Here, we present vFLETCHERS (visible fluorescence-encoded time-domain coherent Raman spectroscopy), which operates in the visible excitation region and overcomes the previous limitation of detectable fluorophores. vFLETCHERS employs a non-collinear optical parametric amplifier as a femtosecond excitation source. As a proof-of-concept demonstration, we acquired low-frequency Raman spectra (<1000 cm-1) of solutions containing commercial fluorophores with the absorption peaks in the 600-700 nm region. These results highlight the potential of vFLETCHERS as a versatile multiplexed imaging technique, opening up new opportunities for research in biology.
KEYWORDS: Surface enhanced Raman spectroscopy, Chemical fiber sensors, Biological and chemical sensing, Sensors, In situ remote sensing, Chemical analysis, Biosensing, Stretchable circuits, Sensor technology, Nanolithography
Wearable sensor technology is a powerful tool, but conventional wearable sensors cannot perform simultaneous chemical sensing of multiple biomarkers in biofluids such as sweat and saliva because they are typically sensitive to only one type of chemical in an analyte at a time. Here we present a wearable dual-surface substrate for in situ surface-enhanced Raman spectroscopy (SERS). The substrate is composed of a gold nanomesh structure that can be tailored into any shape and attached to virtually any surface. Notedly, SERS can be performed on both surfaces of the substrate, highly effective for multiplexed in situ chemical sensing of biofluids.
High-speed Raman spectroscopy has enabled high-dimensional phenotyping of cells. Among these, time-domain Raman spectroscopy, such as Fourier-transform coherent anti-Stokes Raman scattering (FT-CARS), has unique capabilities such as high spectral acquisition rate, broadband spectral sensitivity in the fingerprint region, and nonresonant-background-free spectral acquisition. In this talk, we will present our recent achievements in FT-CARS spectroscopy for life science, including super-multiplex flow cytometry, high-speed dual-band spectroscopy, and broadband Raman-activated cell sorting.
Fluorescence-encoded time-domain coherent Raman spectroscopy (FLETCHERS) is a fluorescence-encoded vibrational spectroscopy technique that boasts both high sensitivity and specificity in the probing of molecular vibrations in the lower fingerprint region. However, to date, all presented data presented so far has been from flowing samples which does not sufficiently demonstrate the method’s applicability for monitoring biological and chemical processes. To amend this, we demonstrate here FLETCHERS imaging of low-concentration samples with an expanded spectral window achieved by utilizing spatial filtering and the confocal collection of fluorescence-encoded light.
Hyperspectral imaging (HSI) is a powerful tool widely used for various scientific and industrial applications due to its ability to provide rich spatiospectral information. However, in exchange for multiplex spectral information, its image acquisition rate is lower than that of conventional imaging, with up to a few colors. In particular, HSI in the infrared region and using nonlinear optical processes is impractically slow because the three-dimensional (3D) data cube must be scanned in a point-by-point manner. In this study, we demonstrate a framework to improve the spectral image acquisition rate of HSI by integrating time-domain HSI and compressed sensing. Specifically, we simulated broadband coherent Raman imaging at a record high frame rate of 25 frames per second (fps) with 100 pixels × 100 pixels, which is 10 × faster than that of previous work, based on an experimentally feasible sampling scheme utilizing 3D Lissajous scanning.
High-speed Raman spectroscopy has enabled label-free characterization of molecules in cells and materials in a space- and time-resolved manner. Among these, time-domain Raman spectroscopy (TDRS) techniques, such as Fourier-transform coherent anti-Stokes Raman scattering (FT-CARS) and impulsive stimulated Raman scattering (ISRS) spectroscopies, have unique capabilities such as high spectral acquisition rates, broadband spectral sensitivity in the fingerprint region, and nonresonant-background-free spectral acquisition. With a few exceptions, most TDRS studies have focused only on the fingerprint region (200 – 1800 cm-1) because the ultrashort pulses typically used for ultrabroadband (200 – 3200 cm-1) spectral acquisitions are difficult to generate and handle. For example, detecting Raman peaks above 3000 cm-1 necessitates a pulse duration of < 10 fs, which demands an expensive laser source and careful dispersion control. Furthermore, with sub-10-fs pulses, Raman detection sensitivity in the fingerprint is compromised because the spectral power density is diluted in the spectrally broad ultrashort pulse.
The present research demonstrates FT-CARS spectroscopy covering both the fingerprint and CH-stretching regions by employing synchronized mode-locked Ti:Sapphire and Yb-doped fiber lasers as the light source. With this method, we show that ultra-broadband FT-CARS spectra can be obtained without using sub-10-fs pulses, which significantly mitigates experimental complexity. More importantly, ultra-broadband Raman detection can be achieved in this scheme without compromising the sensitivity in the fingerprint region, unlike previous ultrashort-pulse approaches. The present method will significantly broaden the application range of TDRS for biomedical and material science research.
