Andrew H. Marcus

Fluorescence-detected two-dimensional electronic coherence spectroscopy by acousto-optic phase modulation

Two-dimensional electronic coherence spectroscopy (ECS) is an important method to study the coupling between distinct optical modes of a material system. Such studies often involve excitation using a sequence of phased ultrashort laser pulses. In conventional approaches, the delays between pulse temporal envelopes must be precisely monitored or maintained. Here, we introduce a new experimental scheme for phase-selective nonlinear ECS, which combines acousto-optic phase modulation with ultrashort laser excitation to produce intensity modulated nonlinear fluorescence signals. We isolate specific nonlinear signal contributions by synchronous detection, with respect to appropriately constructed references. Our method effectively decouples the relative temporal phases from the pulse envelopes of a collinear train of four sequential pulses. We thus achieve a robust and high signal-to-noise scheme for phase-selective ECS to investigate the resonant nonlinear optical response of photoluminescent systems. We demonstrate the validity of our method using a model quantum three-level system-atomic Rb vapor. Moreover, we show how our measurements determine the resonant complex-valued third-order susceptibility.

Conformation of self-assembled porphyrin dimers in liposome vesicles by phase-modulation 2D fluorescence spectroscopy

By applying a phase-modulation fluorescence approach to 2D electronic spectroscopy, we studied the conformation-dependent exciton coupling of a porphyrin dimer embedded in a phospholipid bilayer membrane. Our measurements specify the relative angle and separation between interacting electronic transition dipole moments and thus provide a detailed characterization of dimer conformation. Phase-modulation 2D fluorescence spectroscopy (PM-2D FS) produces 2D spectra with distinct optical features, similar to those obtained using 2D photon-echo spectroscopy. Specifically, we studied magnesium meso tetraphenylporphyrin dimers, which form in the amphiphilic regions of 1,2-distearoyl-sn-glycero-3-phosphocholine liposomes. Comparison between experimental and simulated spectra show that although a wide range of dimer conformations can be inferred by either the linear absorption spectrum or the 2D spectrum alone, consideration of both types of spectra constrain the possible structures to a “T-shaped” geometry. These experiments establish the PM-2D FS method as an effective approach to elucidate chromophore dimer conformation.

Coherent two-dimensional photocurrent spectroscopy in a PbS quantum dot photocell

Recently there has been growing interest in the role of coherence in electronic dynamics. Coherent multidimensional spectroscopy has been used to reveal coherent phenomena in numerous material systems. Here we utilize a recent implementation of coherent multidimensional spectroscopy--two-dimensional photocurrent spectroscopy--in which we detect the photocurrent from a PbS quantum dot photocell resulting from its interactions with a sequence of four ultrafast laser pulses. We observe sub-picosecond evolution of two-dimensional spectra consistent with multiple exciton generation. Moreover, a comparison with two-dimensional fluorescence spectra of the quantum dots demonstrates the potential of two-dimensional photocurrent spectroscopy to elucidate detailed origins of photocurrent generating electronic state coherence pathways. Since the measurement is based on detecting the photocell current in situ, the method is well suited to study the fundamental ultrafast processes that affect the function of the device. This opens new avenues to investigate and implement coherent optimization strategies directly within devices.

Cytoskeletal-assisted dynamics of the mitochondrial reticulum in living cells

Subcellular organelle dynamics are strongly influenced by interactions with cytoskeletal filaments and their associated motor proteins, and lead to complex multiexponential relaxations that occur over a wide range of spatial and temporal scales. Here we report spatio-temporal measurements of the fluctuations of the mitochondrial reticulum in osteosarcoma cells by using Fourier imaging correlation spectroscopy, over time and distance scales of 10−2 to 103 s and 0.5–2.5 μm. We show that the method allows a more complete description of mitochondrial dynamics, through the time- and length-scale-dependent collective diffusion coefficient D(k,τ), than available by other means. Addition of either nocodazole to disrupt microtubules or cytochalasin D to disassemble microfilaments simplifies the intermediate scattering function. When both drugs are used, the reticulum morphology of mitochondria is retained even though the cytoskeletal elements have been de-polymerized. The dynamics of the organelle are then primarily diffusive and can be modeled as a collection of friction points interconnected by elastic springs. This study quantitatively characterizes organelle dynamics in terms of collective cytoskeletal interactions in living cells.

