Ultrafast Vibrational Spectroscopy of the Eumelanin pigment

<p>Solar ultraviolet (UV) radiation is a highly toxic carcinogen prevalent in our environment. Eumelanin pigment is a photo-stable biopolymer naturally produced in the skin's pigmentary system, providing the skin a unique photo-protection mechanism against exposure to UV radiation. The large macro-molecule rapidly dissipates 99 % of incident UV photons as thermal energy on ultra-fast femtosecond (fs) - picosecond (ps) time scales, before damage can occur to the underlying cells. The fundamental nature of eumelanin's structure and its vibrational energy dissipation mechanism is not yet fully understood, with complexities in the molecule's highly disordered chemical structure, and the ultrafast time-scale on which the energy dissipation occurs, rendering its characterisation elusive. </p><p><br></p> <p>Indeed, the absorption spectrum of eumelanin gives little away, rising monotonically in wavelength towards the UV - quite unusual in organic polymers. It is proposed that due to the highly disordered structure of the molecule, multiple chromophores of overlapping energies may be selectively excited with differing irradiation wavelengths. This theory is further supported by eumelanin’s transient absorption signatures and its wavelength dependant photo-luminescence spectra, however the fundamental non-radiative relaxation pathways are not yet understood. </p> <p><br></p><p>To bridge this knowledge gap, we present here the application of femtosecond stimulated Raman spectroscopy (FSRS) to eumelanin pigment. FSRS reveals ultra-fast vibrational dynamics on fs – ps time scales, allowing excited state vibrational pathways to be mapped, providing essential structural information of this intriguing molecule. </p> <p><br></p><p>Following the introduction of eumelanin’s known photo-physical and structural properties as presented in chapter 1, an introduction to FSRS is presented in chapter 2. The build method and optical construction of the FSRS experiment are presented in chapter 4 including a novel bandwidth compression method used to generate a narrow-band Raman pump using frequency-domain nonlinear optics, presented in chapter 3. Here, using a 1.5 kHz, 800 nm Ti:Sapphire pulsed laser at a power of 3 W, conversion efficiencies of up to 30 % are achieved, generating intense second harmonic Raman pump pulses centred at 400 nm with <20 cm-1 bandwidths. Additionally, the well-known spatial filtering technique is used to generate tuneable, narrow-band pulses centered at the fundamental 800 nm laser pulse. </p> <p><br></p><p>The application of FSRS to the study of eumelanin’s indole subunits, 5-6-dihydroxyindole (DHI) and its carboxylated form, 5-6-dihydroxyindole-2-carboxylic acid (DHICA) (and their oligomers) are presented in chapter 5. These studies provide direct evidence for excited state proton transfer (ESPT) in DHICA, and a reference for the interpretation of the complex eumelanin macro-molecule, as well as its dynamic vibrational signatures, which are discussed in chapter 6. Here, vibrational dynamics are resolved on From these FSRS studies, ESPT – proposed as a relaxation pathway in eumelanin’s subunit DHICA – is demonstrated. Direct vibrational relaxation measurement mapped using density functional theory (DFT) provides strong evidence of excited state de-activation of DHICA via this mechanism. Further, eumelanin’s vibrational mode deactivation pathways are presented, providing evidence of specific mode excitations of the macro-molecule upon photo-excitation in both the UV (267 nm) and visible (400 nm) regions. </p><p><br></p><p>Resolving eumelanin’s excited state vibrational modes and kinetics using FSRS provides dynamic structural information of eumelanin's thermal energy transfer system, including that of its indole subunits. Determining FSRS signatures as a function of excitation wavelength in both the visible and UV regions, reveals insights into this highly efficient UV absorbing material. </p>

