Systems and methods for fluorescence optical rotary dispersion from interfacial biomolecular assemblies

Abstract

The invention provides systems and methods for fluorescence optical rotary dispersion from interfacial biomolecular assemblies.

Description

[0001] SYSTEMS AND METHODS FOR FLUORESCENCE OPTICAL ROTARY DISPERSION FROM INTERFACIAL BIOMOLECULAR ASSEMBLIES

[0002] Related Application

[0003] The present application claims the benefit of and priority to U.S. provisional patent application serial number 63 / 655,068, filed June 2, 2024, the content of which is incorporated by reference herein in its entirety.

[0004] Government Support

[0005] This invention was made with government support under 2004046 and 2305178 awarded by National Science Foundation. The government has certain rights in the invention.

[0006] Field of the Invention

[0007] The invention provides systems and methods for fluorescence optical rotary dispersion from interfacial biomolecular assemblies.

[0008] Background

[0009] Biological interfaces are critical structural components in living systems, playing a pivotal role in facilitating essential cellular functions. Lipid membranes, for instance, provide the necessary compartmentalization that is crucial for various cellular processes. These membranes enable chemical communication through surface-bound receptors, despite their nominally impermeable nature. The development of biosensors frequently relies on the surfaceimmobilization of targets, which enhances the detection and quantification of biological molecules. Similarly, affinity chromatography utilizes surface associations to achieve the selective purification of target biopolymers, a process fundamental to many biochemical and clinical applications. However, despite the critical importance of these biological interfaces, the current analytical toolkit for selectively analyzing chemical structure-function relationships at these buried, biologically relevant interfaces remains limited, particularly within complex mixtures. Summary

[0010] The invention provides instrumentation and methods to perform chiral-specific F-ORD measurements and assess the merits of these theoretical predictions. Monomolecular thin films of (S) naproxen as a model system were prepared by dip-coating, followed by F-ORD measurements performed using a purpose-built spectrometer, designed in-house. Results of F- ORD measurements are interpreted in combination with ab initio electronic structure calculations and analytical modeling of the F-ORD response with no adjustable parameters. The agreement between theory and experiment is critically assessed, together with the potential scope of use for F-ORD spectroscopic analysis of molecular interactions at biological interfaces.

[0011] In certain aspects, the invention provides methods for analyzing a molecule that involve producing a monomolecular film on opposing sides of a substrate, wherein the monomolecular film comprises a target molecule, using a spectroscopy system to conduct fluorescence optical rotary dispersion (F-ORD) on the target molecule in the monomolecular film on the substrate, receiving F-ORD data acquired by spectroscopy system to a processing system, and applying one or more F-ORD processing algorithms via the processing system to obtain interface-selective measurements of the target molecule.

[0012] In certain embodiments, analzying comprising probing chemical interactions of the target molecule at biological interfaces. In certain embodiments, the chemical interactions are fully electric dipole-allowed. In certain embodiments, the analyzing allows for chiral-specific responses. In certain embodiments, analyzing allows for generating information for biologically relevant interfacial interactions. In certain embodiments, the method is applied to enable realtime analysis of target binding to surface-immobilized biosensors.

[0013] In certain embodiments, the method is applied for advancing chromatography of chiral compounds by leveraging intrinsic autofluorescence of the target molecule. In certain embodiments, the method is applied for improving understanding and supporting real-time monitoring in affinity chromatography. In certain embodiments, the method is applied for characterizing a protein corona that develops on nanoparticle surfaces upon physiological introduction. In certain embodiments, the method is applied to analyze heterogeneous nucleation and growth of homochiral crystals.

[0014] In other aspects, the invention provides fluorescence optical rotary dispersion (F-ORD) systems that include a light source that generates light; a grating monochromator for fluorescence excitation; a short pass filter; a ultraviolet (UV) fused silica lens; a UV Gian polarizer; a photoelastic modulator (PEM); a second collimating lens; and a UV fused silica single quarter-wave Fresnel Rhomb.

[0015] In certain embodiments, the grating monochromator for fluorescence excitation is between a range of 190 - 300 nanometer (nm). In certain embodiments, the short pass filter is a 300nm short pass filter to allow for transmission of light at wavelengths blue shifted of 300 nm. In certain embodiments, the blue shifted light is subsequently directed through the UV fused silica lens and the UV Gian polarizer. In certain embodiments, the UV Gian polarizer is set at 0° angle relative to the light source to collimate and horizontally polarize the light.

[0016] In certain embodiments, the PEM performs polarization modulation, wherein the PEM is oriented at a 45° angle relative to the plane of the light source with a resonant modulation frequency of If = 50 kHz. In certain embodiments, light emerging from the PEM is directed through the second collimating lens and the UV fused silica single quarter-wave Fresnel Rhomb to induce broad-band quarter wave retardance across the UV spectral range employed for excitation.

[0017] In certain embodiments, addition of the quarter-wave retarder following the PEM resulted in polarization modulation with exclusively linearly polarized light, in which the polarization axis was rapidly modulated rather than the sense of circularity. In certain embodiments, the light source is a 300W, 15A Xenon lamp. In certain embodiments, the system further comprises a motorized stage, wherein for azimuthal scanning about an optical axis, a sample is mounted on the motorized rotation stage.

[0018] Brief Description of the Drawings

[0019] FIG. 1 panel A provides a structure of (S)-naproxen. FIG. 1 panel B provides definitions of Euler angles. FIG. 1 panel C provides depiction of geometric contributions to surface-specific and chiral-specific F-ORD, with chiral steric interactions dictating the net ensemble-averaged clockwise or counterclockwise rotation of the emission transition moment (red) relative to that of excitation (blue) upon projection onto the surface plane. The arrows in (panel A) indicate anticipated absorption and emission transition moments for (S) naproxen based on quantum chemical calculations of electronic structure. FIG. 2 shows an F-ORD instrument schematic. Unpolarized light from an arc lamp is passed through a monochromator and a short pass fdter (SPF) before collimation with a lens (LI). The horizontally polarized component selected using a UV-Glan polarizer was passed through a photoelastic modulator (PEM) for polarization modulation. Subsequent passage through a second lens (L2) and a Fresnel rhomb broadband quarter wave retarder produced linear polarization with the axis of orientation modulated in time. ORD was recorded from the difference in transmission of fluorescence through a horizontal UV-Glan polarizer for detection of excitation light with a preferred axis rotated clockwise vs. counterclockwise about the horizontal axis.

