Search

The dopaminergic system is essential for the function of the brain in health and disease. Therefore, detailed studies focused on unraveling the mechanisms involved in dopaminergic signaling are required. However, the lack of probes that mimic dopamine in living tissues, owing to the neurotransmitter's small size, has hampered analysis of the dopaminergic system. The current study aimed to overcome this limitation by developing alkyne-tagged dopamine compounds (ATDAs) that have a minimally invasive and uniquely identifiable alkyne group as a tag. ATDAs were established as chemically and functionally similar to dopamine and readily detectable by methods such as specific click chemistry and Raman scattering. The ATDAs developed here were verified as analogue probes that mimic dopamine in neurons and brain tissues, allowing the detailed characterization of dopamine dynamics. Therefore, ATDAs can act as safe and versatile tools with wide applicability in detailed studies of the dopaminergic system. Furthermore, our results suggest that the alkyne-tagging approach can also be applied to other small-sized neurotransmitters to facilitate characterization of their dynamics in the brain.

Imaging of biological cells and tissues with subcellular spatial resolution is important in biology and medicine because it allows us to explore the dynamics of cells and to diagnose the structure of tissues. Among various optical imaging modalities, laser microscopy with fluorescent staining is a powerful method for this purpose. However, it still suffers from the limited applicability and cytotoxicity of a staining process. Stimulated Raman scattering (SRS) microscopy is an emerging technique of biological imaging based on molecular vibrational contrast, offering new opportunities of biomedical microscopy. In this paper, we introduce the principle and applications of SRS microscopy and discuss various methods of spectral imaging with SRS microscopy. Then, we present our multicolor SRS microscope, which employs a fast wavelength-tunable picosecond Yb fiber laser and a picosecond Ti:sapphire laser. In particular, we describe the technical details of the Yb fiber oscillator, synchronization electronics, high-speed wavelength-tunable filter, video-rate microscope setup and signal detection electronics including the photodetector and the lock-in amplifier. We hope that this paper will be informative for those interested in the application of ultrafast lasers to coherent Raman microscopy including SRS microscopy.

Epidermal keratinocyte (KC) differentiation, which involves the process from proliferation to cell death for shedding the outermost layer of skin, is crucial for the barrier function of skin. Therefore, in dermatology, it is important to elucidate the epidermal KC differentiation process to evaluate the symptom level of diseases and skin conditions. Previous dermatological studies used staining or labelling techniques for this purpose, but they have technological limitations for revealing the entire process of epidermal KC differentiation, especially when applied to humans. Here, we demonstrate label-free visualization of three-dimensional (3D) intracellular morphological changes of ex vivo human epidermis during epidermal KC differentiation using stimulated Raman scattering (SRS) microscopy. Specifically, we observed changes in nuclei during the initial enucleation process in which the nucleus is digested prior to flattening. Furthermore, we found holes left behind by improperly digested nuclei in the stratum corneum, suggesting abnormal differentiation. Our findings indicate the great potential of SRS microscopy for discrimination of the degree of epidermal KC differentiation.