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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,β-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.

Microbes, especially microalgae, have recently been of great interest for developing novel biofuels, drugs, and biomaterials. Imaging-based screening of live cells can provide high selectivity and is attractive for efficient bio-production from microalgae. Although conventional cellular screening techniques use cell labeling, labeling of microbes is still under development and can interfere with their cellular functions. Furthermore, since live microbes move and change their shapes rapidly, a high-speed imaging technique is required to suppress motion artifacts. Stimulated Raman scattering (SRS) microscopy allows for label-free and high-speed spectral imaging, which helps us visualize chemical components inside biological cells and tissues. Here we demonstrate high-speed SRS imaging, with temporal resolution of 0.14 seconds, of intracellular distributions of lipid, polysaccharide, and chlorophyll concentrations in rapidly moving Euglena gracilis, a unicellular phytoflagellate. Furthermore, we show that our method allows us to analyze the amount of chemical components inside each living cell. Our results indicate that SRS imaging may be applied to label-free screening of living microbes based on chemical information.

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.

We present a picosecond laser source based on a gain-switched laser diode (GS-LD) that can be applied to stimulated Raman scattering (SRS) microscopy. A 1.06-μm GS-LD was used to generate 14-ps pulses at a repetition rate of 38 MHz. The GS-LD was driven by 200-ps electrical pulses, which were triggered through a toggle flip-flop (T-FF). As a result, the GS-LD pulses were subharmonically synchronized to Ti:sapphire laser (TSL) pulses at a repetition rate of 76 MHz. We investigated the timing jitter of GS-LD pulses and found it to be less than 2.5 ps. We also show that the trigger delay can be less sensitive to the optical power of TSL pulses by controlling the threshold voltage of the T-FF. As a result, GS-LD pulses sufficiently overlapped with TSL pulses even when we scanned the wavelength of the TSL pulses. We demonstrate the SRS imaging 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.

The timing jitter of 13-ps gain-switched laser diode pulses at 1.06 μm is measured to be 3.9 ps, which is further reduced by external injection. We successfully demonstrate stimulated Raman imaging using the pulses.

Wavelength-tunable pulses with a constant group delay are successfully generated through spectral filtering of broadband pulses in a modified 4-f configuration. This technique effectively improves the reproducibility of spectral data in stimulated Raman spectral 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.