[0020] FIG. 3 panels A-B show Experimental demonstration of F-ORD (panel A) at the If modulation frequency, and the depolarization amplitude (panel B) at the 2f frequency.

[0021] FIG. 4 shows azimuthal dependence of the F-ORD response. F-ORD remained like signed for all azimuthal angles investigated, consistent with uniaxial symmetry and incompatible with in-plane ordering.

[0022] FIG. 5 is an illustration showing an exemplary data analysis module for implementing the systems and methods of the invention in certain embodiments.

[0023] FIG. 6 panels A-B shows UV-Visible absorbance spectrum (panel A), together with the measured CD spectrum of (S)-naproxen, which probes the imaginary part of the circular differential polarizability (panel B), overlaid with the calculated real part describing ORD, determined from a Kramers-Kronig transformation.

[0024] FIG. 7 shows excitation and emission spectra for fluorescence spectroscopy of (S)- naproxen solutions in ethanol.

[0025] Detailed Description

[0026] Only a small number of methods are capable of providing the necessary interfaceselectivity to analyze interactions at biological interfaces effectively. This limitation underscores the need for advanced techniques that can provide more precise and selective analysis to better understand the complex interactions occurring at these vital interfaces.

[0027] The growing need for additional characterization tools for interfacial chirality has been made even more prominent by observations of surprisingly strong chiral-induced spin selectivity (CISS) for electron transmission across surfaces decorated by ultrathin chiral films. Spin resolved electrons have the potential to play enabling roles in quantum computing platforms, with remarkably high degrees of electron spin polarization achieved by passage through ultrathin homochiral thin organic films. These effects are often attributed to spin-orbit coupling (SOC) within the interfacial layers that is much larger than expected based on SOC interactions for isotropic organic molecular assemblies.3, 5, 6 However, the mechanism of action remains an open scientific question, limited in part by the paucity of available methods with sufficient sensitivity to enable chiral spectroscopic analysis of interfacial films in some cases spanning only a single molecular layer.

[0028] Even-ordered nonlinear optical interactions, such as second harmonic generation (SHG) and sum-frequency generation (SFG) spectroscopy, have proven remarkably effective in probing the local chemical composition and structure at biological interfaces. Despite these achievements, SHG and SFG are predominantly utilized for fundamental research rather than routine analyses. This limited application is partly due to the inherent complexity of ultrafast frequency conversion and the need for precise experimental setups. Typically, measurements are conducted with incident beams angled relative to the surface normal to access the Z-polarized fields required by symmetry for electric-dipole allowed SHG and SFG in uniaxial surface assemblies This geometric arrangement is essential to obtain the desired sensitivity and specificity in the analysis of planar biological interfaces, but poses significant practical constraints in implementation.

[0029] Circumnavigation of the limitations imposed by experimental geometry in three-wave mixing techniques (e.g., SHG and SFG) has been proposed through the use of chiral-specific fourwave mixing methods, including two-dimensional infrared (2DIR) and coherent Raman spectroscopy (CRS). Theoretically, four-wave mixing can generate polarization combinations that are both chiral-specific and interface-specific. This feat is achieved through nonzero tensor elements that interrogate only in-plane optical fields (X, Y), which are consequently symmetry allowed for measurements with the optical axis propagating along the interface normal. Early theoretical work by others demonstrated that chiral-specific four-wave mixing is symmetry allowed from the chiral orientational ordering of nominally achiral and uncoupled chromophores, similar to achiral blades in a chiral propeller structure. The chiral-specific terms, resulting from purely electric dipole interactions, have the potential to match their achiral counterparts in amplitude and be significantly larger than analogous coherent linear optical interactions.

[0030] However, it is believed that no definitive experimental demonstration of these chiral- specific coherent four- wave mixing signals has been reported. The challenges in experimental realization for coherent four-wave mixing likely stem from the relatively low number of molecular oscillators within a single oriented monolayer, leading to significant background interference from bulk allowed four-wave mixing signals and the quadratic scaling of signal strength with interfacial molecular number density in coherent optical processes.

[0031] Intriguingly, the mathematical framework originally developed for describing coherent four-wave mixing nonlinear spectroscopy has been shown to map onto incoherent contributions to linear spectroscopic methods in uniaxial assemblies, including Rayleigh and spontaneous Raman scattering and fluorescence. Others have reported observation of large fluorescence- detected circular dichroism from histograms of individual surface-immobilized fluorophores, but the origin of the observed chiroptical activity remains contested.18-20 In early work, absorption spectroscopy was predicted to support chiral specific observables fully within the electric dipole approximation when performed in a self heterodyned experimental geometry, in which the scattered optical signal is detected. These predictions were very recently confirmed experimentally in a dark-field circular dichroism (CD) spectroscopy design to isolate the low- angle scattered signal.

[0032] Building on this collective early body of theoretical work for both coherent four-wave mixing and incoherent 2-wave mixing, others have bridged the two frameworks in prediction of large chiral-specific and interface-specific F-ORD in uniaxial molecular ensembles. Within the electric dipole approximation, fluorescence within an isolated molecule can be describe by two vectors: the excitation and emission transition moments. By definition, any two vectors define a plane, which necessarily possesses mirror plane symmetry and the absence of chirality within the electric dipole approximation. However, the absence of chirality within the fluorophore itself does not remove the possibility of electric dipole-allowed chiral-specific observables upon consideration of the molecular orientation distribution (by analogy with achiral blades arranged to produce chiral propeller-like structures). A pictorial depiction of these proposed geometric contributions to chiral-specific fluorescence is provided in FIG. 1 panels A-C. These predictions suggest strategies to perform interface-specific fluorescence optical rotary dispersion (F-ORD) detection of chiral assemblies. However, just as in the case of coherent four-wave mixing, these predictions of chiral-specific and interface-selective F-ORD have defied experimental confirmation in prior work.

