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Molecular Vibrational Imaging by Coherent Raman Scattering
Published in Shoogo Ueno, Bioimaging, 2020
Yasuyuki Ozeki, Hideaki Kano, Naoki Fukutake
Coherent Raman scattering microscopy including CARS microscopy and SRS microscopy allows for sensitive detection of molecular-vibrational signature of cells and tissues, and widens its range of applications to label-free imaging, metabolic imaging, and ultramultiplex or supermultiplex imaging. CARS microscopy can obtain broadband Raman spectra in the fingerprint region, and the CH-stretching and OH-stretching regions (>3000 cm−1), thus allowing for label-free imaging of cells and tissues with high specificity. SRS microscopy can be used for sensitive detection of vibrational signatures in a relatively narrow wavenumber region (~300 cm−1), allowing for high-speed label-free imaging of cells and tissues, as well as the monitoring of metabolic activities and multiplexed imaging with Raman-detectable labeling molecules.
Overview of Second- and Third-Order Nonlinear Optical Processes for Deep Imaging
Published in Lingyan Shi, Robert R. Alfano, Deep Imaging in Tissue and Biomedical Materials, 2017
Murugkar Sangeeta, Robert W. Boyd
Two-photon-excited fluorescence (TPEF) or two-photon microscopy has been extensively applied for biological imaging over the past couple of decades [2, 3]. In TPEF, a single femtosecond pulsed laser beam is tightly focused in the specimen such that two low energy, near-IR photons are simultaneously absorbed by a fluorophore and then emitted as one photon at a higher frequency than the incident light. Along with TPEF, nonlinear optical imaging techniques such as second harmonic generation (SHG) and coherent Raman scattering (CRS) have experienced continued growth in the technology and applications over the past decade. In SHG, two photons with the same frequency interacting with a nonlinear optical material are effectively “combined” to generate new photons at twice the frequency of the incident light [4]. CRS, which refers to both stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS), is a technique to enhance spontaneous Raman scattering. The spontaneous Raman effect involves incoherent excitation of molecular vibrations. It is inherently weak in nature with the typical photon conversion efficiencies for Raman being lower than 1 in 1018. This results in long data acquisition times of 100 ms to 1 s per pixel and does not permit fast chemical imaging of a living system [5]. In contrast, CRS involves nonlinear Raman scattering such that molecular vibrations are driven coherently, in phase through stimulated excitation by two synchronized femtosecond (or picosecond) pulsed lasers. This translates to a reduction of the image acquisition time from many minutes or hours required in confocal Raman microscopy to only a couple of seconds using coherent Raman scattering microscopy [6, 7]. CARS and SRS have been shown to be promising techniques for the chemically selective imaging of lipids, proteins, and DNA in skin, brain, and lung tissue. TPEF and SHG enable selective imaging of auto-fluorescent proteins in tissue and of noncentrosymmetric structures such as fibrillar collagen, respectively [8]. We note that in many of these studies the illumination method of choice is one based on the nonlinear optical process of supercontinuum generation (SCG).
Oxybutynin nanosuspension gel for enhanced transdermal treatment for overactive bladder syndrome
Published in Pharmaceutical Development and Technology, 2022
Yuze Sheng, Shuang Zhang, Jiawei Ling, Chenlu Hu, Zhenhai Zhang, Huixia Lv
However, there was still an illogical phenomenon uninterpreted that the retention amount of OXY in SC of OXY-NG group had no statistical difference with that of OXY-CG group at 24 h. For this, an assumption was proposed that the most of intact OXY-NS could hardly penetrate into the SC or just penetrate into several outermost layers of cells, not the whole SC, but in the form of free OXY molecules. It has been reported by R. Alvarez-Román that visualization of the distribution of fluorescent polystyrene nanoparticles, diameters 20 and 200 nm, across porcine skin revealed that polystyrene nanoparticles were limited to the surface of the SC without penetration through it, accumulating in the follicle preferentially (Alvarez-Roman et al. 2004). Polymeric nanoparticles ranging in diameter from 20 to 200 nm were observed to penetrate only into the surface layers with a depth of 2–3 μm approximately of the SC, which was possibly due to the infiltration along fissures in the stratum disjunctum and no time-dependent penetration of nanoparticles was observed, even when the SC was tape-stripped (Campbell et al. 2012). Confocal laser scanning microscopy (CLSM) imaging showed that a portion of curcumin hybrid nanosuspensions could only penetrate into the SC instead of deeper layers but were easier to fill in hair follicles (Shi et al. 2020). It is common sense that SC is composed of corneocytes filled with keratin and keratin-filaggrin complex and intercorneocyte lipids assembled in parallel head to head and tail to tail, producing 5–7 nm lipophilic pathway and 3 nm hydrophilic route (Baroli 2010). By contrast, the separation of corneocyte clusters with a few micronmeters in distance and hair follicles were more attractive places for OXY-NS. It is deserved to figure out the distribution and penetration depth of OXY-NS in skin. So far, distinguishing intact nanoparticles and free drugs was still a tough task. Recently, an aggregation-caused quenching (ACQ) probe has been utilized to explore the fate of nanoparticles in vivo, which is a potential strategy despite the potential occurrence of false positives (Ma et al. 2017; Shen C et al. 2017; Qi et al. 2019; Shi et al. 2020). Our following work is to probe how deep can intact OXY-NS penetrate into skin by Coherent Raman Scattering Microscopy.
Site-specific drug delivery in the skin for the localized treatment of skin diseases
Published in Expert Opinion on Drug Delivery, 2019
Yang Chen, Xun Feng, Shengnan Meng
The vasoconstrictor assay has been recommended by the FDA for topically applied glucocorticoids. For all other topical drug products whose rate-limiting penetration barrier is the SC and the concentration in the underlying epidermis is related to that in the SC, the DPK/tape striping method has been proposed to measure topical bioavailability/bioequivalence study. By quantifying the drug content on each tape strip and determining the SC depth via total epidermal water loss measurements, the parameters including K (the SC-vehicle partition coefficient of the drug) and D/L2 (a first-order rate constant for drug transport through the SC) can be determined according to the Fick’s second law. They can be used to calculate the Cmax (the maximum concentration of drug in the skin), Tmax (the time at which Cmax is achieved), and area under the concentration–time curve (AUC) [213]. Different from DPK, the microdialysis is an in vivo sampling technique that can allows real-time and continuous monitoring of the extracellular concentration of drugs and their metabolites in the dermis and hypodermis, which is more invasive and suitable for the quantification of drugs in the deeper skin layers. It can be used in the case of skin barrier perturbation or skin disease, and is independent of the penetration route. The major disadvantages with this method lie in the variability in probe insertion and probe manufacture, as well as in the limited use with lipophilic and highly protein-bound drugs. Despite that, microdialysis is expected to gain more recognition among different regulatory authorities for future topical drug research [214]. In addition, imaging techniques such as confocal scanning microscopy are also important for the investigation of drug delivery in the skin. It can facilitate the distribution and penetration of drugs in deeper skin layers, and also visualize the route of skin penetration. However, this method requires the use of dye to substitute the non-fluorescent drug to investigate the penetration behavior, which may not reflect the actual scenario of drug penetration [215]. In contrast to confocal scanning microscopy, coherent Raman scattering microscopy was recently developed to provide label-free analysis with a high spatial and temporal resolution for dermal drug delivery [216], and the confocal Raman spectroscopy has also been investigated for its ability to probe drug disposition in the skin [217]. In addition, novel imaging technologies based on mass spectrometry, e.g. matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) are also explored for the determination of drug distribution profiles in the skin [218].