i0 = transition dipole moments from ground (0) to each excited state (m and n). In Eq. (3), m_i0 refers to the transition moment to state _{i∈ { n, m }} , with n and m referencing the two excited states contributing to the doublet, q is the relative to the m-transition moment, and y is the twist angle of the plane containing the m and n transition moments. Unlike CD in isotropic systems, the CD illustrated by Eq. (3) in uniaxial assemblies does not require coupling between the two transition moments (e.g., to generate magnetic dipole or electric quadrupole contributions). Geometric arrangement alone is sufficient to produce nonzero orientational averages via Eq. (3). [0053] The orientational averages in Eq. Error! Reference source not found. illustrate the origin of the interface-specificity and chiral-specificity of the nonreciprocal CD in uniaxial assemblies. In the first term, the CD is maximized for an internal angle of 45^o between the two transition moments and for tilt and twist angles of 90 degrees, in which the plane of the PRF-70689-02 chromophore parallels the surface plane. In the absence of chirality driving interfacial assembly, both clockwise and counterclockwise relative arrangements of the two transition moments would be equally likely, resulting in a zero-valued orientational average in y. A 180^o rotation of the sample about the X or Y surface axes will invert the sign of y and change from clockwise to counter-clockwise rotation (or vice versa), giving rise to an implicit dependence on polar order. The need for polar order is arguably more explicit in the second term in Eq. (3), which averages to zero for an even distribution in q and inversion upon 180^o rotation about X or Y. Similarly, the chiral-specificity in the second term is arguably also more explicitly intuitive, rising from “propeller-like” twist angle y arrangements of the plane defined by the two orthogonal transition moments. [0054] Referring back to FIGs.1B and 1C, three replicate measurements were performed across different days and samples. During each day of measurements, the beam block was positioned to maximize the extinction of the coherently transmitted beam. An extinction of 2.14 was observed throughout most of the near-UV regime, with the quality of extinction reducing to 1.31 in far UV. Loss in extinction in the blue end of the spectrum is attributed to dispersion in the refractive optics used to reimage the slit, which likely shifted to focal plane away from the dark-field aperture position. Use of reflective optics, such as spherical or parabolic mirrors reduces such chromic aberrations. [0055] As discussed above, representative dark-field extinction measurements are shown in FIG. 2, which is a graph of CD measured in mdeg vs. wavelength in nm for a microcrystalline naproxen thin film oriented at the silica/air interface. Consistent with the expectations for the response described by Eqs. (1)-(3), the spectra exhibited substantial enhancements in the nonreciprocal CD activity when measured in a dark-field configuration (i.e., only incoherent component). The major features in the CD spectra were not substantially impacted by rotation of the sample about the surface normal. Notably, the nonreciprocal CD spectrum exhibited prominent features between 260 nm – 340 nm, attributed to absorption features localized primarily on the planar aromatic naphthalene ring motif, and enhancement of the transitions around 200−230 nm spanning the carboxyllic acid group across the chiral carbon. These same features are relatively weak in the isotropic CD response due to the intrinsic planarity of the PRF-70689-02 electronic transitions localized to the aromatic moiety. However, deviations from planarity are not required for the nonreciprocal CD response, consistent with the original description by Hecht and Barron and the subsequent theoretical work by Turner and coworkers. [0056] Complementary measurements performed using a confocal aperture are shown in FIGs. 3A, 3B, and 3C along with isotropic CD measurements. FIG.3A is a graph of CD measurements in mdeg vs. slit width in mm providing integrated peak difference of the CD spectrum from 250 to 350 nm, indicating a loss in low-frequency contributions dominating the nonreciprocal response. FIGs.3B and 3C are representative sum and difference CD spectra with a wide 3.3 mm slit width, and a 0.9 mm slit width, respectively. The isotropic CD spectrum is overlaid in both FIG.3B and FIG.3C for comparison. The larger slit widths indicated in the “rejected” region of FIG.3A correspond to apertures limited by the physical dimensions of the beam path rather than the slit width. In excellent agreement with theoretical predictions, introduction of a confocal aperture resulted in substantial suppression of the nonreciprocal response. CD spectroscopy in the confocal configuration reduced the incoherent nonreciprocal CD by 3.4-fold and facilitated isolation of the reciprocal CD that is dependent more directly on intrinsic molecular chirality. Reciprocal CD spectra approaching those expected in isotropic media can, in principle, be obtained by summation of the spectra acquired in the two sample orientations. In practice, departure from an isotropic orientation distribution