[0033] In the work herein, homochiral monomolecular thin films adsorbed to fused silica were observed to produce optical rotary dispersion more than seven orders of magnitude greater than anticipated by conventional origins of ORD (i.e., optical rotation of the excitation beam). Optical rotation of — 6° was observed in the principal axis of fluorescence emission relative to that of excitation for ultrathin dip-coated (S) naproxen films, with the sign of the rotation inverting for films of (R) naproxen. The chiral-specific F-ORD responses were non-reciprocal, inverting in sign upon flipping of the sample orientation (i.e., source-facing vs. detector-facing), providing selectivity to chiral molecules oriented at the interface. The interface selectivity, chiral selectivity, and large magnitude of the response are in excellent agreement with a fully electric dipole allowed orientational mechanism for fluorescence in uniaxial systems, with ah initio calculations for naproxen with a simple orientation distribution at the interface recovering the signs and magnitudes of the experimental observations with no adjustable parameters. These observations elevate fluorescence as a novel chiral-specific probe with both exquisite sensitivity to chirality and interface-specificity akin to that normally reserved for even-ordered nonlinear optical interactions, including second harmonic and sum-frequency spectroscopy. Further, the broad experimental access to fluorescence opens the possibility for surface-specific spectroscopy of chiral interfaces that might be otherwise challenging to interrogate using coherent nonlinear optical methods.

[0034] Mathemtical Framework

[0035] The mathmetical foundation for F-ORD has been detailed in previous work (Deng et al., Journal of Physical Chemistry Letters 2016, 7 (21), 4248-4252) and is briefly summarized here to frame the experiment observables. The intensity I of / / -polarized fluorescence ( / / = horizontal, H, or vertical, F) as a function of the incident polarization rotation angle y for linearly polarized excitation light depends on the following three trigonometric polynomial coefficients A - C.

[0036] In Eq. (1), the Bn coefficient is chiral-specific, disappearing in achiral uniaxial ensembles exhibiting C°°v, D°°, or D°°h symmetry within the electric dipole approximation. For chiral uniaxial symmetries of C°° symmetry, the coefficient BH can be connected directly back to the molecular orientation distribution at the interface through the following relation.

[0037] The angleL>in Eq. (2) indicates the internal angle within the molecule between the transition ''' J? 0 moments for absorption a ■ and fluorescence emission ’ . The Euler angles and ■ describe polar tilt and twist angles, respectively, illustrated in FIG. 1 panels A-C. Uniaxial symmetry imposes a uniform distribution in the third Euler angle describing azimuthal orientation about the interface normal, Unlike chiral phenomena in isotropic media, the chiroptical signals arising in uniaxial systems and described in Eq. (2) are fully electric dipole- allowed and there potentially of comparable magnitude as their achiral counterparts. The expression for the chiral response in Eq. (2) arises from orientational ordering alone and holds in the limit of negligible intermolecular coupling between planar chromophores, which are defined entirely by the absorption and emission transition moment vectors.

[0038] The magnitude of the F-ORD signal can be interpreted in the context of a linear intensity difference (LID) measurement most analogous to circular intensity difference measurements with circularly polarized excitation. In LID measurements, the polarized fluorescence intensity is measured for +45° and -45° linear polarizations, with the difference in the detected intensity directly connected to the F-ORD rotation angle.

[0039] 0 = 54,7°. £ = 90sBy inspection of Eq. (2), a maximal BH coefficients is achieved when and " , which was found previously to yield an upper theoretical limit of LID <0.808.

[0040] Alternatively, this same three-parameter polynomial expression can be recast in terms of frequency-dependent harmonics using trigonometric identities to yield the following expression.

[0041] In this formulation, an= (An + Cn) / 2, bn= (An- Cn) / 2, and cn= Bn / 2, where n ={H, I7} and Bv = - BH.

[0042] For the experimental configuration shown in FIG. 2, a peak retardance of the PEM of yields a time-dependence of ' given by the following expression.

[0043] Substitution into Eq. (4) yields a time-dependent intensity scaling with sine and cosine harmonics of in Eq. (5).

[0044] Cosines of sine functions exclusively contribute to even harmonics in time, while sines of sine functions contribute only to odd harmonics. As such, selective detection of the odd harmonics enables isolation of the chiral-specific contributions from the time-dependent intensity. A Taylor series expansion of the sinusoidal contributions to Eq. (4) yields the following expression for the chiral-specific intensity.

[0045] Analogous expressions can also be generated for the time-dependence of the achiral response, which depends exclusively on the even harmonics of the modulation frequency fo.

[0046]

[0047] From Eq. (3), a maximum in the LID with an amplitude of ~ 1 corresponds to a ~ 45° rotation between excitation and the principal axis of emission. If one chooses a reference point corresponding to coparallel excitation and emission neglecting depolarization (e.g., from orientational diffusion), in this limit Cn« An and LID = BH I AH. In this limit, the F-ORD rotation angle "FORD can be defined from the ratio of If to 2f modulation amplitude rlf / 2f

[0048] < beari •n , suc

[0049] Expressing the optical rotation in terms of rlf'2f enables the use of identical detection schemes (e g., lock-in amplification) for both modulation harmonics, removing scaling factors for the total intensity for calculations based on the average fluorescence intensity for LID determination.

[0050] Systems and methods

[0051] Monomolecular films of enantiopure naproxen samples were prepared by dip-coating hydrophilic fused silica microscope slides (Esco Optics) from a solution of 0.7 mg / mL (S)- naproxen solution (98% purity; Sigma-Aldrich) in toluene (Mallinckrodt Chemicals) at room temperature. Microscope slides were maintained in the solution for approximately 20 s and let to dry for 15-20 min before any experimental data acquisition. The slides were cut to have dimensions of 5.0 cm by 1.2 cm to coincide with the spectrometer sample chamber (used to confirm a reciprocal response) and surface-treated by submersion in a “piranha” solution consisting of four parts concentrated sulfuric acid (with more than 51% acid - Mallinckrodt Chemicals) to one part 30% hydrogen peroxide (Fisher Chemical) at room temperature for 30 min prior to dip-coating. Dip coating was performed using a custom instrument built in-house consisting of an electrically controlled motorized stage operated at a withdrawal rate of ~14 mm / min. Thickness for the dip-coated films was evaluated by dual-color ellipsometry using a Gaertner LI 16SF Variable Angle Stokes Ellipsometer. Samples for ellipsometry analysis consisted of silicon wafers with a native oxide overlayer, undergoing surface treatment similar to that described above for UV-fused silica slides. Following dip-coating, one of the two surfaces was subsequently cleaned with a solvent-soaked wipe to remove the sample.

