Cyano-Hydrol green derivatives: Expanding the 9-cyanopyronin-based resonance Raman vibrational palette.

9-cyanopyronin is a promising scaffold that exploits resonance Raman enhancement to enable sensitive, highly multiplexed biological imaging. Here, we developed cyano-Hydrol Green (CN-HG) derivatives as resonance Raman scaffolds to expand the color palette of 9-cyanopyronins. CN-HG derivatives exhibit sufficiently long wavelength absorption to produce strong resonance Raman enhancement for near-infrared (NIR) excitation, and their nitrile peaks are shifted to a lower frequency than those of 9-cyanopyronins. The fluorescence of CN-HG derivatives is strongly quenched due to the lack of the 10th atom, unlike pyronin derivatives, and this enabled us to detect spontaneous Raman spectra with high signal-to-noise ratios. CN-HG derivatives are powerful candidates for high performance vibrational imaging.

Visualization of Modified Bisarylbutadiyne-Tagged Small Molecules in Live-Cell Nuclei by Stimulated Raman Scattering Microscopy.

Visualizing the distribution of small-molecule drugs in living cells is an important strategy for developing specific, effective, and minimally toxic drugs. As an alternative to fluorescence imaging using bulky fluorophores or cell fixation, stimulated Raman scattering (SRS) imaging combined with bisarylbutadiyne (BADY) tagging enables the observation of small molecules closer to their native intracellular state. However, there is evidence that the physicochemical properties of BADY-tagged analogues of small-molecule drugs differ significantly from those of their parent drugs, potentially affecting their intracellular distribution. Herein, we developed a modified BADY to reduce deviations in physicochemical properties (in particular, lipophilicity and membrane permeability) between tagged and parent drugs, while maintaining high Raman activity in live-cell SRS imaging. We highlight the practical application of this approach by revealing the nuclear distribution of a modified BADY-tagged analogue of JQ1, a bromodomain and extra-terminal motif inhibitor with applications in targeted cancer therapy, in living HeLa cells. The modified BADY, methoxypyridazyl pyrimidyl butadiyne (MPDY), revealed intranuclear JQ1, while BADY-tagged JQ1 did not show a clear nuclear signal. We anticipate that the present approach combining MPDY tagging with live-cell SRS imaging provides important insight into the behavior of intracellular drugs and represents a promising avenue for improving drug development.

Stimulated Raman scattering microscopy reveals a unique and steady nature of brain water dynamics.

The biological activities of substances in the brain are shaped by their spatiotemporal dynamics in brain tissues, all of which are regulated by water dynamics. In contrast to solute dynamics, water dynamics have been poorly characterized, owing to the lack of appropriate analytical tools. To overcome this limitation, we apply stimulated Raman scattering multimodal multiphoton microscopy to live brain tissues. The microscopy system allows for the visualization of deuterated water, fluorescence-labeled solutes, and cellular structures at high spatiotemporal resolution, revealing that water moves faster than fluorescent molecules in brain tissues. Detailed analyses demonstrate that water, unlike solutes, diffuses homogeneously in brain tissues without differences between the intra- and the extracellular routes. Furthermore, we find that the water dynamics are steady during development and ischemia, when diffusions of solutes are severely affected. Thus, our approach reveals routes and uniquely robust properties of water diffusion in brain tissues.

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.

Deuterium- and Alkyne-Based Bioorthogonal Raman Probes for In Situ Quantitative Metabolic Imaging of Lipids within Plants.

Plants can rapidly respond to different stresses by activating multiple signaling and defense pathways. The ability to directly visualize and quantify these pathways in real time using bioorthogonal probes would have practical applications, including characterizing plant responses to both abiotic and biotic stress. Fluorescence-based labels are widely used for tagging of small biomolecules but are relatively bulky and with potential effects on their endogenous localization and metabolism. This work describes the use of deuterium- and alkyne-derived fatty acid Raman probes to visualize and track the real-time response of plants to abiotic stress within the roots. Relative quantification of the respective signals could be used to track their localization and overall real-time responses in their fatty acid pools due to drought and heat stress without labor-intensive isolation procedures. Their overall usability and low toxicity suggest that Raman probes have great untapped potential in the field of plant bioengineering.

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.

Protocol to image deuterated propofol in living rat neurons using multimodal stimulated Raman scattering microscopy.