Surface-enhanced Raman spectroscopy (SERS) is a powerful tool for vibrational spectroscopy, but is compromised by its low reproducibility, uniformity, biocompatibility, and durability. This is because it depends on “hot spots” for high signal enhancement. Here we report our experimental demonstration of a plasmon-free nanostructure composed of a two-dimensional array of porous carbon nanowires as a SERS substrate for highly sensitive, biocompatible, and reproducible SERS. Specifically, the substrate provides not only high signal enhancement, but also high reproducibility and fluorescence quenching capability. We experimentally demonstrated these excellent properties with various molecules such as rhodamine 6G (R6G), β-lactoglobulin, and glucose.
Raman optical activity (ROA) is a powerful tool for identifying the absolute conformational information and behavior of chiral molecules in aqueous solutions, but suffers from low sensitivity. Here we report our development of a silicon nanodisk array that tailors a chiral field to significantly increase the interaction between the excitation light and chiral molecules via exploiting a dark mode. Specifically, we used the array with pairs of chemical and biological enantiomers to show >100x enhanced chiral light-molecule interaction with negligible artifacts for ROA measurements. Our silicon nanodisk array opens a cost-effective way for conformational analysis of trace chiral molecules.
Dual-comb spectroscopy, in which two synchronized mode-locked lasers are used as light sources of Fourier-transform spectroscopy, attracts much attention by its high spectral resolution and broad spectral bandwidth. However, there exists a trade-off between the spectral acquisition rate and the signal-to-noise ratio or the spectral bandwidth. Here, we overcome this trade-off by employing a mode-locked laser, whose repetition rate is rapidly modulated, together with a fixed-repetition-rate mode-locked laser. Specifically, we performed characterization of molecular vibrations through time-domain coherent Raman spectroscopy. In this demonstration, a high-spectral acquisition rate of 100,000 spectra/s was achieved with a broad bandwidth of 200 – 1400 cm-1.
High-speed broadband Raman spectroscopy (>1000 cm^−1 bandwidth) provides label-free molecular vibrational information with fine temporal resolution, making it valuable for biomedical applications such as vibrational imaging or detecting transient molecular dynamics. Current techniques for high-speed broadband Raman spectroscopy, such as Fourier-transform coherent anti-Stokes Raman scattering (FT-CARS) spectroscopy, can probe the Raman “fingerprint” region (500-1800 cm^−1), but lack sensitivity in the low-frequency or THz region (<200 cm^−1, <6 THz), preventing measurement of rich intermolecular vibrational information. Here we demonstrate a technique combining FT-CARS spectroscopy-like optical filtering with Sagnac interferometry for simultaneous acquisition of THz and fingerprint Raman spectra at 24,000 spectra/sec.
We present Raman image-activated cell sorting (RIACS), a technology that performs real-time coherent Raman image-based sorting of single live cells with a throughput of up to ~100 cells per second. Specifically, the fast image acquisition of the RIACS is enabled by multicolor stimulated Raman scattering (SRS) imaging based on a pulse-pair-resolved wavelength-switchable laser. To show the broad utility of the RIACS, we show its applicability to diverse cell types and sizes. The technology is highly versatile and holds promise for numerous applications that are previously difficult or undesirable with fluorescence-based technologies.
Raman flow cytometry is a promising approach for large-scale single-cell analysis in a label-free manner. However, due to the limited sensitivity of Raman spectral measurements, its application range is still limited compared to fluorescence-based flow cytometry. Here we present the developments of a high-throughput Raman-based spectroscopic flow cytometer and a Raman-activated cell sorter realized by integrating state-of-the-art coherent Raman techniques and acoustofluidic devices for cell manipulation in a flow stream. We demonstrate analysis and sorting of microalgal cells based on their bioproducts’ contents such as palamyron, starch, and carotenoids.
Broadband Raman spectroscopy (detection bandwidth >1000 cm − 1) is a valuable and widely used tool for understanding samples via label-free measurements of their molecular vibrations. Two important Raman spectral regions are the chemically specific “fingerprint” (200 to 1800 cm − 1) and “low-frequency” or “terahertz” (THz) (<200 cm − 1; <6 THz) regions, which mostly contain intramolecular and intermolecular vibrations, respectively. These two regions are highly complementary; broadband simultaneous measurement of both regions can provide a big picture comprising information about molecular structures and interactions. Although techniques for acquiring broadband Raman spectra covering both regions have been demonstrated, these methods tend to have spectral acquisition rates <10 spectra / s, prohibiting high-speed applications, such as Raman imaging or vibrational detection of transient phenomena. Here, we demonstrate a single-laser method for ultrafast (24,000 spectra / s) broadband Raman spectroscopy covering both THz and fingerprint regions. This is achieved by simultaneous detection of Sagnac-enhanced impulsive stimulated Raman scattering (SE-ISRS; THz-sensitive) and Fourier-transform coherent anti-Stokes Raman scattering (FT-CARS; fingerprint-sensitive). With dual-detection impulsive vibrational spectroscopy, the SE-ISRS signal shows a >500 × enhancement of <6.5 THz sensitivity compared with that of FT-CARS, and the FT-CARS signal shows a >10 × enhancement of fingerprint sensitivity above 1000 cm − 1 compared with that of SE-ISRS.