[0052] Custom instrumentation built in-house to support F-ORD spectroscopy is shown in FIG. 2, in which a 300W, 15A Xenon lamp (LOT Oriel) was coupled with a grating monochromator (LOT Oriel Omni X- 1509) for fluorescence excitation between a range of 190 - 300 nm. The beam was directed through a 300nm short pass filter (Asahi Spectra XUS0300) to allow for the transmission of light at wavelengths blue shifted of 300 nm and was subsequently directed through a UV fused silica lens (Thorlabs LA4725) and a UV Gian polarizer (Thorlabs GLB10- UV) set at 0° angle relative to the laser table to collimate and horizontally polarize the light. Polarization modulation was performed using a photoelastic modulator (PEM, Hinds Instruments) oriented at a 45° angle relative to the plane of the table with a resonant modulation frequency of If = 50 kHz. Light emerging from the PEM was directed through a second collimating lens (Thorlabs LA4725) and a UV fused silica single quarter-wave Fresnel Rhomb (EKSMA Optics) to induce broad-band quarter wave retardance across the UV spectral range employed for excitation. The addition of a quarter-wave retarder following the PEM resulted in polarization modulation with exclusively linearly polarized light, in which the polarization axis was rapidly modulated rather than the sense of circularity. The time-dependent polarization states at key locations in the optical path are depicted in Fig. 2. For azimuthal scanning about the optical axis, samples were mounted on a motorized rotation stage (Newport Corporation SR50CC). The transmitted UV fluorescence was collected in transmission with a 15x, 0.4 NA Cassegrain reflective microscope objective (Newport - MKS Instruments). The signal was then passed through a second UV Gian polarizer (Thorlabs GLB10-UV) set at 0° (horizontal, coplanar with the laser table) and filtered with a 355 / 40 nm fluorescence bandpass filter (Semrock FL004467) before it was collected by a photomultiplier tube (PMT) module (Hamamatsu R6095P-01). Data acquisition with wavelength and / or sample rotation was performed using custom software written in-house in MATLAB. In the sample rotation studies, spectral scans were acquired for each 30° angle of azimuthal rotation, with the sample rotating from 0° to 360°. Following amplification using a preamplifier (ORTEC 9305), the modulation amplitudes from the PMT were digitized using two Stanford Research Systems analog lock-in amplifiers (SR860 at the fundamental If modulation frequency and SR810 at the 2f frequency). Calibration of the Gian polarizer rotation angles measured in the absence of a sample was performed by minimization of the If modulation amplitude of the transmitted ultraviolet beam with a high dynamic range UV-sensitive PMT (Hamamatsu, R2078) powered by a high-voltage source (KEPCO, BHK 2000-20MG). The use of a Cassegrain reflective objective for fluorescence detection centered along the optical axis served both to provide achromic collection of the low angle fluorescence and as a spatial filter with the central obscuration reducing direct transmission of the excitation beam.

[0053] Fluorescence optical rotation measurements

[0054] Fluorescence optical rotation measurements using the instrument depicted in FIG. 2 were acquired for naproxen thin films at normal incidence, representative results of which are shown in FIG. 3 panels A-B. Polarization-dependent fluorescence excitation spectra are shown for both source-facing and detector-facing orientations of the sample in the instrument. As can be seen in the figure, the spectra acquired by modulation at the If frequency for the two different orientations are close to mirror images, similar in magnitude and spectral dependence but opposite in sign. Complementary measurements are also shown for the 2f modulation, which in accordance with the polarization-dependence described in FIG. 2 reports on the depolarization amplitude (i.e., the difference in fluorescence for coparallel vs. cross-polarized excitation). Consistent with expectations, the 2f amplitude is reciprocal, yielding trends identical in sign and similar in magnitude for the two different sample orientations. Slides prepared with naproxen on both the source-facing and detector-facing surfaces yielded minimal residual If amplitudes, consistent with the summation of the blue and red curves in FIG. 3 panel A. The minor amplitude difference in the 2f response for source-facing versus detector-facing orientations is tentatively attributed to difference in Fresnel factors for the excitation and fluorescence frequencies in the two different configurations.

[0055] Prior to attribution of the results in FIG. 3 panels A-B to F-ORD, a series of control experiments was performed to assess the potential contributions from possible competing artefacts. First and foremost, azimuthal rotation studies were performed to assess the significance of interferences from in-plane orientational order. Linear dichroism from in-plane orientational order along an angle tilted relative to the horizontal axis would also produce a large F-ORD amplitude unrelated to interfacial chirality. Furthermore, such an in-plane ordering would have the potential to produce a nonreciprocal response for the horizontal polarization upon sample rotation about the vertical axis, as was performed herein. However, such an artifact would require the axis of ordering to be tilted relative to the axis of sample rotation and dip-coating, which is unexpected. To assess the contributions from linear dichroism, F-ORD measurements were made as a function of azimuthal sample rotation about the optical axis, the results of which are shown in FIG. 4. For ORD dominated by linear dichroism, the sign of the response must invert every 90° of azimuthal rotation in order to average to zero according to the pattern for linear dichroism indicated in FIG. 4. In contrast, the theoretical foundation for chiral-specific F- ORD is built around the assumption of uniaxial symmetry, consistent with a response that is independent of azimuthal rotation. The experimentally measured F-ORD for excitation from 288 nm to 294 nm and detected at 335-375 nm retains the same sign and a similar magnitude throughout the full 360° azimuthal rotation, in excellent agreement with the predictions for chiral-specific F-ORD and in stark contrast to the expectations for spurious linear dichroism arising from in-plane ordering.

[0056] Recent “dark-field” studies of naproxen thin films have reported large nonreciprocal CD spectroscopy from the scattered incoherent component of the transmitted beam, which could also potentially contribute to the observed F-ORD in uniaxial assemblies. Although the Frensel rhomb is designed to operate as a quarter wave retarder from 210 - 400 nm, subtle but nonzero dispersion within the fused silica could potentially introduce weak ellipticity in the optical beam, with the corresponding possibility of interferences from CD in the excitation beam. To quantify these contributions, the If modulated signal in the transmitted UV beam was measured with the UV sensitive PMT used for optical calibration. No detectable If signal could be observed in the transmitted excitation beam, despite the significantly higher signal to noise of transmission detection relative to the fluorescence signal. Furthermore, the sample produced no detectable CD using a conventional CD spectrometer (Jasco J- 1500). These observations are consistent with the substantial differences in sample preparation; the prior study was designed around oriented microcrystalline samples producing significant optical scatter, while the present study is investigating fluorescence from sub-monolayer ultrathin interfacial films. In such limits, the mechanism accessed in the dark-field study is not expected to significantly contribute.

