Fluorescence-detected Circular Dichroism Research Articles

(Invited) Advanced Carbon Nanotube Fluorescence Spectrometry for Novel Applications

Instrumental advances in near-IR fluorescence spectroscopy are enabling new types of measurements involving single-wall carbon nanotubes (SWCNTs). Two unique systems will be described. The first is a two-dimensional fluorescence-detected circular dichroism (FDCD) spectrometer. In this, SWCNT samples are excited by a spectrally selected supercontinuum laser beam that is switched between left- and right-circular polarization in an electro-optic modulator. Near-infrared sample fluorescence emitted in the backward direction is captured and directed to a scanning monochromator with a cooled InGaAs single-channel detector. After amplification and high precision digitization, the modulated signal component is extracted by computer-based phase sensitive detection. The system can measure a sample’s E22 circular dichroism in four spectral modes: 1) conventional FDCD, with scanned visible excitation wavelength and spectrally integrated (zero-order grating) emission detection; 2) Emission-specific FDCD, with scanned visible excitation wavelengths and selected emission wavelength; 3) Emission-scanned FDCD, with selected visible excitation wavelength and scanned emission wavelengths; 4) Excitation-Emission FDCD, with excitation and emission wavelengths both scanned to give two-dimensional data sets. This instrument can spectroscopically resolve enantiomer signals from a single (n,m) species in a racemic SWCNT sample.In a parallel project, developments in SWCNT fluorescence spectrometry are advancing nanotube-based strain measurement technology toward commercialization. Because SWCNT emission wavelengths vary systematically with axial strain, nanotubes in a thin coating on a specimen can serve as optically interrogated strain gauges. We apply this effect to measure strain maps through hyperspectral imaging of SWCNT fluorescence. A rotated band pass filter is used to capture a set of images in multiple spectral slices, from which a custom computer program deduces strain at each of ~105 image pixels and compiles strain maps. We will describe how this apparatus has evolved from a lab prototype into a compact portable system that can make measurements in industrial settings.

Polarization Resolved Fluorescence Detection of Carbon Nanotube Corona Phases

Circular dichroism measures how chiral particles absorb right- and left- circularly polarized light differently to distinguish pairs of enantiomers. It can also elucidate differential photoabsorption of chiral fluorescent species via their emissions. However, this method has generally been restricted to bulk samples. Hence, these methods conceal information on the specific chiroptical response of a single particle. Furthermore, circular dichroism has been restricted in time- resolution as well as spectral range. In this work, we observed fluorescence detected circular dichroism (FDCD) of individually deposited single walled carbon nanotube (SWNT) corona. A custom-built setup compromising a linear polarizer (LP) and a quarter waveplate (QWP) was used to induce circularly polarized light (CPL). The light polarization state at the sample plane was analyzed using Stoke’s parameter. Experimental results showed that linear dichroism of the dichroic filter inside the microscope changes the polarization state of the incoming beam drastically, and thus inducing elliptically polarized light at the sample plane. A liquid crystal variable retarded (LCVR) was used to correct the polarization state of light and yield CPL at the sample plane. Singly-dispersed SWNT with different wrapping polymers were deposited onto a glass substrate. Alternating cycles of right- and left-handed circularly polarized light was delivered to excite the sample. The fluorescence intensities of each individual SWNT were tracked. The dissymmetry (g) parameters: g = 2(IL-IR)/(IL+IR) (where I L and I R are the measured fluorescence intensities with left and right circularly polarized light, respectively) were analyzed from the fluorescent signals to understand photoabsorption differences. A non-zero value of dissymmetry parameter provides information on how the corona modulates SWNT photoluminescence, an outcome not observable by bulk characterization methods. This observation can potentially enable the measurement of chiral species local interactions without a separations process.

Fluorescence detected circular dichroism (FDCD) of a stereodynamic probe.

The use of chiroptical techniques in combination with stereodynamic probes is becoming one of the leading strategies for chiral sensing. While in most of the reported studies circular dichroism (CD) is the adopted spectroscopic technique, examples regarding the use of vibrational CD (VCD), circularly polarized luminescence (CPL), and Raman optical activity (ROA) are emerging as innovative tools. In this communication, an anthracene-decorated tris(2-pyridylmethyl)amine zinc complex (TPMA) is reported for its capability to act as a chiral sensor using either CD or fluorescence detected circular dichroism (FDCD). The latter technique offers the unique possibility to determine the enantiomeric excess of a series of carboxylic acids at sensor concentrations down to 0.1 μM. Limitations and possibilities opened by the use of this methodology, in particular regarding the specificity of the probe in the presence of another contaminant, are discussed.