is expected to result in departures from the isotropic CD spectrum due to preferred orientation effects. However, the peak locations should still be accurately recovered. [0057] The results in FIGs.2, 3A, 3B, and 3C represent compelling direct experimental evidence supporting the early theoretical predictions for surface-specific CD spectroscopy of ordered assemblies. Acquisition of CD spectra in the presence of the dark-field mask produced major enhancements in the nonreciprocal CD response, consistent with preferential suppression of the coherent non-reciprocal CD contributions. Consistent with the nonreciprocity and in agreement with the prior theoretical predictions, these results support the inherent surface-selectivity of the CD spectroscopy performed in this dark-field configuration. In the absence of interfacial ordering, the two opposing nonreciprocal responses arising from optical scattering will collectively sum to zero. Consistent with this expectation, prior transmission CD measurements PRF-70689-02 of isotropic microcrystalline naproxen suspensions produced CD spectra nearly identical to that from naproxen solutions. [0058] Building on this agreement between theory and experiment, these measurements of low- angle scattering performed at normal incidence provide a foundation for experimental extension to more diverse sample orientations and detection configurations. The low-angle scattering considered in this work allowed simplification of the chiral-specific response by consideration of just the X and Y polarization components of the optical fields accessible for an incident beam along the interface normal. Furthermore, local-field correction factors can be assumed to be identical for the X and Y polarization components for normal incidence in uniaxial systems, substantially simplifying the optical modeling. Extension to more diverse sample geometries has the potential to substantially expand the scope of applications by accessing additional chiral- specific and surface-specific polarization combinations. The theoretical framework developed by Davis et al. includes the full set of polarization-dependent chiral-specific contributions allowed by symmetry within the electric dipole approximation. These tensors and tensor products provide a rigorous structure for extension of the measurements described herein to different geometric sample orientations and configurations, expanding the potential scope of interfaces accessible for chiral analysis by optical scattering. [0059] In addition to isolation of the scattered, “apparent” signal by dark-field CD, these results also demonstrate the converse experimental utility of the confocal detection geometry for isolation of the isotropic-like CD. In instances for which the intrinsic molecular chiral response is desired, integration of a confocal aperture represents a straightforward modification to preferentially enhance the reciprocal response and suppress the incoherent nonreciprocal CD response. [0060] The inherent surface-specificity in the nonreciprocal CD obtained in the dark-field geometry sets the stage for potential use of incoherent CD to selectively probe molecular organization at interfaces. Specifically, the symmetry requirements of the nonreciprocal response share similarities with those in second-order nonlinear optical methods, such as second harmonic and sum-frequency generation, which are routinely used for interface-selective spectroscopy. In this context, the dark-field CD configuration demonstrated herein supports the further PRF-70689-02 development of incoherent CD spectroscopy as a potentially new method for surface analysis of chiral interfaces. Such measurements could inform on bioassay design and studies of structure within biological membranes and biomimetic interfaces. Interfacial interactions are particularly important in bioassay characterization and development (enzyme-linked immunosorbent assays). Interfacial interactions are also expected to play a significant role in protein crystallization, aggregation, and denaturation. [0061] It should be appreciated that the wavelength of the circularly polarized light according to the present disclosure can range from about 190 nm to upper visible range of about 700 nm. However, the approach described herein is extendable to the visible and mid-IR with proper lens material selection (e.g., calcium fluoride) and minor additions to the positioning of the dark-field and confocal masks. For example, to use calcium fluoride across the entire visible and mid infrared regime, a fine positioning/ piezo element are needed to be added to the lenses and darkfield mask to compensate for chromic aberration. It should also be appreciated that selections of lenses with higher numerical apertures and shorter focal lengths will increase the collected scattering angles in the darkfield detection, and introduction of precision hardware in the slit mask would improve coherent isolation in confocal detection. [0062] Those having ordinary skill in the art will recognize that numerous modifications can be made to the specific implementations described above. The implementations should not be limited to the particular limitations described. Other implementations may be possible.