[0057] The F-ORD spectral response is distinct from the ultraviolet-visible (UV-Vis) absorbance spectra previously reported for both isotropic solutions and microcrystalline thin films, but remarkably similar to fluorimetry measurements of naphthalene solutions. Notably, the previously reported UV-Vis absorbance spectra contain large features at -235 nm, blue-shifted significantly from the peaks shown in FIG. 3 panels A-B. These high-energy absorption bands are attributed to dissociative states, which in turn do not relax through internal conversion to lower-lying electronic states that contribute to fluorescence emission. Consistent with this expectation, these strong features blue-shifted of 250 nm are absent in the fluorimetry measurements of naproxen solutions performed with detection red-shifted of 300 nm. Excitationscans in fluorimetry yield a strong peak at -293 nm, in excellent agreement with the spectral features accessed in F-ORD measurements, the results of which are summarized in the Examples herein.

[0058] Quantum chemical calculations were performed to assist in interpretation of the F-ORD spectral response for naproxen monolayer films, the results of which are summarized in FIG. 1 panels A-C. Emission is assumed to originate from the lowest energy excited electronic state, with the transition moment direction indicated by the red arrow. The absorption band at -293 nm is assumed to couple to the next highest energy excited electronic state with a calculated energy of 273nm and a transition moment given by the blue arrow, yielding an internal angle between the two vectors of <5 = 18°. Considering just these two electric dipole allowed transitions and neglecting any interm olecular electronic coupling, the predicted F-ORD response can be calculated for a given presumed molecular orientation distribution from Eq. 2.

[0059] Results of those calculations for an orientation distribution in which the OH bond axis of the carboxylic acid group is oriented anti-parallel with the interface normal are shown in FIG. 1 panerls A-C. Although the true orientation distribution is unknown, it is reasonable to conjecture a molecular orientation in which the hydrophilic portion of naproxen preferentially orients toward the hydrophilic silica interface when deposited from a hydrophobic solution. This configuration produces a tilt angle of 0 = 54° and a twist angle of * / / = 107° for a source-facing sample orientation and 0 = 186° and \| / = 287° relative to the optical axis for a sample oriented towards the detector.

[0060] Using these presumed angles together with a calculated value of 8 = 18° allows approximation of the anticipated electric-dipole allowed F-ORD response with no adjustable parameters. Predictions from the quantum chemical modeling for an assumed preferred molecular orientation are in quite good qualitative agreement with the experimental observations, despite the obvious oversimplification of the naproxen / surface orientation distribution. This presumed molecular orientation for (S)-naproxen yields a predicted LID of -7%, a rotation angle of FORD = -2°, and a 1 f / 2f ratio of -20% for a sample in a source-facing orientation. From inspection of FIG. 2, the measured ratio was -62%. Although differing by a factor of -3 in magnitude, the ab initio calculations nevertheless recover the correct absolute sign and approximate magnitude of the response. Several possible mechanisms could explain the quantitative differences. Most notably, deviations away from such an artificially sharp orientation distribution could substantially impact both the numerator and denominators in impacting the experimental ratio. Furthermore, the quantum chemical calculations were performed in the limit of a frozen sample. In practice, molecular reorientation and excited state dynamics are known to evolve over timescales comparable to the fluorescence lifetime and collectively contribute to fluorescence depolarization.

[0061] In such instances, the difference amplitude for coparallel vs. cross-polarized fluorescence in the denominator would be smaller than predicted by ab initio calculations, and the ratio correspondingly larger than predicted. Finally, subtle inaccuracies in the quantum chemical calculations could produce underestimates of the internal angle between the excitation and emission transition moments. Higher values of 6 generally correspond to increases in the numerator with minimal change in the denominator in Eq. (3).

[0062] The measured value of -62% for the normalized chiral-specific 1 f / 2f ratio and the corresponding QFORD optical rotation angle of the principal emission axis is notably well outside the range of responses anticipated from classical bulk optical rotation based on nonzero magnetic dipole and / or electric quadrupole interactions. Estimates of the latter were evaluated by calculation of the ORD expected from bulk (isotropic) circular birefringence. Although no literature values could be found for the ORD of naproxen at the excitation wavelength of -280 nm, bulk-allowed ORD is directly coupled to absorbance CD through Kramers-Kronig relations. The details of the Kramers-Kronig transformation of the CD spectrum of naproxen are shown in the Examples. The bulk ORD of (S)-naproxen at 293 nm was calculated from the CD spectrum to produce a rotation coefficient of -780 mdeg M’1cm’1. For a monolayer with an approximate surface coverage of ~1014molecules / cm2, the anticipated ORD rotation angle from passage through a naproxen monolayer based on this cross-section is — 1.3 x 10'4mdeg. For comparison, the rotation angle from F-ORD can be estimated from the lf / 2f ratio from Eq. (8). The observed ratio of rlf2f= -62% corresponds to a clockwise rotation angle of -6° between the principal excitation and emission axes from a single molecular monolayer. This observation equates to an enhancement factor of more than seven orders of magnitude relative to standard bulk mechanisms for ORD within the shorter excitation wavelength.

[0063] Interestingly, the dominant features for 293 nm excitation in the F-ORD measurements shown in FIG. 3 panels A-B produce negligibly weak isotropic CD. This observation is consistent with electronic transitions localized to the nominally planar (and therefore achiral) conjugated ring structure. Importantly, F-ORD does not require chirality within the chromophore within the electric dipole approximation, provided that the plane of the chromophore adopts a chiral surface orientation in the tilt and twist angles (e.g., from chirality within the steric interactions driving interfacial ordering). This fundamental difference between intramolecular structural chirality and intermolecular orientational chirality highlight the highly complementary nature of the two chiralspecific spectroscopic observables. The results of this study suggest a potential re-interpretation of long-standing conflicting reports in single molecule circular dichroism fluorescence studies. In 2006, large fluorescence detected circular dichroism (FDCD) dissymmetry parameters of g = ±0.2 in the population averages were reported in single molecule fluorescence studies of surface-immobilized fluorophores. It was subsequently argued that these effects may have arisen from a combination of polarization changes by optics in the beampath (e.g., dichroic mirrors) coupled with spurious linear dichroism in the sample. Consistent with this conjecture, the ensemble-averaged CID was significantly suppressed upon attention to ensure circularly polarized light at the sample plane.