Ultrafast Vibrational Spectroscopy of the Eumelanin pigment

<p>Solar ultraviolet (UV) radiation is a highly toxic carcinogen prevalent in our environment. Eumelanin pigment is a photo-stable biopolymer naturally produced in the skin's pigmentary system, providing the skin a unique photo-protection mechanism against exposure to UV radiation. The large macro-molecule rapidly dissipates 99 % of incident UV photons as thermal energy on ultra-fast femtosecond (fs) - picosecond (ps) time scales, before damage can occur to the underlying cells. The fundamental nature of eumelanin's structure and its vibrational energy dissipation mechanism is not yet fully understood, with complexities in the molecule's highly disordered chemical structure, and the ultrafast time-scale on which the energy dissipation occurs, rendering its characterisation elusive. </p><p><br></p> <p>Indeed, the absorption spectrum of eumelanin gives little away, rising monotonically in wavelength towards the UV - quite unusual in organic polymers. It is proposed that due to the highly disordered structure of the molecule, multiple chromophores of overlapping energies may be selectively excited with differing irradiation wavelengths. This theory is further supported by eumelanin’s transient absorption signatures and its wavelength dependant photo-luminescence spectra, however the fundamental non-radiative relaxation pathways are not yet understood. </p> <p><br></p><p>To bridge this knowledge gap, we present here the application of femtosecond stimulated Raman spectroscopy (FSRS) to eumelanin pigment. FSRS reveals ultra-fast vibrational dynamics on fs – ps time scales, allowing excited state vibrational pathways to be mapped, providing essential structural information of this intriguing molecule. </p> <p><br></p><p>Following the introduction of eumelanin’s known photo-physical and structural properties as presented in chapter 1, an introduction to FSRS is presented in chapter 2. The build method and optical construction of the FSRS experiment are presented in chapter 4 including a novel bandwidth compression method used to generate a narrow-band Raman pump using frequency-domain nonlinear optics, presented in chapter 3. Here, using a 1.5 kHz, 800 nm Ti:Sapphire pulsed laser at a power of 3 W, conversion efficiencies of up to 30 % are achieved, generating intense second harmonic Raman pump pulses centred at 400 nm with <20 cm-1 bandwidths. Additionally, the well-known spatial filtering technique is used to generate tuneable, narrow-band pulses centered at the fundamental 800 nm laser pulse. </p> <p><br></p><p>The application of FSRS to the study of eumelanin’s indole subunits, 5-6-dihydroxyindole (DHI) and its carboxylated form, 5-6-dihydroxyindole-2-carboxylic acid (DHICA) (and their oligomers) are presented in chapter 5. These studies provide direct evidence for excited state proton transfer (ESPT) in DHICA, and a reference for the interpretation of the complex eumelanin macro-molecule, as well as its dynamic vibrational signatures, which are discussed in chapter 6. Here, vibrational dynamics are resolved on From these FSRS studies, ESPT – proposed as a relaxation pathway in eumelanin’s subunit DHICA – is demonstrated. Direct vibrational relaxation measurement mapped using density functional theory (DFT) provides strong evidence of excited state de-activation of DHICA via this mechanism. Further, eumelanin’s vibrational mode deactivation pathways are presented, providing evidence of specific mode excitations of the macro-molecule upon photo-excitation in both the UV (267 nm) and visible (400 nm) regions. </p><p><br></p><p>Resolving eumelanin’s excited state vibrational modes and kinetics using FSRS provides dynamic structural information of eumelanin's thermal energy transfer system, including that of its indole subunits. Determining FSRS signatures as a function of excitation wavelength in both the visible and UV regions, reveals insights into this highly efficient UV absorbing material. </p>

Improved spectral resolution of the femtosecond stimulated Raman spectroscopy achieved by the use of the 2nd-order diffraction method

Prolongation of the picosecond Raman pump laser pulse in the femtosecond stimulated Raman spectroscopy (FSRS) setup is essential for achieving the high spectral resolution of the time-resolved vibrational Raman spectra. In this work, the 2nd-order diffraction has been firstly employed in the double-pass grating filter technique for realizing the FSRS setup with the sub-5 cm−1 spectral resolution. It has been experimentally demonstrated that our new FSRS setup gives rise to a highly-resolved Raman spectrum of the excited trans-stilbene, which is much improved from those reported in the literatures. The spectral resolution of the present FSRS system has been estimated to be the lowest value ever reported to date, giving Δν = 2.5 cm−1.

Erbium-Doped Fiber Amplification Assisted Multi-Wavelength Brillouin-Raman Fiber Laser

Multi-wavelength fiber laser based on Brillouin scattering in optical fiber has the potential of application in dense wavelength division multiplexing (DWDM) system. To enhance the performance of the fiber lasers, researchers proposed usages of erbium, or Raman amplification techniques. In an earlier work, it was reported that extracting residual Raman pump out of the laser cavity improves the performance of a multi-wavelength Raman fiber laser. In this paper, we proposed a setup utilizing the residual Raman pump to pump an erbium-doped fiber in multi-wavelength Brillouin-Raman fiber laser. Results show that the additional erbium-doped fiber is capable of amplifying the propagating Brillouin Stokes by more than 15-dB. This in turn helps in achieving lower stimulated Brillouin threshold and subsequently allow for higher number of Brillouin Stokes lines to be generated.

Excited State Structural Evolution of a GFP Single-Site Mutant Tracked by Tunable Femtosecond-Stimulated Raman Spectroscopy

Tracking vibrational motions during a photochemical or photophysical process has gained momentum, due to its sensitivity to the progression of reaction and change of environment. In this work, we implemented an advanced ultrafast vibrational technique, femtosecond-stimulated Raman spectroscopy (FSRS), to monitor the excited state structural evolution of an engineered green fluorescent protein (GFP) single-site mutant S205V. This mutation alters the original excited state proton transfer (ESPT) chain. By strategically tuning the Raman pump to different wavelengths (i.e., 801, 539, and 504 nm) to achieve pre-resonance with transient excited state electronic bands, the characteristic Raman modes of the excited protonated (A*) chromophore species and intermediate deprotonated (I*) species can be selectively monitored. The inhomogeneous distribution/population of A* species go through ESPT with a similar ~300 ps time constant, confirming that bridging a water molecule to protein residue T203 in the ESPT chain is the rate-limiting step. Some A* species undergo vibrational cooling through high-frequency motions on the ~190 ps time scale. At early times, a portion of the largely protonated A* species could also undergo vibrational cooling or return to the ground state with a ~80 ps time constant. On the photoproduct side, a ~1330 cm−1 delocalized motion is observed, with dispersive line shapes in both the Stokes and anti-Stokes FSRS with a pre-resonance Raman pump, which indicates strong vibronic coupling, as the mode could facilitate the I* species to reach a relatively stable state (e.g., the main fluorescent state) after conversion from A*. Our findings disentangle the contributions of various vibrational motions active during the ESPT reaction, and offer new structural dynamics insights into the fluorescence mechanisms of engineered GFPs and other analogous autofluorescent proteins.