Capturing Structural Snapshots during Photochemical Reactions with Ultrafast Raman Spectroscopy: From Materials Transformation to Biosensor Responses

Chemistry studies the composition, structure, properties, and transformation of matter. A mechanistic understanding of the pertinent processes is required to translate fundamental knowledge into practical applications. The current development of ultrafast Raman as a powerful time-resolved vibrational technique, particularly femtosecond stimulated Raman spectroscopy (FSRS), has shed light on the structure–energy–function relationships of various photosensitive systems. This Perspective reviews recent work incorporating optical innovations, including the broad-band up-converted multicolor array (BUMA) into a tunable FSRS setup, and demonstrates its resolving power to watch metal speciation and photolysis, leading to high-quality thin films, and fluorescence modulation of chimeric protein biosensors for calcium ion imaging. We discuss advantages of performing FSRS in the mixed time–frequency domain and present strategies to delineate mechanisms by tracking low-frequency modes and systematically modifying chemical structures with specific functional groups. These unique insights at the chemical-bond level have started to enable the rational design and precise control of functional molecular machines in optical, materials, energy, and life sciences

Correlated Molecular Structural Motions for Photoprotection after Deep-UV Irradiation

Exposure to ultraviolet (UV) light could cause photodamage to biomolecular systems and degrade optoelectronic devices. To mitigate such detrimental effects from the bottom up, we strategically select a photosensitive molecule pyranine and implement femtosecond electronic and Raman spectroscopies to elucidate its ultrafast photoprotection mechanisms in solution. Our results show that pyranine undergoes excited-state proton transfer in water, while this process is blocked in methanol regardless of excitation wavelengths (267, 400 nm). After 267 nm irradiation, the molecule relaxes from a higher lying electronic state into a lower lying singlet state with a <300 fs time constant, followed by solvation events. Transient Raman marker bands exhibit different patterns of intensity dynamics and frequency shift that elucidate the real-time interplay among conformational motions, photochemical reaction, and vibrational cooling after excitation. More energetic photons are revealed to selectively enhance certain relaxation pathways. These mechanistic findings offer new guidelines to improve the UV tolerance and stability of the engineered functional molecules in materials and life sciences

Correlated Molecular Structural Motions for Photoprotection after Deep-UV Irradiation

Exposure to ultraviolet (UV) light could cause photodamage to biomolecular systems and degrade optoelectronic devices. To mitigate such detrimental effects from the bottom up, we strategically select a photosensitive molecule pyranine and implement femtosecond electronic and Raman spectroscopies to elucidate its ultrafast photoprotection mechanisms in solution. Our results show that pyranine undergoes excited-state proton transfer in water, while this process is blocked in methanol regardless of excitation wavelengths (267, 400 nm). After 267 nm irradiation, the molecule relaxes from a higher lying electronic state into a lower lying singlet state with a <300 fs time constant, followed by solvation events. Transient Raman marker bands exhibit different patterns of intensity dynamics and frequency shift that elucidate the real-time interplay among conformational motions, photochemical reaction, and vibrational cooling after excitation. More energetic photons are revealed to selectively enhance certain relaxation pathways. These mechanistic findings offer new guidelines to improve the UV tolerance and stability of the engineered functional molecules in materials and life sciences

Antibiotic susceptibility of <i>B. subtilis</i> strains.

a<p>Minimal inhibitory concentrations (MIC) determined from three biological replicates; where a range of concentrations is given, results varied between replicates.</p

VanS_B-VanR_B-dependent induction of P<i>_vanYB</i> by vancomycin in <i>B. subtilis</i>.

<p>Both the P<i>_vanYB</i>-<i>lux</i> reporter construct pCF133 and the P<i>_xylA</i>-<i>vanR_BS_B</i> expression construct pCF132 were introduced into <i>B. subtilis</i> strain TMB1518 (unmarked deletion of <i>bceRS-bceAB</i>, <i>psdRS-psdAB</i>, <i>yxdJK-yxdLM-yxeA</i>). Cultures growing exponentially either (A) in the absence of xylose or (B) in the presence of 0.2% (w/v) xylose were challenged at t = 0 min with 0.01 μg ml⁻¹ (open squares), 0.025 μg ml⁻¹ (grey circles), 0.05 μg ml⁻¹ (solid circles), 0.25 μg ml⁻¹ (solid squares) vancomycin, or left untreated (open circles). Luminescence normalized to optical density (RLU/OD) was monitored over 60 min. Results are shown as the mean and standard deviation of three biological replicates.</p

Functionality of the reporter systems on solid media.

