Label-free stimulated Raman scattering microscopy visualizes changes in intracellular morphology during human epidermal keratinocyte differentiation.

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.

Chemically-activatable alkyne-tagged probe for imaging microdomains in lipid bilayer membranes.

A chemically-activatable alkynyl steroid analogue probe has been synthesized for visualizing the lipid raft membrane domains by Raman microscopy. The Raman probe, in which ring A of its steroid backbone is replaced with an alkynyl group, was designed to enable activation of the alkyne signal through the Eschenmoser-Tanabe fragmentation reaction of the oxidized cholesterol precursor in lipid bilayer membranes. The alkynyl steroid analogue was observed to form liquid-ordered raft-like domains on a model giant-liposome system in a similar manner as cholesterol, and the large alkyne signal of the accumulated probe at 2120 cm-1 was mapped on the microdomains with a Raman microscope. The alkyne moiety of the probe was confirmed to be converted from the α,ß-epoxy ketone group of its precursor by reaction with p-toluensulfonyl hydrazine under a mild condition. Through the reaction, the alkyne signal of the probe was activated on the lipid bilayer membrane of liposomes. Furthermore, the signal activation of the probe was also detected on living cells by stimulated Raman scattering microscopy. The ring-A-opened alkyne steroid analogue, thus, provides a first chemically-activatable Raman probe as a promising tool for potentially unravelling the intracellular formation and trafficking of cholesterol-rich microdomains.

In situ visualization of intracellular morphology of epidermal cells using stimulated Raman scattering microscopy.

Visualization of epidermal cells is important because the differentiation patterns of keratinocytes (KCs) are considered to be related to the functions and condition of skin. Optical microscopy has been widely used to investigate epidermal cells, but its applicability is still limited because of the need for sample fixation and staining. Here, we report our staining-free observation of epidermal cells in both tissue and culture by stimulated Raman scattering (SRS) microscopy that provides molecular vibrational contrast. SRS allowed us to observe a variety of cellular morphologies in skin tissue, including ladder-like structures in the spinous layer, enucleation of KCs in the granular layer, and three-dimensional cell column structures in the stratum corneum. We noticed that some cells in the spinous layer had a brighter signal in the cytoplasm than KCs. To examine the relevance of the observation of epidermal layers, we also observed cultured epidermal cells, including KCs at various differentiation stages, melanocytes, and Langerhans cell-like cells. Their SRS images also demonstrated various morphologies, suggesting that the morphological differences observed in tissue corresponded to the cell lineage. These results indicate the possible application of SRS microscopy to dermatological investigation of cell lineages and types in the epidermis by cellular-level analysis.

Generation of synchronized picosecond pulses by a 1.06-µm gain-switched laser diode for stimulated Raman scattering microscopy.

We propose that a gain-switched laser diode (GS-LD) can be used as a picosecond laser source for stimulated Raman scattering (SRS) microscopy. We employed a 1.06-µm GS-LD to generate ~13-ps pulses at a repetition rate of 38 MHz and amplified them to >100 mW with Yb-doped fiber amplifiers. The GS-LD was driven by 200-ps electrical pulses, which were triggered through a toggle flip-flop (T-FF) so that the GS-LD pulses were synchronized to Ti:sapphire laser (TSL) pulses at a repetition rate of 76 MHz. We found the timing jitter of GS-LD pulses to be approximately 2.7 ps in a jitter bandwidth of 7 MHz. We also show that the delay of electrical pulses can be less sensitive to the optical power of TSL pulses by controlling the threshold voltage of the T-FF. We demonstrate the SRS imaging of polymer beads and of HeLa cells with GS-LD pulses and TSL pulses, proving that GS-LD is readily applicable to SRS microscopy as a compact and stable pulse source.