[0064] However, prior studies fail to adequately explain the origins of the chiral sensitivity observed experimentally if attributed to parasitic linearly polarized contributions. In prior theoretical work, some of us conjectured that F-ORD may well have driven the original observations, in which the spurious linearly polarized components may have contributed significantly to the FDCD observations through F-ORD. The present work provides compelling experimental evidence supporting that previous hypothesis and bridging the otherwise conflicting observations by prior groups. The interface and chiral specificity in F-ORD demonstrated herein open new opportunities for probing chemical interactions at biological interfaces. Through F-ORD, interface-selective measurements can, in principle, be performed with interface-selectivity rivaling even-ordered nonlinear optical measurements. Because the interactions are fully electric dipole-allowed, the chiral-specific response has the potential to be on the same order as the achiral response, providing high sensitivity for biologically relevant interfacial interactions. F- ORD has the potential to enable real-time analysis of target binding to surface-immobilized biosensors, advancing chromatography of chiral compounds by leveraging intrinsic autofluorescence, improving understanding and supporting real-time monitoring in affinity chromatography, and characterizing the protein corona that develops on nanoparticle surfaces upon physiological introduction. Moreover, the insights gained from F-ORD might contribute to our understanding of heterogeneous nucleation and growth of homochiral crystals, which in turn are hypothesized as sources for chiral enrichment setting the stage for the emergence of life. Collectively, these applications suggest a broad range of promising impacts of F-ORD across diverse disciplines of biological research.

[0065] System Architecture

[0066] In certain embodiments, the systems and methods of the invention can be carried out using automated systems and computing devices. Specifically, aspects of the invention described herein can be performed using any type of computing device, such as a computer, that includes a processor, e.g., a central processing unit, or any combination of computing devices where each device performs at least part of the process or method. In some embodiments, systems and methods described herein may be controlled using a handheld device, e.g., a smart tablet, or a smart phone, or a specialty device produced for the system.

[0067] Systems and methods of the invention can be performed using software, hardware, firmware, hardwiring, or combinations of any of these. Features implementing functions can also be physically located at various positions, including being distributed such that portions of functions are implemented at different physical locations (e.g., imaging apparatus in one room and host workstation in another, or in separate buildings, for example, with wireless or wired connections). Processors suitable for the execution of computer program include, by way of example, both general and special purpose microprocessors, and any one or more processor of any kind of digital computer. Generally, a processor will receive instructions and data from a read-only memory or a random access memory or both. The essential elements of computer are a processor for executing instructions and one or more memory devices for storing instructions and data. Generally, a computer will also include, or be operatively coupled to receive data from or transfer data to, or both, one or more mass storage devices for storing data, e.g., magnetic, magneto-optical disks, or optical disks. Information carriers suitable for embodying computer program instructions and data include all forms of non-volatile memory, including by way of example semiconductor memory devices, (e.g., EPROM, EEPROM, solid state drive (SSD), and flash memory devices); magnetic disks, (e.g., internal hard disks or removable disks); magnetooptical disks; and optical disks (e.g., CD and DVD disks). The processor and the memory can be supplemented by, or incorporated in, special purpose logic circuitry.

[0068] To provide for interaction with a user, the subject matter described herein can be implemented on a computer having an I / O device, e.g., a CRT, LCD, LED, or projection device for displaying information to the user and an input or output device such as a keyboard and a pointing device, (e.g., a mouse or a trackball), by which the user can provide input to the computer. Other kinds of devices can be used to provide for interaction with a user as well. For example, feedback provided to the user can be any form of sensory feedback, (e.g., visual feedback, auditory feedback, or tactile feedback), and input from the user can be received in any form, including acoustic, speech, or tactile input.

[0069] The subject matter described herein can be implemented in a computing system that includes a back-end component (e.g., a data server), a middleware component (e.g., an application server), or a front-end component (e.g., a client computer having a graphical user interface or a web browser through which a user can interact with an implementation of the subject matter described herein), or any combination of such back-end, middleware, and frontend components. The components of the system can be interconnected through network by any form or medium of digital data communication, e.g., a communication network. For example, the reference set of data may be stored at a remote location and the computer communicates across a network to access the reference set to compare data derived from the female subject to the reference set. In other embodiments, however, the reference set is stored locally within the computer and the computer accesses the reference set within the CPU to compare subject data to the reference set. Examples of communication networks include cell network (e.g., 3G or 4G), a local area network (LAN), and a wide area network (WAN), e.g., the Internet.

[0070] The subject matter described herein can be implemented as one or more computer program products, such as one or more computer programs tangibly embodied in an information carrier (e.g., in a non-transitory computer-readable medium) for execution by, or to control the operation of, data processing apparatus (e.g., a programmable processor, a computer, or multiple computers). A computer program (also known as a program, software, software application, app, macro, or code) can be written in any form of programming language, including compiled or interpreted languages (e.g., C, C++, Perl), and it can be deployed in any form, including as a stand-alone program or as a module, component, subroutine, or other unit suitable for use in a computing environment. Systems and methods of the invention can include instructions written in any suitable programming language known in the art, including, without limitation, C, C++, Perl, Java, ActiveX, HTML5, Visual Basic, or JavaScript.

[0071] A computer program does not necessarily correspond to a file. A program can be stored in a file or a portion of file that holds other programs or data, in a single file dedicated to the program in question, or in multiple coordinated files (e.g., files that store one or more modules, sub-programs, or portions of code). A computer program can be deployed to be executed on one computer or on multiple computers at one site or distributed across multiple sites and interconnected by a communication network.

[0072] A file can be a digital file, for example, stored on a hard drive, SSD, CD, or other tangible, non-transitory medium. A file can be sent from one device to another over a network (e.g., as packets being sent from a server to a client, for example, through a Network Interface Card, modem, wireless card, or similar).

[0073] Writing a file according to the invention involves transforming a tangible, non-transitory computer-readable medium, for example, by adding, removing, or rearranging particles (e.g., with a net charge or dipole moment into patterns of magnetization by read / write heads), the patterns then representing new collocations of information about objective physical phenomena desired by, and useful to, the user. In some embodiments, writing involves a physical transformation of material in tangible, non-transitory computer readable media (e.g., with certain optical properties so that optical read / write devices can then read the new and useful collocation of information, e.g., burning a CD-ROM). In some embodiments, writing a file includes transforming a physical flash memory apparatus such as NAND flash memory device and storing information by transforming physical elements in an array of memory cells made from floatinggate transistors. Methods of writing a file are well-known in the art and, for example, can be invoked manually or automatically by a program or by a save command from software or a write command from a programming language.