Single-molecule FRET and linear dichroism studies of DNA breathing and helicase binding at replication fork junctions

Significance Unique single-molecule fluorescence techniques were used to monitor DNA “breathing” at and near the junctions of model DNA replication forks on biologically relevant microsecond-to-millisecond time scales. Experiments performed in the absence and presence of helicase complexes addressed the role of these fluctuations in helicase function during DNA replication. These studies simultaneously monitored single-molecule Förster resonance energy transfer and single-molecule fluorescence linear dichroism of “internal” Cy3/Cy5 labels placed rigidly into the DNA backbones at positions near the fork junction. Our results showed significant breathing at the fork junction that was greatly augmented by the presence of weakly bound helicase, followed by still larger fluctuations and strand separation after full duplex DNA unwinding by the complete tightly bound and processive helicase complex. DNA “breathing” is a thermally driven process in which base-paired DNA sequences transiently adopt local conformations that depart from their most stable structures. Polymerases and other proteins of genome expression require access to single-stranded DNA coding templates located in the double-stranded DNA “interior,” and it is likely that fluctuations of the sugar–phosphate backbones of dsDNA that result in mechanistically useful local base pair opening reactions can be exploited by such DNA regulatory proteins. Such motions are difficult to observe in bulk measurements, both because they are infrequent and because they often occur on microsecond time scales that are not easy to access experimentally. We report single-molecule fluorescence experiments with polarized light, in which tens-of-microseconds rotational motions of internally labeled iCy3/iCy5 donor–acceptor Förster resonance energy transfer fluorophore pairs that have been rigidly inserted into the backbones of replication fork constructs are simultaneously detected using single-molecule Förster resonance energy transfer and single-molecule fluorescence-detected linear dichroism signals. Our results reveal significant local motions in the ∼100-μs range, a reasonable time scale for DNA breathing fluctuations of potential relevance for DNA–protein interactions. Moreover, we show that both the magnitudes and the relaxation times of these backbone breathing fluctuations are significantly perturbed by interactions of the fork construct with a nonprocessive, weakly binding bacteriophage T4-coded helicase hexamer initiation complex, suggesting that these motions may play a fundamental role in the initial binding, assembly, and function of the processive helicase–primase (primosome) component of the bacteriophage T4-coded DNA replication complex.

Wave packet interferometry and quantum state reconstruction by acousto-optic phase modulation.

Studies of wave packet dynamics often involve phase-selective measurements of coherent optical signals generated from sequences of ultrashort laser pulses. In wave packet interferometry (WPI), the separation between the temporal envelopes of the pulses must be precisely monitored or maintained. Here we introduce a new (and easy to implement) experimental scheme for phase-selective measurements that combines acousto-optic phase modulation with ultrashort laser excitation to produce an intensity-modulated fluorescence signal. Synchronous detection, with respect to an appropriately constructed reference, allows the signal to be simultaneously measured at two phases differing by 90 degrees. Our method effectively decouples the relative temporal phase from the pulse envelopes of a collinear train of optical pulse pairs. We thus achieve a robust and high signal-to-noise scheme for WPI applications, such as quantum state reconstruction and electronic spectroscopy. The validity of the method is demonstrated, and state reconstruction is performed, on a model quantum system--atomic Rb vapor. Moreover, we show that our measurements recover the correct separation between the absorptive and dispersive contributions to the system susceptibility.