<p>Strains of <i>B. subtilis</i> harbouring the P<i>_bcrA</i>-<i>lacZ</i> reporter (A and B) or the P<i>_bcrA</i>-<i>luxABCDE</i> reporter (C and D) were grown on agar plates containing 0 μg ml⁻¹ (A and C), 0.3 μg ml⁻¹ (D) or 1 μg ml⁻¹ (B) bacitracin. (A, B) Blue colouration due to reporter induction is depicted by the dark grey shading of bacterial growth. Sector 1, SGB40 (BcrR⁺, BceAB⁺); sector 2, SGB36 (BcrR⁺, BcrAB⁺); sector 3, SGB43 (BcrR⁺); sector 4, SGB42 (BcrR⁺, BceAB⁺, BcrAB⁺). Plates contained 200 μg ml⁻¹ X-Gal. (C, D) Plates inoculated with SGB237 (BcrR⁺) were photographed under white light (left sub-panels), followed by detection of luminescence in the dark (right sub-panels); the same sector of the agar plates is shown in both sub-panels.</p

Plasmids and strains used in this study.

a<p>Bac, bacitracin; cm, chloramphenicol; fs, fusidic acid; kan, kanamycin; mls, macrolide-lincosamide-streptogramin B group antibiotics; rif, rifampin; spc, spectinomycin; van, vancomycin; r, resistant.</p

Primers used in this study.

a<p>Restriction sites are underlined; overlaps to other primers for PCR fusions are shown by lower case letters.</p

Monitoring Photochemical Reaction Pathways of Tungsten Hexacarbonyl in Solution from Femtoseconds to Minutes

Metal–organic complexes are widely used across disciplines for energy and biological applications, however, their photophysical and photochemical reaction coordinates remain unclear in solution due to pertaining molecular motions on ultrafast time scales. In this study, we apply transient absorption and tunable femtosecond stimulated Raman spectroscopy (FSRS) to investigate the UV photolysis of tungsten hexacarbonyl and subsequent solvent binding events. On the macroscopic time scale with UV lamp irradiation, no equilibrated intermediate is observed from W­(CO)₆ to W­(CO)₅(solvent), corroborated by vibrational normal mode calculations. Upon 267 nm femtosecond laser irradiation, the excited-state absorption band within ∼400500 nm exhibits distinct dynamics in methanol, tetrahydrofuran, and acetonitrile on molecular time scales. In methanol, solvation of the nascent pentacarbonyl–solvent complex occurs in ∼8 ps and in tetrahydrofuran, 13 ps which potentially involves the associative oxygen-donating ligand rearrangement reaction. In contrast, a stimulated emission feature above 480 nm emerges after ∼1 ps in acetonitrile with a nitrogen-donating ligand. These structural dynamics insights demonstrate the combined resolving power of ultrafast electronic and stimulated Raman spectroscopy to elucidate photochemistry of functional organometallic complexes in solution. The delineated reaction pathways in relation to ligand nucleophilicity and solvent reorientation time provide the rational design principles for solution precursors in nanowrite applications

BcrR-dependent induction of P<i>_bcrA</i> by bacitracin in <i>B. subtilis</i>.

<p>The P<i>_bcrA</i>-<i>lacZ</i> reporter construct pES601 was introduced into different strains of <i>B. subtilis</i> producing either BcrR or BcrR and BcrAB. The relevant genes for bacitracin transporters in each strain are given at the top right of each graph. (A) SGB40; wild-type (WT) <i>B. subtilis</i> with BcrR. (B) SGB43; <i>bceAB</i>::kan mutant with BcrR. (C) SGB36; <i>bceAB</i>::kan mutant with BcrR and BcrAB. (D) SGB42; wild-type <i>B. subtilis</i> with BcrR and BcrAB. Cultures growing exponentially in the presence of 0.2% (w/v) xylose were challenged with different concentrations of bacitracin as indicated for 30 min, and β-galactosidase activities, expressed in Miller Units (MU), were determined. Results are shown as the mean plus standard deviation of three to four biological replicates.</p