Claims

PRF-70689-02 Claims: 1. A circular dichroism (CD) spectroscopy system, comprising: a source of circularly polarized light having a predetermined wavelength shone along a predetermined projection axis; a film which is translucent to the predetermined wavelength with chiral molecules disposed thereon placed in a plane perpendicular to the predetermined projection axis, light exiting the film represents coherent and incoherent components; a focusing lens having a predetermined focal length placed a first distance away from the film such that the coherent component of light focuses at a predetermined focal point based on the predetermined focal length of the focusing length; a beam block disposed at the predetermined focal point to block substantially all of the coherent component of light from the film; and an optical detector configured to receive substantially only the incoherent light from the film and generate a signal representing chirality of the chiral molecules. 2. The CD spectroscopy system of claim 1, wherein the predetermined wavelength is between about 190 nm and about 700 nm. 3. The CD spectroscopy system of claim 1, wherein the film is made of S-naproxen. 4. The CD spectroscopy system of claim 1, wherein the source of circularly polarized light is based on a system, comprising: a light source providing light at a plurality of wavelengths; a selective wavelength device adapted to receive light the plurality of wavelengths and output light at a selective wavelength; a linear polarization filter adapted to receive light at the selective wavelength and output a linearly polarized light at the selective wavelength; and a circular polarization filter adapted to receive the linearly polarized light and at the selective wavelength and output a circularly polarized light. 5. The CD spectroscopy system of claim 4, wherein the selective wavelength device is a diffraction grating. PRF-70689-02 6. The CD spectroscopy system of claim 5, wherein light from the light source is provided to the diffraction grating via one or more mirrors, including a parabolic mirror. 7. The CD spectroscopy system of claim 5, wherein light output from the source of circular polarized light is provided from the diffraction grating via one or more mirrors, including a parabolic mirror. 8. The CD spectroscopy system of claim 1, wherein the film is rotated about an axis perpendicular to the projection axis from a normal position to a flipped position 180 rotated from the normal position to thereby generate signals associated with normal and flipped orientations. 9. The CD spectroscopy system of claim 8, wherein the film rotation is based on continuous basis there by providing a plurality of signals associated with normal and flipped orientations, thus allowing an averaging of said signals. 10. The CD spectroscopy system of claim 8, wherein a difference in signals associated with normal and flipped orientations is about 10× higher than if both coherent and incoherent signals were registered by the optical detector. 11. A circular dichroism (CD) spectroscopy system, comprising: a source of circularly polarized light having a predetermined wavelength shone along a predetermined projection axis; a film which is translucent to the predetermined wavelength with chiral molecules disposed thereon placed in a plane perpendicular to the predetermined projection axis, light exiting the film represents coherent and incoherent components; a focusing lens having a predetermined focal length placed a first distance away from the film such that the coherent component of light focuses at a predetermined focal point based on the predetermined focal length of the focusing length; a slit disposed at the predetermined focal point to block substantially all of the incoherent component of light from the film; and an optical detector configured to receive substantially only the coherent light from the film and a portion of the incoherent light exiting the slit and generate a signal representing chirality of the chiral molecules. PRF-70689-02 12. The CD spectroscopy system of claim 11, wherein the predetermined wavelength is between about 190 nm and about 700 nm. 13. The CD spectroscopy system of claim 11, wherein the film is made of S-naproxen. 14. The CD spectroscopy system of claim 11, wherein the source of circularly polarized light is based on a system, comprising: a light source providing light at a plurality of wavelengths; a selective wavelength device adapted to receive light the plurality of wavelengths and output light at a selective wavelength; a linear polarization filter adapted to receive light at the selective wavelength and output a linearly polarized light at the selective wavelength; and a circular polarization filter adapted to receive the linearly polarized light and at the selective wavelength and output a circularly polarized light. 15. The CD spectroscopy system of claim 14, wherein the selective wavelength device is a diffraction grating. 16. The CD spectroscopy system of claim 15, wherein light from the light source is provided to the diffraction grating via one or more mirrors, including a parabolic mirror. 17. The CD spectroscopy system of claim 15, wherein light output from the source of circular polarized light is provided from the diffraction grating via one or more mirrors, including a parabolic mirror. 18. The CD spectroscopy system of claim 11, wherein the film is rotated about an axis perpendicular to the projection axis from a normal position to a flipped position 180 rotated from the normal position to thereby generate signals associated with normal and flipped orientations. 19. The CD spectroscopy system of claim 18, wherein the film rotation is based on continuous basis there by providing a plurality of signals associated with normal and flipped orientations, thus allowing an averaging of said signals. 20. The CD spectroscopy system of claim 18, wherein a summation of signals associated with normal and flipped orientations is substantially similar in form as compared to a PRF-70689-02 signal received from an isotropic CD spectroscopy from a sample in a vessel having chiral molecules based solely on coherent light. 21. The CD spectroscopy system of claim 11, wherein the slit is between about 3.34 mm and about 0.86 mm.