Propofol is a widely used anesthetic important in clinics, but like many other bioactive molecules, it is too small to be tagged and visualized by fluorescent dyes. Here, we present a protocol to visualize deuterated propofol in living rat neurons using stimulated Raman scattering (SRS) microscopy with carbon-deuterium bonds serving as a Raman tag. We describe the preparation and culture of rat neurons, followed by optimization of the SRS system. We then detail neuron loading and real-time imaging of anesthesia dynamics. For complete details on the use and execution of this protocol, please refer to Oda et al.1.

Activatable Raman Probes Utilizing Enzyme-Induced Aggregate Formation for Selective Ex Vivo Imaging.

Detecting multiple enzyme activities simultaneously with high spatial specificity is a promising strategy to investigate complex biological phenomena, and Raman imaging would be an excellent tool for this purpose due to its high multiplexing capabilities. We previously developed activatable Raman probes based on 9CN-pyronins, but specific visualization of cells with target enzyme activities proved difficult due to leakage of the hydrolysis products from the target cells after activation. Here, focusing on rhodol bearing a nitrile group at the position of 9 (9CN-rhodol), we established a novel mechanism for Raman signal activation based on a combination of aggregate formation (to increase local dye concentration) and the resonant Raman effect along with the bathochromic shift of the absorption, and utilized it to develop Raman probes. We selected the 9CN-rhodol derivative 9CN-JCR as offering a suitable combination of increased stimulated Raman scattering (SRS) signal intensity and high aggregate-forming ability, resulting in good retention in target cells after probe activation. By using isotope-edited 9CN-JCR-based probes, we could simultaneously detect ß-galactosidase, γ-glutamyl transpeptidase, and dipeptidyl peptidase-4 activities in live cultured cells and distinguish cell regions expressing target enzyme activity in Drosophila wing disc and fat body ex vivo.

9-Cyano-10-telluriumpyronin Derivatives as Red-light-activatable Raman Probes.

Photoactivatable fluorescence probes can track the dynamics of specific cells or biomolecules with high spatiotemporal resolution, but their broad absorption and emission peaks limit the number of wavelength windows that can be employed simultaneously. In contrast, the narrower peak width of Raman signals offers more scope for simultaneous discrimination of multiple targets, and therefore a palette of photoactivatable Raman probes would enable more comprehensive investigation of biological phenomena. Herein we report 9-cyano-10-telluriumpyronin (9CN-TeP) derivatives as photoactivatable Raman probes whose stimulated Raman scattering (SRS) intensity is enhanced by photooxidation of the tellurium atom. Modification to increase the stability of the oxidation product led to a julolidine-like derivative, 9CN-diMeJTeP, which is photo-oxidized at the tellurium atom by red light irradiation to afford a sufficiently stable oxidation product with strong electronic pre-resonance, resulting in a bathochromic shift of the absorption spectrum and increased SRS intensity.

Complete characterization of picosecond optical pulses by the offset frequency intensity modulation.

We present a method for characterizing the intensity waveform, spectrum, frequency chirp, and spectral phase of picosecond pulses at a moderate repetition rate of ∼100 MHz. The proposed method exploits the intensity modulation at ∼10 GHz, which is slightly offset from the integer multiple of the repetition rate of the pulses. The modulated pulses are split into two, and one is measured by an optical spectrum analyzer, whose output is detected by a lock-in amplifier, while the other is directly detected by a photodiode and its output is used as a reference signal of the lock-in amplifier. In the experiment, we demonstrate the measurement of picosecond Ti:sapphire laser pulses to investigate frequency chirp induced by self-phase modulation. We anticipate that the proposed method will be useful for the characterization of various types of picosecond pulses.

Stimulated Raman scattering spectroscopy with quantum-enhanced balanced detection.