Dual-comb coherent anti-Stokes Raman scattering (DC-CARS) spectroscopy is an effective tool for high-speed acquisition of vibrational spectra in the fingerprint region. DC-CARS spectroscopy also provides high spectral resolution by virtue of its ability to cover a large time delay between the pump and probe pulses, from two optical frequency combs with slightly different, fixed pulse repetition rates. However, less than 1% of the incident pulse energy is used to acquire the CARS signal because the repetition interval of the laser pulses (<1 ns) is much longer than the coherence lifetime of molecular vibrations (~3 ps). This results in a low spectral acquisition rate and a low signal-to-noise ratio. Here, we introduce a novel method for DC-CARS spectroscopy with a nearly 100% energy efficiency using a “quasi”-dual-comb laser. Specifically, one of the repetition rates of the two lasers is rapidly modulated by controlling the pumping intensity of a Ti:sapphire laser, so that the group delay between two pulses is shorter than the coherence lifetime of molecular vibrations, while the group delay is monitored with two-color interferometry to calibrate the time-domain CARS signal. With this method, we realized a spectral acquisition rate of 100,000 spectra/s, which is 10x higher than conventional DC-CARS spectroscopy. Due to its ~100% energy efficiency, sensitivity at this record high acquisition rate is even higher than conventional DC-CARS spectroscopy operating at a slower acquisition rate of 10,000 spectra/s. Our method holds promise for diverse applications in fields ranging from materials science to life science, such as high-throughput screening, flow cytometry, and live-cell imaging.
Fluorescence-encoded Raman spectroscopy has become increasingly more popular by virtue of its high chemical specificity and sensitivity. However, current fluorescence-encoding methods are narrowband and lack sensitivity in the low wavenumber region which if addressed could further enhance these methods. To overcome these limitations, we propose and experimentally demonstrate a novel broadband method for fluorescence-encoded Raman spectroscopy, termed fluorescence-encoded time-domain coherent Raman spectroscopy (FLETCHERS), which is capable of probing molecular vibrations in the lower fingerprint region (200 – 750 cm-1 ) with sample concentrations as dilute as 100 nM and laser powers as low as 20 mW.
We present a high-throughput multimodal Raman-fluorescence flow cytometer that produces simultaneous Raman and fluorescence excitation spectra at a demonstrated throughput of 100 events per second. Our method combines Fourier-transform coherent anti-Stokes Raman scattering with Fourier-transform two-photon excitation fluorescence, which produces broadband Raman spectra that span the biological fingerprint region and single-detector multicolor fluorescence via spectral analysis. As a proof-of-principle demonstration, we perform multimodal label-free analysis of stress response in the microalga Haematococcus pluvialis by Raman measurement of its secondary metabolite astaxanthin and two-color fluorescence measurement of chlorophyll and fluorescent chlorophyll catabolites under different stress conditions.
KEYWORDS: Raman spectroscopy, Raman scattering, Spectroscopy, Chemical analysis, Signal to noise ratio, Intelligence systems, Biological research, Chemistry, Biology, Medicine
By virtue of its chemical specificity and label-free nature, Raman spectroscopy is a ubiquitous tool in analytical chemistry which has recently found increasing interest in biology and medicine, as technical improvements steadily enable applications. In particular, the low-frequency region of the Raman spectrum, which contains rich information about intermolecular interactions and higher-order structure, has promise for biological applications. However, detection of low-frequency modes remains a challenge with conventional techniques for Raman spectroscopy. Here, we propose and experimentally demonstrate Sagnac-enhanced impulsive stimulated Raman scattering (SE-ISRS), a method for time-domain Raman spectroscopy that provides highly sensitive low-frequency Raman spectra at all probe frequencies.
We introduce two newly developed methods for high-throughput vibrational flow cytometry, namely Fourier-transform coherent anti-Stokes Raman scattering flow cytometry and stimulated Raman scattering imaging flow cytometry, which cover the fingerprint and high-frequency regions with high throughputs of >1,000 cells/s and >100 cells/s, respectively. With these methods, we also show large-scale single-cell analysis of diverse types of live cells (e.g., microbes, cancer cells, blood cells) based on carbohydrates, proteins, chlorophyll, carotenoids, and lipids with unique capabilities that are not possible with fluorescence-based flow cytometry. We also discuss a novel class of opportunities that can be offered by high-throughput vibrational flow cytometry.
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