Extrinsic chirality in metasurfaces: Traditional and unconventional experiments – INVITED

Plasmonic nanostructures with achiral, but asymmetric shapes can exhibit chiro-optical phenomena at the nanoscale, given that the nanostructure-light interaction symmetry is broken. Such behaviour is defined as extrinsic chirality, and it is induced by properly arranging the experimental set-up. We show measurement techniques for extrinsic chirality in low-cost, asymmetric samples with nanostructures organized in metasurfaces. We employ widely tuneable chiro-optical characterization of transmission and reflection, as well as the circular polarization degree of the transmitted signal; near-infrared range (680-1080nm) and oblique incidence allow for the detection of resonant features in extrinsic chirality. Other, unconventional experiments use photo-thermal consequences of chirality governed absorption in metasurfaces. Photo-acoustic spectroscopy directly gives circular dichroism as a differential absorption of the left and right circular polarizations exciting the sample. Photo-deflection spectroscopy gives additional information of diffraction phenomena governed by the extrinsic chirality. We showed that these techniques can monitor the extrinsic chiral behaviour of the hybrid plasmonic metamaterials. Moreover, they can be used in combination with fluorescence-detected circular dichroism to measure the emission properties of fluorescent materials.

Base-Stacking Heterogeneity in RNA Resolved by Fluorescence-Detected Circular Dichroism Spectroscopy.

RNA plays a critical role in many biological processes, and the structures it adopts are intimately linked to those functions. Among many factors that contribute to RNA folding, van der Waals interactions between adjacent nucleobases stabilize structures in which the bases are stacked on top of one another. Here, we utilize fluorescence-detected circular dichroism spectroscopy (FDCD) to investigate base-stacking heterogeneity in RNA labeled with the fluorescent adenine analogue 2-aminopurine (2-AP). Comparison of standard (transmission-detected) CD and FDCD spectra reveals that in dinucleotides, 2-AP fluorescence is emitted almost exclusively by unstacked molecules. In a trinucleotide, some fluorescence is emitted by a population of stacked and highly quenched molecules, but more than half originates from a minor ∼10% population of unstacked molecules. The combination of FDCD and standard CD measurements reveals the prevalence of stacked and unstacked conformational subpopulations as well as their relative fluorescence quantum yields.

Probing conformational subpopulations of RNA and DNA using fluorescence-detected circular dichroism spectroscopy

Fluorescence and circular dichroism (CD) spectroscopy are two of the most widely used techniques for probing nucleic acids. RNA and DNA can adopt a variety of different structures, and the power of bulk spectroscopy would be greatly enhanced if spectra could be extracted individually for certain conformational subpopulations. To do this, we utilized fluorescence-detected circular dichroism (FDCD) spectroscopy, which measures the difference in fluorescence intensity when a molecule is excited by left- or right-handed circularly polarized light.

Signal and noise analysis for chiral structured illumination microscopy.

Recently, chiral structured illumination microscopy has been proposed to image fluorescent chiral domains at sub-wavelength resolution. Chiral structured illumination microscopy is based on the combination of structured illumination microscopy, fluorescence-detected circular dichroism, and optical chirality engineering. Since circular dichroism of natural chiral molecules is typically weak, the differential fluorescence is also weak and can be easily buried by the noise, hampering the fidelity of the reconstructed images. In this work, we systematically study the impact of the noise on the quality and resolution of chiral domain images obtained by chiral SIM. We analytically describe the signal-to-noise ratio of the reconstructed chiral SIM image in the Fourier domain and verify our theoretical calculations with numerical demonstrations. Accordingly, we discuss the feasibility of chiral SIM in different experimental scenarios and propose possible strategies to enhance the signal-to-noise ratio for samples with weak circular dichroism.

Fluorescence detected circular dichroism (FDCD) for supramolecular host-guest complexes.

Fluorescence-detected circular dichroism (FDCD) spectroscopy is applied for the first time to supramolecular host–guest and host–protein systems and compared to the more known electronic circular dichroism (ECD). We find that FDCD can be an excellent choice for common supramolecular applications, e.g. for the detection and chirality sensing of chiral organic analytes, as well as for reaction monitoring. Our comprehensive investigations demonstrate that FDCD can be conducted in favorable circumstances at much lower concentrations than ECD measurements, even in chromophoric and auto-emissive biofluids such as blood serum, overcoming the sensitivity limitation of absorbance-based chiroptical spectroscopy. Besides, the combined use of FDCD and ECD can provide additional valuable information about the system, e.g. the chemical identity of an analyte or hidden aggregation phenomena. We believe that simultaneous FDCD- and ECD-based chiroptical characterization of emissive supramolecular systems will be of general benefit for characterizing fluorescent, chiral supramolecular systems due to the higher information content obtained by their combined use.

