Super-resolution vibrational imaging based on photoswitchable Raman probe.

Super-resolution vibrational microscopy is promising to increase the degree of multiplexing of nanometer-scale biological imaging because of the narrower spectral linewidth of molecular vibration compared to fluorescence. However, current techniques of super-resolution vibrational microscopy suffer from various limitations including the need for cell fixation, high power loading, or complicated detection schemes. Here, we present reversible saturable optical Raman transitions (RESORT) microscopy, which overcomes these limitations by using photoswitchable stimulated Raman scattering (SRS). We first describe a bright photoswitchable Raman probe (DAE620) and validate its signal activation and depletion characteristics when exposed to low-power (microwatt level) continuous-wave laser light. By harnessing the SRS signal depletion of DAE620 through a donut-shaped beam, we demonstrate super-resolution vibrational imaging of mammalian cells with excellent chemical specificity and spatial resolution beyond the optical diffraction limit. Our results indicate RESORT microscopy to be an effective tool with high potential for multiplexed super-resolution imaging of live cells.

Three-Dimensional Analysis of Water Dynamics in Human Skin by Stimulated Raman Scattering.

The stratum corneum (SC), the outermost layer of the skin, has an important function to provide a barrier against dry environments. To evaluate the barrier function and the skin condition, it is crucial to investigate the ability of SC to absorb and retain water. In this study, we demonstrate stimulated Raman scattering (SRS) imaging of three-dimensional SC structure and water distribution when water is absorbed into dried SC sheets. Our results show that the process of water absorption and retention is dependent on the specific sample and can be spatially heterogeneous. We also found that acetone treatment leads to spatially homogeneous retention of water. These results suggest the great potential of SRS imaging in diagnosing skin conditions.

Imaging the uptake of deuterated methionine in Drosophila with stimulated Raman scattering.

Introduction: Visualizing small individual biomolecules at subcellular resolution in live cells and tissues can provide valuable insights into metabolic activity in heterogeneous cells, but is challenging. Methods: Here, we used stimulated Raman scattering (SRS) microscopy to image deuterated methionine (d-Met) incorporated into Drosophila tissues in vivo. Results: Our results demonstrate that SRS can detect a range of previously uncharacterized cell-to-cell differences in d-Met distribution within a tissue at the subcellular level. Discussion: These results demonstrate the potential of SRS microscopy for metabolic imaging of less abundant but important amino acids such as methionine in tissue.

Preparation of pH-sensitive anionic liposomes designed for drug delivery system (DDS) application.

We prepared pH-sensitive anionic liposomes composed solely of anionic bilayer membrane components that were designed to promote efficient release of entrapped agents in response to acidic pH. The pH-sensitive anionic liposomes showed high dispersion stability at neutral pH, but the fluidity of the bilayer membrane was enhanced in an acidic environment. These liposomes were rather simple and were composed of dimyristoylphosphatidylcholine (DMPC), an anionic bilayer membrane component, and polyoxyethylene sorbitan monostearate (Tween 80). In particular, the present pH-sensitive anionic liposomes showed higher temporal stability than those of conventional DMPC/DPPC liposomes. We found that pHsensitive properties strongly depended on the molecular structure, pKa value, and amount of an incorporated anionic bilayer membrane component, such as sodium oleate (SO), dimyristoylphosphatidylserine (DMPS), or sodium ß-sitosterol sulfate (SS). These results provide an opportunity to manipulate liposomal stability in a pH-dependent manner, which could lead to the formulation of a high performance drug delivery system (DDS).