Heterogeneous distribution of pyruvate dehydrogenase in the matrix of mitochondria

A fusion protein between GFP and the E1alpha subunit of the pyruvate dehydrogenase (PDH) complex was created and shown to assemble into functional PDH complexes using immunoprecipitation and activity assays. The expression of this GFP-E1alpha chimera is specific to mitochondria and results in two different fluorescence patterns. These patterns have been distinguished by immunolabeling experiments using monoclonal antibodies against PDH subunits and GFP. The bright, localized fluorescent spots represent the assembled form of the GFP-E1alpha in PDH complexes. The uniform, dim fluorescence is given by the unassembled chimera free to diffuse throughout the mitochondrial reticulum. This study reveals a discrete, heterogeneous distribution of PDH complexes in the matrix of mitochondria, both in cells with normal and reduced levels of PDH. The uneven arrangement of PDH complexes is maintained over time and most likely reflects the structural and metabolic compartmentalization of mitochondria.

Solution conformation of 2-aminopurine dinucleotide determined by ultraviolet two-dimensional fluorescence spectroscopy

We have observed the conformation-dependent electronic coupling between the monomeric subunits of a dinucleotide of 2-aminopurine (2-AP), a fluorescent analog of the nucleic acid base adenine. This was accomplished by extending two-dimensional fluorescence spectroscopy (2D FS) - a fluorescence-detected variation of 2D electronic spectroscopy - to excite molecular transitions in the ultraviolet (UV) regime. A collinear sequence of four ultrafast laser pulses centered at 323 nm was used to resonantly excite the coupled transitions of 2-AP dinucleotide. The phases of the optical pulses were continuously swept at kilohertz frequencies, and the ensuing nonlinear fluorescence was phase-synchronously detected at 370 nm. Upon optimization of a point-dipole coupling model to our data, we found that in aqueous buffer the 2-AP dinucleotide adopts an average conformation in which the purine bases are non-helically stacked (center-to-center distance R12 = 3.5 Å ± 0.5 Å, twist angle θ12 = 5° ± 5°), which differs from the conformation of such adjacent bases in duplex DNA. These experiments establish UV-2D FS as a method for examining the local conformations of an adjacent pair of fluorescent nucleotides substituted into specific DNA or RNA constructs, which will serve as a powerful probe to interpret, in structural terms, biologically significant local conformational changes within the nucleic acid framework of protein-nucleic acid complexes.

Control of nuclear centration in the C. elegans zygote by receptor-independent Gα signaling and myosin II

Mitotic spindle positioning in the Caenorhabditis elegans zygote involves microtubule-dependent pulling forces applied to centrosomes. In this study, we investigate the role of actomyosin in centration, the movement of the nucleus–centrosome complex (NCC) to the cell center. We find that the rate of wild-type centration depends equally on the nonmuscle myosin II NMY-2 and the Gα proteins GOA-1/GPA-16. In centration- defective let-99(−) mutant zygotes, GOA-1/GPA-16 and NMY-2 act abnormally to oppose centration. This suggests that LET-99 determines the direction of a force on the NCC that is promoted by Gα signaling and actomyosin. During wild-type centration, NMY-2–GFP aggregates anterior to the NCC tend to move further anterior, suggesting that actomyosin contraction could pull the NCC. In GOA-1/GPA-16–depleted zygotes, NMY-2 aggregate displacement is reduced and largely randomized, whereas in a let-99(−) mutant, NMY-2 aggregates tend to make large posterior displacements. These results suggest that Gα signaling and LET-99 control centration by regulating polarized actomyosin contraction.

Compressed Sensing for Multidimensional Spectroscopy Experiments

Compressed sensing is a processing method that significantly reduces the number of measurements needed to accurately resolve signals in many fields of science and engineering. We develop a two-dimensional variant of compressed sensing for multidimensional spectroscopy and apply it to experimental data. For the model system of atomic rubidium vapor, we find that compressed sensing provides an order-of-magnitude (about 10-fold) improvement in spectral resolution along each dimension, as compared to a conventional discrete Fourier transform, using the same data set. More attractive is that compressed sensing allows for random undersampling of the experimental data, down to less than 5% of the experimental data set, with essentially no loss in spectral resolution. We believe that by combining powerful resolution with ease of use, compressed sensing can be a powerful tool for the analysis and interpretation of ultrafast spectroscopy data.