Flash Memory: Photochemical Imprinting of Neuronal Action Potentials onto a Microbial Rhodopsin

We developed a technique, “flash memory”, to record a photochemical imprint of the activity state—firing or not firing—of a neuron at a user-selected moment in time. The key element is an engineered microbial rhodopsin protein with three states. Two nonfluorescent states, D1 and D2, exist in a voltage-dependent equilibrium. A stable fluorescent state, F, is reached by a photochemical conversion from D2. When exposed to light of a wavelength λwrite, population transfers from D2 to F, at a rate determined by the D1 ⇌ D2 equilibrium. The population of F maintains a record of membrane voltage which persists in the dark. Illumination at a later time at a wavelength λread excites fluorescence of F, probing this record. An optional third flash at a wavelength λreset converts F back to D2, for a subsequent write–read cycle. The flash memory method offers the promise to decouple the recording of neural activity from its readout. In principle, the technique may enable one to generate snapshots of neural activity in a large volume of neural tissue, e.g., a complete mouse brain, by circumventing the challenge of imaging a large volume with simultaneous high spatial and high temporal resolution. The proof-of-principle flash memory sensors presented here will need improvements in sensitivity, speed, brightness, and membrane trafficking before this goal can be realized

Bright and Fast Multicoloured Voltage Reporters via Electrochromic FRET

Genetically encoded fluorescent reporters of membrane potential promise to reveal aspects of neural function not detectable by other means. We present a palette of multicoloured brightly fluorescent genetically encoded voltage indicators with sensitivities from 8–13% ΔF/F per 100 mV, and half-maximal response times from 4–7 ms. A fluorescent protein is fused to an archaerhodopsin-derived voltage sensor. Voltage-induced shifts in the absorption spectrum of the rhodopsin lead to voltage-dependent nonradiative quenching of the appended fluorescent protein. Through a library screen, we identify linkers and fluorescent protein combinations that report neuronal action potentials in cultured rat hippocampal neurons with a single-trial signal-to-noise ratio from 7 to 9 in a 1 kHz imaging bandwidth at modest illumination intensity. The freedom to choose a voltage indicator from an array of colours facilitates multicolour voltage imaging, as well as combination with other optical reporters and optogenetic actuators.Chemistry and Chemical BiologyEngineering and Applied SciencesPhysic

Femtosecond Coherence and Quantum Control of Single Molecules at Room Temperature

Quantum mechanical phenomena, such as electronic coherence and entanglement, play a key role in achieving the unrivalled efficiencies of light-energy conversion in natural photosynthetic light-harvesting complexes, and triggered the growing interest in the possibility of organic quantum computing. Since biological systems are intrinsically heterogeneous, clear relations between structural and quantum-mechanical properties can only be obtained by investigating individual assemblies. However, single-molecule techniques to access ultrafast coherences at physiological conditions were not available so far. Here we show by employing femtosecond pulse-shaping techniques that quantum coherences in single organic molecules can be created, probed, and manipulated at ambient conditions even in highly disordered solid environments. We find broadly distributed coherence decay times for different individual molecules giving direct insight into the structural heterogeneity of the local surroundings. Most importantly, we induce Rabi-oscillations and control the coherent superposition state in a single molecule, thus performing a basic femtosecond single-qubit operation at room temperature

Nanoscale Coherent Control. Ultrafast dynamics of single molecules, individual light harvesting complexes and discrete nanoantennas at room temperature.

Ultrafast pulses allow observation of molecular dynamics with femtosecond time resolution through pump probe experiments. However, averaging over an ensemble of molecules tends to wash out phase sensitive information, necessary to probe quantum effects, due to the intrinsic inhomogeneity in molecular conformations, orientations and interactions that lead to unique potential energy landscapes for each molecule. It is therefore important to go beyond the ensemble average when looking at quantum dynamics of organic systems at room temperature, and resolve the behaviour of specific molecules on an individual basis. In this thesis, we show the creation, manipulation and observation of ultrafast coherent effects in single molecules at room temperature, and resolve a certain measure of environmental influence on the specific dynamics of each molecule. Moreover, we apply this insight to investigate a functional light harvesting biosystem, and lay the basis for a technique that has the time and space resolution required to observe these systems in vivo. In chapter 1, we introduce the concepts and techniques the research in this thesis is built on. In chapter 2, we treat the possibility of controlling ultrafast pulses at the high-NA diffraction limit, and come to conclusions about the procedure to follow there that hold for all pulse-shaping experiments. We show in proof of principle experiments that we can control the ultrafast characteristics of optical pulses in nanometric excitation volumes. In chapters 3 and 4 we report the creation, detection and control of ultrafast quantum dynamics in single organic molecules at room temperature. We show that manipulation of superposition states is possible in these systems within a coherence dephasing time of ~50 fs. This leads to the first observation of rabi-oscillations in room temperature single molecules, to ultrafast operation of an organic qubit, and to the creation of non-stationary superposition states (vibrational wavepackets). We probe the influence of the local environment on the composition and dynamics of these wavepackets and show we can optimize the state preparation protocol for each individual molecule in its own nanoenvironment, leading to high fidelity coherent control. In these chapters we lay out the proof of principle work of detecting the quantumdynamics of a complex system in interaction with its environment at room temperature. In chapter 5 we discuss application of these techniques to the investigation of long lived coherence in photosynthetic systems. We show that electronic coherence between different rings of the LH2 system persists to time scales of 100s of femtoseconds at room temperature. Moreover we show that the energy transfer pathways in LH2 adapt to environmentally induced changes in the molecule and that the nature of the transfer remains coherent for each pathway, providing strong evidence that coherent energy transfer is the optimum process for energy transfer in photosynthesis. Finally, in chapter 6 we take the technical development one step further and report on the creation of a framework based on plasmonic antennas that allows for control of the amplitude-phase characteristics in nanometric sized hotspot fields. We show for the first time that the ultrafast characteristics of plasmonic hotspots can directly be engineered through design of the plasmonic system and experimentally demonstrate two much-anticipated examples: a sub-diffraction resolution phase shaper and an ultrafast plasmonic switch for pump probe experiments. The results presented in this thesis form the first creation and observation of ultrafast coherent dynamics in individual molecular systems at room temperature. This is a necessary step to be able to do true quantum tomography in complex systems, resolve the influence of the environment on molecular dynamics, and investigate the physics that determines evolutionary optimization and functionality in biomolecules