[0074] Suitable computing devices typically include mass memory, at least one graphical user interface, at least one display device, and typically include communication between devices. The mass memory illustrates a type of computer-readable media, namely computer storage media. Computer storage media may include volatile, nonvolatile, removable, and non-removable media implemented in any method or technology for storage of information, such as computer readable instructions, data structures, program modules, or other data. Examples of computer storage media include RAM, ROM, EEPROM, flash memory, or other memory technology, CD-ROM, digital versatile disks (DVD) or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, Radiofrequency Identification tags or chips, or any other medium which can be used to store the desired information and which can be accessed by a computing device.

[0075] As one skilled in the art would recognize as necessary or best-suited for performance of the methods of the invention, a computer system or machines of the invention include one or more processors (e.g., a central processing unit (CPU) a graphics processing unit (GPU) or both), a main memory and a static memory, which communicate with each other via a bus.

[0076] In an exemplary embodiment shown in FIG. 5, system 200 can include a computer 249 (e.g., laptop, desktop, or tablet). The computer 249 may be configured to communicate across a network 209. Computer 249 includes one or more processor 259 and memory 263 as well as an input / output mechanism 254. Where methods of the invention employ a client / server architecture, steps of methods of the invention may be performed using server 213, which includes one or more of processor 221 and memory 229, capable of obtaining data, instructions, etc., or providing results via interface module 225 or providing results as a file 217. Server 213 may be engaged over network 209 through computer 249 or terminal 267, or server 213 may be directly connected to terminal 267, including one or more processor 275 and memory 279, as well as input / output mechanism 271. System 200 or machines according to the invention may further include, for any of I / O 249, 237, or 271 a video display unit (e.g., a liquid crystal display (LCD) or a cathode ray tube (CRT)). Computer systems or machines according to the invention can also include an alphanumeric input device (e.g., a keyboard), a cursor control device (e.g., a mouse), a disk drive unit, a signal generation device (e.g., a speaker), a touchscreen, an accelerometer, a microphone, a cellular radio frequency antenna, and a network interface device, which can be, for example, a network interface card (NIC), Wi-Fi card, or cellular modem.

[0077] Memory 263, 279, or 229 according to the invention can include a machine-readable medium on which is stored one or more sets of instructions (e.g., software) embodying any one or more of the methodologies or functions described herein. The software may also reside, completely or at least partially, within the main memory and / or within the processor during execution thereof by the computer system, the main memory and the processor also constituting machine-readable media. The software may further be transmitted or received over a network via the network interface device.

[0078] Incorporation by Reference

[0079] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure, including to the Supplementary. The Supplementary, and all other such documents are hereby incorporated herein by reference in their entirety for all purposes.

[0080] Equivalents

[0081] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein.

[0082] EXAMPLES

[0083] Example 1 : Instrument calibration protocol

[0084] Subtle inaccuracies in optical orientation have the potential to result in significant background interferences in F-ORD analysis. A combination of simulations and experiments was performed to aid in instrument calibration. Prior to F-ORD analyses, characterization and optimization of the optical beampath was performed by measurement of the transmitted beam. For transmission analysis, the Cassegrain objective was removed from the beampath, and the visible photomultiplier tube (PMT) was replaced by a “solar-blind” UV PMT (Hamamatsu R2078). The PEM was positioned at a fixed angle of +45°. The Fresnel rhomb was placed on a kinematic prism mount with small-angle control for optimization, and the two UV-Glan polarizers were mounted in precision rotation stages with micrometer controls for fine adjustment. Rotation of the two Gian laser polarizers was optimized to minimize the relative amplitude of the If signal, first measured on an oscilloscope, then subsequently on a lock-in amplifier for fine tuning.

[0085] Modeling of the beampath was performed using a script written in-house in Matlab based on Jones matrices and Jones vectors 1 for calculation of the time-dependent intensity of the transmitted beam. Use of the script enabled analysis of the sensitivity of the instrument to alignment errors. The simulations suggested low sensitivity of the If modulation amplitude on the rotation angle of the first Gian, with inaccuracies manifesting primarily as unmodulated DC background intensities. Similarly, subtle orientational errors or departures from quarter-wave retardance of the Fresnel rhomb resulted in correspondingly subtle DC amplitudes in the transmitted beam, but no substantial background at either the If or 2f frequencies. In contrast, the orientation of the second Gian polarizer placed just before the detector was critical, with subtle inaccuracies resulting in large If modulated background responses. Notably, the relative orientation between the PEM and the detection polarizer was much more important than the absolute, in effect resulting in the fixed orientation angle of the PEM defining the relative orientations of all other optics in the beam path. The results of these simulations were in excellent agreement with qualitative observations of sensitivity assessed during calibration and optimization of the UV transmission.

[0086] Example 2: Ab initio predictions of lf / 2f F-ORD ratio

[0087] Calculations of the experimental observables from ab initio calculations of electronic structure were performed systematically working back from observables to molecular structure and orientation. From Eq. 1-8 in the main manuscript, the key F-ORD measurements can be collectively expressed in terms of the polynomial coefficients AH, BH, and CH for the horizontally polarized detection geometry adopted in the instrument schematic. These polynomial coefficients are related to the internal angle 5 between the excitation and emission transition moments, which is generally nonzero for excited state relaxation through internal conversion (i.e., emission from a different electronic excited state surface than excitation). In systems exhibiting negligible rotational dynamics over the excited state lifetime, the orientational averages bridging the molecular frame to the surface frame can be evaluated collectively. Expressions for each of the nine orientational averages in Equation (SI.3) for a uniform distribution in <$) can be evaluated explicitly. Modeled after procedures described in Davis t al. (Selection Rules and Symmetry Relations for Four-Wave Mixing Measurements of Uniaxial Assemblies. J. Phys. Chem. B 2008, 112, 5834-5848), the content of which is incorporated by reference herein in its entirety, the set of orientational averages contributing to the achiral coefficients AH and CH are given by the following expressions.

[0088] The orientational averages for the chiral-specific contribution in BH are given by the following expressions.

[0089] The electric dipole allowed chiral response arises from the nonzero orientational averages in Equations (SI.11) and (SI.12).

[0090] For an internal molecular coordinate system defined by the emission and absorption transition moments as depicted in FIG. 1 panels A-C, the expressions in Eqs. (SI.4)

[0091] - (SI.12) can be evaluated in the limit of a narrow distribution about the mean tilt and twist angles, 0 and \| / , respectively. In turn, those defined the values of the polynomial coefficients AH

[0092] - CH.