Chiral Structured Illumination Microscopy

We propose a chiral imaging modality based on optical chirality engineering, fluorescence-detected circular dichroism and structured illumination microscopy. In this method, the optical chirality of the illumination is structured and the circular dichroism dependent fluorescence is detected. With image reconstruction, the spatial distribution of chiral domains can be obtained at sub-diffraction limited resolution. We theoretically demonstrate this method and discuss the feasibility using an optical chirality engineering approach based on far-field optics.

Fluorescence-Detected Circular Dichroism of a Chiral Molecular Monolayer with Dielectric Metasurfaces.

Strong enhancement of molecular circular dichroism (CD) has the potential to enable efficient asymmetric photolysis, a method of chiral separation that has conventionally been impeded by insufficient yield and low enantiomeric excess. Here, we study experimentally how predicted enhancements in optical chirality density near resonant silicon nanodisks boost CD. We use fluorescence-detected circular dichroism (FDCD) spectroscopy to measure indirectly the differential absorption of circularly polarized light by a monolayer of optically active molecules functionalized to silicon nanodisk arrays. Importantly, the molecules and nanodisk antennas have spectrally coincident resonances, and our fluorescence technique allows us to deconvolute absorption in the nanodisks from the molecules. We find that enhanced FDCD signals depend on nanophotonic resonances, in good agreement with simulated differential absorption and optical chirality density, while no signal is detected from molecules adsorbed on featureless silicon surfaces. These results verify the potential of nanophotonic platforms to be used for asymmetric photolysis with lower energy requirements.

The Role of Fiber Agglomeration in Formation of Perylene-Based Fiber Networks

The agglomeration of self-assembled fibers of π-conjugated molecules is crucial to the formation of low-molecular-weight supramolecular gels. However, identification of the in situ spectroscopic signatures of fiber agglomeration has remained a challenging endeavor. Here, we use a combination of chiroptical techniques, namely circular dichroism (CD), magnetic circular dichroism (MCD), and fluorescence-detected circular dichroism (FDCD), to investigate the agglomeration of self-assembled fibers of a chiral low-molecular-weight gelator, l -alanine-substituted perylene imide bis( n-butyl ) ester (PIBE). Surprisingly, we find that agglomerated PIBE fibers exhibit an opposite CD signature in comparison to the isolated PIBE fibers. In contrast, the MCD and FDCD responses do not change during the agglomeration process, revealing that the local structure in the individual fibers is unperturbed. Using Brownian dynamics simulations, we conclude that the effective charge on the fibers dictates the agglomeration process and that the final geometry of the agglomerated fibers is marked by crossed nodes. A combination of chiroptical techniques is used to characterize fiber agglomeration The effective charge on the fibers is shown to dictate the agglomeration process Simulations reveal that fiber agglomerates preferentially form crossed nodes In situ characterization of three-dimensional self-assembly processes is a major challenge in the field of supramolecular self-assembly. Here, Sharma et al. use a range of complementary chiroptical techniques and theoretical modeling to characterize three-dimensional agglomeration of self-assembled fibers in solution and demonstrate that agglomeration of helical fibers results in the formation of crossed nodes.

Fluorescence-detected circular dichroism spectroscopy of jet-cooled ephedrine.

The resonant two-photon ionization circular dichroism (R2PICD) spectrum represents the cumulative circular dichroism (CD) of one-photon excitation and the subsequent one-photon ionization, whereas the fluorescence-detected circular dichroism (FDCD) spectra exhibit only the CD of one-photon excitation, similar to conventional CD spectra. We obtained the FDCD spectra of jet-cooled ephedrine (EPD) near the origin band of the S0-S1 transition to measure the CD of one-photon absorption and thus the CD of the ionization process in R2PI in comparison with the R2PICD spectra. The CD effects of the ionization following excitation of the A (0-0) and C (930 cm(-1)) bands in the spectrum are small, whereas those of the B band (530 cm(-1)) are anomalously large, leading to opposite CD signs for the FDCD and R2PICD spectra. Based on the intermediate state-selective fragmentation patterns in the R2PI spectra, this large CD effect is attributed to the state-selective isomerization that occurs after excitation of the B band. By comparing the experimental and theoretical spectra, we determined that the B band corresponds to an asymmetric ring distortion mode that involves torsional motions of the side chain, which may facilitate the isomerization process. This study demonstrates that FDCD spectroscopy combined with R2PICD spectroscopy provides a powerful tool to measure the CD effects of the excitation and ionization processes separately in R2PI and thus probe the structural changes that occur during the ionization process following excitation to an intermediate state.