Quantum-enhanced stimulated Raman scattering (QE-SRS) is a promising technique for highly sensitive molecular vibrational imaging and spectroscopy surpassing the shot noise limit. However, the previous demonstrations of QE-SRS utilized rather weak optical power which hinders from competing with the sensitivity of state-of-the-art SRS microscopy and spectroscopy using relatively high-power optical pulses. Here, we demonstrate SRS spectroscopy with quantum-enhanced balanced detection (QE-BD) scheme, which works even when using high-power optical pulses. We used 4-ps pulses to generate pulsed squeezed vacuum at a wavelength of 844 nm with a squeezing level of -3.28 ± 0.12 dB generated from a periodically-poled stoichiometric LiTaO3 waveguide. The squeezed vacuum was introduced to an SRS spectrometer employing a high-speed spectral scanner to acquire QE-SRS spectrum in the wavenumber range of 2000-2280 cm-1 within 50 ms. Using SRS pump pulses with an average power of 11.3 mW, we successfully obtained QE-SRS spectrum whose SNR was better than classical SRS with balanced-detection by 2.27 dB.

Direct visualization of general anesthetic propofol on neurons by stimulated Raman scattering microscopy.

The consensus for the precise mechanism of action of general anesthetics is through allosteric interactions with GABA receptors in neurons. However, it has been speculated that these anesthetics may also interact with the plasma membrane on some level. Owing to the small size of anesthetics, direct visualization of these interactions is difficult to achieve. We demonstrate the ability to directly visualize a deuterated analog of propofol in living cells using stimulated Raman scattering (SRS) microscopy. Our findings support the theory that propofol is highly concentrated and interacts primarily through non-specific binding to the plasma membrane of neurons. Additionally, we show that SRS microscopy can be used to monitor the dynamics of propofol binding using real-time, live-cell imaging. The strategy used to visualize propofol can be applied to other small molecule drugs that have been previously invisible to traditional imaging techniques.

Phase locking of squeezed vacuum generated by a single-pass optical parametric amplifier.

In high-precision optical measurements, squeezed vacuum states are a promising resource for reducing the shot noise. To utilize a squeezed vacuum, it is important to lock the phase of the local oscillator (LO) to the squeezed light. The coherent control sideband (CCSB) scheme has been established for the precise phase locking, while the previous CCSB scheme was designed for the squeezed vacuum generated with an optical parametric oscillator (OPO). Thus the previous CCSB scheme is not applicable to squeezing by a single-pass optical parametric amplifier (OPA), which is attractive for generating broadband squeezed vacuum states. In this study, we propose a variant of CCSB scheme, which is applicable to the squeezing by single-pass OPA. In this scheme, we inject pump light and frequency-shifted signal light into an OPA crystal in the same way as the previous CCSB scheme. The parametric process in the OPA crystal generates a squeezed vacuum, amplifies the signal light, generates an idler light, and causes the pump depletion reflecting the interference of the amplified signal light and the idler light. Through the lock-in detection of the pump depletion, we can phase-lock the injected signal light to the pump light. Then, after the heterodyne detection of the signal and the idler light, we get the error signal of LO and realize the precise phase locking of LO to the squeezed quadrature. We show the feasibility of the proposed scheme by deriving the signal-to-noise ratio (SNR) of the modulated pump signal. We experimentally demonstrate the proposed scheme on pulsed squeezing by a single-pass OPA.

Probing Methionine Uptake in Live Cells by Deuterium Labeling and Stimulated Raman Scattering.

The small biomolecule methionine (Met) is a fundamental amino acid required for a vast range of biological processes such as protein synthesis, cancer metabolism, and epigenetics. However, it is still difficult to visualize the subcellular distribution of small biomolecules including Met in a minimally invasive manner. Here, we demonstrate stimulated Raman scattering (SRS) imaging of cellular uptake of deuterated methionine (d8-Met) in live HeLa cells by way of comparison to the previously used alkyne-labeled Met analogue─homopropargylglycine (Hpg). We show that the solutions of d8-Met and Hpg have similar SRS signal intensities. Furthermore, by careful image analysis with background subtraction, we succeed in the SRS imaging of cellular uptake of d8-Met with a much greater signal intensity than Hpg, possibly reflecting the increased and minimally invasive uptake kinetics of d8-Met compared with Hpg. We anticipate that d8-Met and other deuterated biomolecules will be useful for investigating metabolic processes with subcellular resolution.

Engineered Mutants of a Marine Photosynthetic Purple Nonsulfur Bacterium with Increased Volumetric Productivity of Polyhydroxyalkanoate Bioplastics.

