Abstract
In haemoglobin the change from the low-spin (LS) hexacoordinated haem to the high spin (HS, S = 2) pentacoordinated domed deoxy-myoglobin (deoxyMb) form upon ligand detachment from the haem ...and the reverse process upon ligand binding are what ultimately drives the respiratory function. Here we probe them in the case of Myoglobin-NO (MbNO) using element- and spin-sensitive femtosecond Fe K
α
and K
β
X-ray emission spectroscopy at an X-ray free-electron laser (FEL). We find that the change from the LS (S = 1/2) MbNO to the HS haem occurs in ~800 fs, and that it proceeds via an intermediate (S = 1) spin state. We also show that upon NO recombination, the return to the planar MbNO ground state is an electronic relaxation from HS to LS taking place in ~30 ps. Thus, the entire ligand dissociation-recombination cycle in MbNO is a spin cross-over followed by a reverse spin cross-over process.
The charge-carrier dynamics of anatase TiO2 nanoparticles in an aqueous
solution were studied by femtosecond time-resolved X-ray absorption spectroscopy using an
X-ray free electron laser in ...combination with a synchronized ultraviolet femtosecond laser
(268 nm). Using an arrival time monitor for the X-ray pulses, we obtained a temporal
resolution of 170 fs. The transient X-ray absorption spectra revealed an ultrafast Ti
K-edge shift and a subsequent growth of a pre-edge structure. The edge shift occurred in
ca. 100 fs and is ascribed to reduction of Ti by localization of generated conduction band
electrons into shallow traps of self-trapped polarons or deep traps at penta-coordinate Ti
sites. Growth of the pre-edge feature and reduction of the above-edge peak intensity occur
with similar time constants of 300–400 fs, which we assign to the structural distortion
dynamics near the surface.
Purely label-free imaging to directly monitor small molecules in a biological organism is still challenging despite recent technical advancements. Time-resolved pump–probe coherent Raman scattering ...microscopy is a promising label-free approach to increase chemical specificity. However, conventional time-resolved methods involve a compromise between three conflicting requirements: high spectral resolution, low background levels, and high sensitivity. Here, we present an advanced spectral-focusing technique using asymmetric pulses produced by nonlinear chirping and demonstrate its performance in pump–probe phase-modulated stimulated Raman scattering microscopy. In addition, we report for the first time a novel frequency-scanning spectral-focusing system using tunable bandpass filters. Our concept uses the filters not only as a frequency allocation tool for the probe pulses but also as a pulse-shaping tool that provides a strong nonlinear chirp. The spectral resolution and signal-to-noise ratio are greatly improved by highly efficient time-resolved detection using asymmetric spectrally focused probe pulses. We achieve a spectral resolution of ∼25 cm−1, a reduced nonresonant background level on the order of 10−8, and a detectable concentration limit of 0.01% dimethyl sulfoxide/water solution (1.5 mM). Using this method, we demonstrate high-contrast imaging of a small-molecule drug in a tissue. These advancements will allow time-resolved coherent Raman microscopy to be used as a practical drug-imaging tool for biomedical sciences.
Background
An assessment of the drug penetration and distribution profiles within the skin is essential in dermatology and cosmetology. Recent advances in label‐free imaging technologies have ...facilitated the direct detection of unlabeled compounds in tissues, with high resolution. However, it remains challenging to provide quantitative time‐course distribution maps of drugs within the complex skin tissue. The present study aims at acquiring the real‐time quantitative skin penetration profiles of topically applied caffeine, by means of a combination of pump–probe phase‐modulated stimulated Raman scattering (PM‐SRS) and confocal reflection microscopy. The recently developed PM‐SRS microscopy is a unique imaging tool that can minimize strong background signals through a pulse‐shaping technique, while providing high‐contrast images of small molecules in tissues.
Materials and methods
Reconstructed human skin epidermis models were used in order to analyze caffeine penetration in tissues. The penetration profiles of caffeine in an aqueous solution, an oil‐in‐water gel, and a water‐in‐oil gel were examined by combining PM‐SRS and confocal reflection microscopy.
Results
The characteristic Raman signal of caffeine was directly detected in the skin model using PM‐SRS. Integrating PM‐SRS and confocal reflection microscopy allowed real‐time concentration maps of caffeine to be obtained from formulation samples, within the skin model. Compared with the conventional Raman detection method, PM‐SRS lowered the background tissue‐oriented signals and supplied high‐contrast images of caffeine.
Conclusion
We successfully established real‐time skin penetration profiles of caffeine from different formulations. PM‐SRS microscopy proved to be a powerful, non‐invasive, and real‐time depth‐profile imaging technique for use in quantitative studies of topically applied drugs.
Skin penetration analysis of topically applied drugs or active compounds is essential in biomedical applications. Stimulated Raman scattering (SRS) microscopy is a promising label-free skin ...penetration analysis tool. However, conventional SRS microcopy suffers from limited signal contrast owing to strong background signals, which prevents its use in low-concentration drug imaging. Here, we present a skin penetration analysis method of topical agents using recently developed phase-modulated SRS (PM-SRS) microscopy. PM-SRS uses phase modulation and time-resolved signal detection to suppress both nonlinear background signals and Raman background signals from a tissue. A proof-of-concept experiment with a topically applied skin moisturizing agent (ectoine) in an in vitro skin tissue model revealed that PM-SRS with 1.7-ps probe delay yields a signal contrast 40 times higher than that of conventional amplitude-modulated SRS (AM-SRS). Skin penetration measurement of a topical therapeutic drug (loxoprofen sodium) showed that the mean drug concentration at the tissue surface layer after 240 min was 47.3 ± 4.8 mM. The proposed PM-SRS microscopy can be employed to monitor the spatial and temporal pharmacokinetics of small molecules in the millimolar concentration regime.
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