A sensitive method based on fluorescence-detected circular dichroism for protein local structure analysis

We report an improved fluorescence-detected circular dichroism (FDCD)-based analytical method that is useful for probing protein three-dimensional structures. The method uses a novel FDCD device with an ellipsoidal mirror that functions on a standard circular dichroism (CD) spectrometer and eliminates all artifacts. Our experiments demonstrated three important findings. First, the method is applicable to any proteins either by using intrinsic fluorescence derived from tryptophan residues or by introducing a fluorescent label onto nonfluorescent proteins. Second, by using intrinsic fluorescence, FDCD spectroscopy can detect a structural change in the tertiary structure of metmyoglobin due to stepwise denaturation on a change in pH. Such changes could not be detected by conventional CD spectroscopy. Third, based on the typical advantages of fluorescence-based analyses, FDCD measurements enable observation of only the target proteins in a solution even in the presence of other peptides. Using our ellipsoidal mirror FDCD device, we could observe structural changes of fluorescently labeled calmodulin on binding with Ca2+ and/or interacting with binding peptides. Because FDCD appears to reflect the protein’s local structure around the fluorophore, it may provide a useful means for “pinpoint analysis” of protein structures.

Dichrometer Errors Resulting from Large Signals or Improper Modulator Phasing

A single-beam spectrometer equipped with a photoelastic modulator can be configured to measure a number of different parameters useful in characterizing chemical and biochemical materials including natural and magnetic circular dichroism, linear dichroism, natural and magnetic fluorescence-detected circular dichroism, and fluorescence polarization anisotropy as well as total absorption and fluorescence. The derivations of the mathematical expressions used to extract these parameters from ultraviolet, visible, and near-infrared light-induced electronic signals in a dichrometer assume that the dichroic signals are sufficiently small that certain mathematical approximations will not introduce significant errors. This article quantifies errors resulting from these assumptions as a function of the magnitude of the dichroic signals. In the case of linear dichroism, improper modulator programming can result in errors greater than those resulting from the assumption of small signal size, whereas for fluorescence polarization anisotropy, improper modulator phase alone gives incorrect results. Modulator phase can also impact the values of total absorbance recorded simultaneously with linear dichroism and total fluorescence.

Size Dependence of Chiroptical Activity in Colloidal Quantum Dots

The synthesis of chiral penicillamine-capped CdS and CdSe quantum dots (QDs) was adjusted to control the size of the nanoparticles. This, together with size separation, allowed for simultaneous tuning of absorption, circular dichroism (CD), and fluorescence on a wide wavelength range. Band edge transitions were accompanied by circular dichroism peaks which red-shifted together with the increase in particle size. The clear correlation between absorption and CD bands as well as between absorption bands and size in semiconductor QDs was used to derive an experimental scaling law for optical activity. The decrease in the intensity of circular dichroism-to-absorption ratio (dissymmetry) with the increase in particle size was stronger than linear, probably exponential. In addition, strong material type dependence was observed. The CD line shape appeared to be sensitive to the nature of the transition and may thus serve as a tool for sorting out the electronic states of the QDs. Fluorescence-detected circular dichroism (FDCD) was introduced as a new probe of optically active fluorescent nanoparticles. The analysis of the size and material dependence of the chiroptical induction effect leads to the conclusion that it is primarily an electronic interaction effect between the adsorbed chiral molecules and the electron-hole states.

Evaluation of Fluorescent Analogs of Deoxycytidine for Monitoring DNA Transitions from Duplex to Functional Structures

Topological variants of single-strand DNA (ssDNA) structures, referred to as “functional DNA,” have been detected in regulatory regions of many genes and are thought to affect gene expression. Two fluorescent analogs of deoxycytidine, Pyrrolo-dC (PdC) and 1,3-diaza-2-oxophenoxazine (tC°), can be incorporated into DNA. Here, we describe spectroscopic studies of both analogs to determine fluorescent properties that report on structural transitions from double-strand DNA (dsDNA) to ssDNA, a common pathway in the transition to functional DNA structures. We obtained fluorescence-detected circular dichroism (FDCD) spectra, steady-state fluorescence spectra, and fluorescence lifetimes of the fluorophores in DNA. Our results show that PdC is advantageous in fluorescence lifetime studies because of a distinct ~2 ns change between paired and unpaired bases. However, tC° is a better probe for FDCD experiments that report on the helical structure of DNA surrounding the fluorophore. Both fluorophores provide complementary data to measure DNA structural transitions.