Polyhydroxyalkanoates (PHAs) are green and sustainable bioplastics that could replace petrochemical synthetic plastics without posing environmental threats to living organisms. In addition, sustainable PHA production could be achieved using marine photosynthetic purple nonsulfur bacteria (PNSBs) that utilize natural seawater, sunlight, carbon dioxide gas, and nitrogen gas for growth. However, PHA production using marine photosynthetic PNSBs has not been economically feasible yet due to its high cost and low productivity. In this work, strain improvement, using genome-wide mutagenesis coupled with high-throughput screening via fluorescence-activated cell sorting, we were able to create Rhodovulum sulfidophilum mutants with enhanced volumetric PHA productivity, with an up to 1.7-fold increase. The best selected mutants (E6 and E6M4) reached the stationary growth phase 1 day faster and accumulated the maximum PHA content 2 days faster than the wild type. Maximizing volumetric PHA productivity before the stationary growth phase is indeed an additional advantage for R. sulfidophilum as a growth-associated PHA producer.

Quantum-enhanced stimulated Raman scattering microscopy in a high-power regime.

Quantum-enhanced stimulated Raman scattering (QESRS) microscopy is expected to realize molecular vibrational imaging with sub-shot-noise sensitivity, so that weak signals buried in the laser shot noise can be uncovered. Nevertheless, the sensitivity of previous QESRS did not exceed that of state-of-the-art stimulated Raman scattering (SOA-SRS) microscopes mainly because of the low optical power (3 mW) of amplitude squeezed light [Nature594, 201 (2021)10.1038/s41586-021-03528-w]. Here, we present QESRS based on quantum-enhanced balanced detection (QE-BD). This method allows us to operate QESRS in a high-power regime (>30 mW) that is comparable to SOA-SRS microscopes, at the expense of 3 dB sensitivity drawback due to balanced detection. We demonstrate QESRS imaging with 2.89 dB noise reduction compared with classical balanced detection scheme. The present demonstration confirms that QESRS with QE-BD can work in the high-power regime, and paves the way for breaking the sensitivity of SOA-SRS microscopes.

Probing the Biogenesis of Polysaccharide Granules in Algal Cells at Sub-Organellar Resolution via Raman Microscopy with Stable Isotope Labeling.

Phototrophs assimilate CO2 into organic compounds that accumulate in storage organelles. Elucidation of the carbon dynamics of storage organelles could enhance the production efficiency of valuable compounds and facilitate the screening of strains with high photosynthetic activity. To comprehensively elucidate the carbon dynamics of these organelles, the intraorganellar distribution of the carbon atoms that accumulate at specific time periods should be probed. In this study, the biosynthesis of polysaccharides in storage organelles was spatiotemporally probed via stimulated Raman scattering (SRS) microscopy using a stable isotope (13C) as the tracking probe. Paramylon granules (a storage organelle of ß-1,3-glucan) accumulated in a unicellular photosynthetic alga, Euglena gracilis, were investigated as a model organelle. The carbon source of the culture medium was switched from NaH12CO3 to NaH13CO3 during the production of the paramylon granules; this resulted in the distribution of the 12C and 13C constituents in the granules, so that the biosynthetic process could be tracked. Taking advantage of high-resolution SRS imaging and label switching, the localization of the 12C and 13C constituents inside a single paramylon granule could be visualized in three dimensions, thus revealing the growth process of paramylon granules. We propose that this method can be used for comprehensive elucidation of the dynamic activities of storage organelles.

Super-multiplex imaging of cellular dynamics and heterogeneity by integrated stimulated Raman and fluorescence microscopy.

Observing multiple molecular species simultaneously with high spatiotemporal resolution is crucial for comprehensive understanding of complex, dynamic, and heterogeneous biological systems. The recently reported super-multiplex optical imaging breaks the "color barrier" of fluorescence to achieve multiplexing number over six in living systems, while its temporal resolution is limited to several minutes mainly by slow color tuning. Herein, we report integrated stimulated Raman and fluorescence microscopy with simultaneous multimodal color tunability at high speed, enabling super-multiplex imaging covering diverse molecular contrasts with temporal resolution of seconds. We highlight this technique by demonstrating super-multiplex time-lapse imaging and image-based cytometry of live cells to investigate the dynamics and cellular heterogeneity of eight intracellular components simultaneously. Our technique provides a powerful tool to elucidate spatiotemporal organization and interactions in biological systems.