[0093] Determination of the appropriate tilt and twist angles was facilitated using the tensor visualization plug-in NLOPredict (Moad et al., NLOPredict: Visualization and Data Analysis Software for Nonlinear Optics. J Comput Chem 2007, 28 (12), 1996-2002), the content of which is incorporated by reference herein in its entirety, within the UCSF Chimera (Pettersen et al., UCSF Chimera - A Visualization System for Exploratory Research and Analysis. J Comput Chem 2004, 25 (13), 1605-1612), the content of which is incorporated by reference herein in its entirety, platform by first updating the parameters that define the tensors and adjusting to correspond to the emission transition moment, defining molecular coordinates according to the definitions in FIG. 1 panels A-C, applying the current orientation to zero-value the Euler angles, then reorienting to a molecular orientation with the OH bond axis of the carboxyl group either coparallel or antiparallel to the out-of-screen Z-axis.

[0094] Example 3: Kramers-Kronig calculations of the contribution from bulk-allowed ORD of the excitation beam

[0095] Calculation of the ellipticity induced in the excitation beam was performed through Kramers- Kronig transformation of the isotropic circular dichroism spectrum of an (S)-naproxen solution in ethanol, both of which are shown in FIG. 6. Calculations were performed for the bluer excitation wavelength, as it is more prone to rotation than the fluorescence emission and therefore represents an upper bound on the anticipated bulk-allowed ORD of the thin film sample. Molar ellipticity in mdegM^cm’1was calculated from the recovered CD spectrum following baseline subtraction, with the corresponding Kramers-Kronig transformation performed using a built-in function in Matlab to recover the real part of the chiral polarizability, which describes ORD. Estimation of the ORD from a single molecular monolayer was performed by assuming an interfacial number density of ~1014molecules / cm2. Multiplication by the molar ellipticity following appropriate units conversion yielded the anticipated rotation angle of -1.3 x 10'4mdeg.

[0096] Example 4: Excitation and emission fluorimetry

[0097] Fluorimetry measurements were performed on solutions of (S)-naproxen in ethanol, the results of which are shown in FIG. 7. For an excitation wavelength of 295 nm, the emission spectrum produced a broad peak centered about 360 nm, in reasonably good agreement with previous reports of maximal emission at ~35O nm. The excitation scan performed for emission at 358 nm yielded peaks around 295 nm. The peak in the fluorescence excitation wavelength was significantly red-shifted from prior reports in strongly acidic chloroform solutions (with a peak around -270 nm), but in excellent agreement with spectral features in both the If and 2f spectra acquired independently with the F-ORD instrument for ultrathin interfacial films. The red-shift relative to studies performed in acidic solutions is tentatively attributed to spectroscopic measurements dominated by the naproxen anion herein, rather than then neutral form studies in the prior work. The pKa of naproxen is -4.15, suggesting a significant degree of dissociation in aqueous solutions, and presumably accordingly in the dip-coated thin surface films prepared at hydrophilic interfaces. The substantial Stokes shift between excitation and emission maxima is consistent with internal conversion between a gateway electronic state with a high absorption cross section and an emissive state with a lower crosssection, in excellent agreement with the quantum chemical calculations of electronic structure.

Claims

What is claimed is:

1. A method for analyzing a molecule, the method comprising: producing a monomolecular film on opposing sides of a substrate, wherein the monomolecular film comprises a target molecule; using a spectroscopy system to conduct fluorescence optical rotary dispersion (F-ORD) on the target molecule in the monomolecular film on the substrate; receiving F-ORD data acquired by spectroscopy system to a processing system; and applying one or more F-ORD processing algorithms via the processing system to obtain interface-selective measurements of the target molecule.

2. The method of claim 1, wherein analzying comprising probing chemical interactions of the target molecule at biological interfaces.

3. The method of claim 2, wherein the chemical interactions are fully electric dipole-allowed.

4. The method of claim 3, wherein the analyzing allows for chiral-specific responses.

5. The method of claim 4, wherein analyzing allows for generating information for biologically relevant interfacial interactions.

6. The method of claim 5, wherein the method is applied to enable real-time analysis of target binding to surface-immobilized biosensors.

7. The method of claim 5, wherein the method is applied for advancing chromatography of chiral compounds by leveraging intrinsic autofluorescence of the target molecule.

8. The method of claim 5, wherein the method is applied for improving understanding and supporting real-time monitoring in affinity chromatography.

9. The method of claim 5, wherein the method is applied for characterizing a protein corona that develops on nanoparticle surfaces upon physiological introduction.

10. The method of claim 5, wherein the method is applied to analyze heterogeneous nucleation and growth of homochiral crystals.

11. A fluorescence optical rotary dispersion (F-ORD) system comprising: a light source that generates light; a grating monochromator for fluorescence excitation; a short pass filter; a ultraviolet (UV) fused silica lens; a UV Gian polarizer; a photoelastic modulator (PEM); a second collimating lens; and a UV fused silica single quarter- wave Fresnel Rhomb.

12. The system of claim 11, wherein the grating monochromator for fluorescence excitation is between a range of 190 - 300 nanometer (nm)13. The system of claim 12, wherein the short pass filter is a 300nm short pass filter to allow for transmission of light at wavelengths blue shifted of 300 nm.

14. The system of claim 13, wherein the blue shifted light is subsequently directed through the UV fused silica lens and the UV Gian polarizer.

15. The system of claim 14, wherein the UV Gian polarizer is set at 0° angle relative to the light source to collimate and horizontally polarize the light.

16. The system of claim 15, wherein the PEM performs polarization modulation, wherein the PEM is oriented at a 45° angle relative to the plane of the light source with a resonant modulation frequency of If = 50 kHz.

17. The system of claim 16, wherein light emerging from the PEM is directed through the second collimating lens and the UV fused silica single quarter-wave Fresnel Rhomb to induce broad-band quarter wave retardance across the UV spectral range employed for excitation.

18. The system of claim 17, wherein addition of the quarter-wave retarder following the PEM resulted in polarization modulation with exclusively linearly polarized light, in which the polarization axis was rapidly modulated rather than the sense of circularity.

19. The system of claim 11, wherein the light source is a 300W, 15A Xenon lamp.

20. The system of claim 11, further comprising a motorized stage, wherein for azimuthal scanning about an optical axis, a sample is mounted on the motorized rotation stage.

Images