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Surface-enhanced Raman scattering (SERS) is an attractive technique in molecular detection with high sensitivity and label-free characteristics. However, its use in protein detection is limited by the large volume of proteins, hindering its approach to the narrow spaces of hotspots. In this study, we fabricated a Au nanoTriangle plate Array on Gel (AuTAG) as an SERS substrate by attaching a Au nanoTriangle plate (AuNT) arrangement on a thermoresponsive hydrogel surface. The AuTAG acts as an actively tunable plasmonic device, on which the interparticle distance is altered by controlling temperature via changes in hydrogel volume. Further, we designed a Gel Filter Trapping (GFT) method as an active protein delivery strategy based on the characteristics of hydrogels, which can absorb water and separate biopolymers through their three-dimensional (3D) polymer networks. On the AuTAGs, fabricated with AuNTs modified with charged surface ligands to prevent the nonspecific adsorption of analytes to particles, the GFT method helped the delivery of proteins to hotspot areas on the AuNT arrangement. This combination of a AuTAG substrate and the GFT method enables ultrahigh sensitivity for protein detection by SERS up to a single-molecule level as well as a wide quantification concentration range of 6 orders due to their geometric advantages.

The advent of image-activated cell sorting and imaging-based cell picking has advanced our knowledge and exploitation of biological systems in the last decade. Unfortunately, they generally rely on fluorescent labeling for cellular phenotyping, an indirect measure of the molecular landscape in the cell, which has critical limitations. Here we demonstrate Raman image-activated cell sorting by directly probing chemically specific intracellular molecular vibrations via ultrafast multicolor stimulated Raman scattering (SRS) microscopy for cellular phenotyping. Specifically, the technology enables real-time SRS-image-based sorting of single live cells with a throughput of up to ~100 events per second without the need for fluorescent labeling. To show the broad utility of the technology, 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.

Time-course analysis of single cells is important to characterize heterogeneous activities of individual cells such as the metabolic response to their environment. Single-cell isolation is an essential step prior to time-course analysis of individual cells by collecting, culturing, and identifying multiple single-cell targets. Although single-cell isolation has been performed by various methods previously, a glass microfluidic device with semiclosed microchannels dramatically improved this process with its simple operation and easy transfer for time-course analysis of identified single cells. This study demonstrates isolating single cells of the highly motile microalgae, Euglena gracilis , by semiclosed microchannels with liquid flow only. The isolated single cells were identified in isolating channels and continuously cultured to track, by Raman microscopy, for the formation of subcellular granules composed of polysaccharide paramylon, a unique metabolite of E. gracilis , generated through photosynthesis. Through low-temperature glass bonding, a thin glass interface was incorporated to the microfluidic device. Thus, the device could perform the direct measurements of cultured single cells at high magnification by Raman microscopy with low background noise. In this study, the first demonstration of sequential monitoring of paramylon biogenesis in a single identified E. gracilis cell is shown.

Flow cytometry is an indispensable tool in biology for counting and analyzing single cells in large heterogeneous populations. However, it predominantly relies on fluorescent labeling to differentiate cells and, hence, comes with several fundamental drawbacks. Here, we present a high-throughput Raman flow cytometer on a microfluidic chip that chemically probes single live cells in a label-free manner. It is based on a rapid-scan Fourier-transform coherent anti-Stokes Raman scattering spectrometer as an optical interrogator, enabling us to obtain the broadband molecular vibrational spectrum of every single cell in the fingerprint region (400 to 1600 cm -1 ) with a record-high throughput of ~2000 events/s. As a practical application of the method not feasible with conventional flow cytometry, we demonstrate high-throughput label-free single-cell analysis of the astaxanthin productivity and photosynthetic dynamics of Haematococcus lacustris .

Microalgae offer great potential for the production of biofuel, but high photosynthetic activity is demanded for the practical realisation of microalgal biofuels. To this end, it is essential to evaluate the photosynthetic activity of single microalgal cells in a heterogeneous population. In this study, we present a method to monitor the photosynthetic activity of microalgae (in particular Euglena gracilis, a microalgal species of unicellular, photosynthetic, flagellate protists as our model organism) at single-cell resolution by Raman spectroscopy with deuterium from deuterium oxide (D 2 O) as a tracking probe. Specifically, we replaced H 2 O in culture media with D 2 O up to a concentration of 20 % without disturbing the growth rate of E. gracilis cells and evaluated C-D bond formation as a consequence of photosynthetic reactions by Raman spectroscopy. We used the probe to monitor the kinetics of the C-D bond formation in E. gracilis cells by incubating them in D 2 O media under light irradiation. Furthermore, we demonstrated Raman microscopy imaging of each single E. gracilis cell to discriminate deuterated cells from normal cells. Our results hold great promise for Raman-based screening of E. gracilis and potentially other microalgae with high photosynthetic activity by using D 2 O as a tracking probe., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)