Conformational Dynamics at the Active Site of α-Chymotrypsin and Enzymatic Activity

The role of dynamical flexibility at the active site of a proteolytic enzyme alpha-chymotrypsin (CHT) has been correlated with its catalytic activity. The temperature-dependent efficiency of catalysis reveals a bell-shaped feature with a peak at 37 degrees C, the typical body temperature of homeothermal animals. The overall structural integrity of the enzyme in our experimental temperature range has been confirmed from dynamic light scattering (DLS) and circular dichroism (CD) studies. We have followed the dynamical evolution at the active site of CHT with temperature using picosecond-resolved fluorescence anisotropy of anthraniloyl probe (covalently attached to the serine-195 residue) and a substrate mimic (inhibitor) proflavin. The conformational dynamics at the active site is found to have a distinct connection with the enzyme functionality. The conformational flexibility of the enzyme is also evidenced from the compressibility studies on the enzyme. The site selective fluorescence detected circular dichroism (FDCD) studies reveal that the conformational flexibility of the enzyme has an effect on the structural perturbation at the active site. We have also proposed the possible implications of the dynamics in the associated energetics.

Four-way-output molecular response system based on the dihydrodibenzo[ c, g]phenanthrene skeleton: modulation of CD and FDCD activity by acid and electron-transfer

Presence or absence of electron-accepting dye chromophores is the key function for ON/OFF switching of fluorescence from the dihydrodibenzo[ c, g]phenanthrene skeleton in 2 2+/ 3. The helical fluorophore in 2 2+ could be generated stereoselectively upon electrolysis of binaphthylic donor 1 through intramolecular chirality transmission. Huge chiroptical signals are easily detected by circular dichroism (CD) and fluorescence-detected CD (FDCD) techniques to realize novel four-way-output response.

Probing the Chiroptical Response of a Single Molecule

Chirally sensitive measurement techniques have generally been restricted to bulk samples. Here, we report the observation of fluorescence-detected circular dichroism (FDCD) from single (bridgedtriarylamine) helicene molecules by using an excitation wavelength (457 nanometers) in the vicinity of an electronic transition that shows circular dichroism in bulk samples. The distributions of dissymmetry (g) parameters by analysis of signals from pure M- and P-type diastereomers are almost perfect mirror images of one another, each spanning a range of both positive and negative values. In addition, we observe a well-defined structure in the histogram of dissymmetry parameters suggestive of specific molecular orientations at the polymer interface. These single-molecule results highlight strong intrinsic circular dichroism responses that can be obscured by cancellation effects in ensemble measurements of a randomly oriented bulk sample.

Excimer Fluorescence as a Tool for Monitoring Protein Domain Dynamics Applied to Actin Conformation Changes Based on Circulary Polarized Fluorescence Spectroscopy

Fluorescence-detected circular dichroism (FDCD) was introduced into the study of protein conformation changes. Actin was used as a model protein which undergoes dynamic conformation changes as it polymerizes. Actin labeled with N-(1-pyrene)iodoacetamide (PIA) showed monomer fluorescence peak at 386 and 410 nm, and excimer fluorescence peak at around 480 nm. Excimer was formed by PIA-dimers labeled to different sites of amino acid residues. New information concerned with actin structural changes were monitored by fluorescence emission spectra excited with left- and right-circulary polarized light at 355 nm. FDCD intensities were shown as the difference in the fluorescence emission DeltaF, where DeltaF=(F (L)-F (R))/(F (L)+F (R)) denoting F (L) and F (R) as emissions obtained by excitation with left- and right-circulary polarized light. When solvent conditions of PIA-actin were changed by addition of NaCl, TFE, or ATP, DeltaF showed sensitive responses to these compounds. From the analysis of DeltaF (M) and DeltaF (E) which represent the peaks of DeltaF at the monomer- and excimer-emission band, the information concerned with the actin intrastructural changes were obtained. This method based on monitoring the excimer fluorescence with FDCD could be used for other proteins to extract finer structural changes that cannot be detected by the normal fluorescence spectroscopy.