Journal: Nature Communications

Article Title: Ultra-bright Raman dots for multiplexed optical imaging

doi: 10.1038/s41467-021-21570-0

Figure Lengend Snippet: a 20 nm polystyrene (PS) nanoparticles are first swelled in organic solvents such as tetrahydrofuran (THF), and small-molecule Raman probes are allowed to diffuse into the nanoparticles, followed by shrinking the nanoparticles to entrap the Raman probes to generate Rdots. The dense packing of Raman probes inside nanoparticles makes Rdots ultra bright. b For easy biofunctionalization, Rdots are first conjugated with a mixture of amine-PEG8-alcohol and amine-PEG16-acid through EDC/NHS coupling to react with abundant carboxyl groups on the surface. Such combination of the two PEG chains can help reduce the non-specific binding and aggregation. Then IgG or other amine bearing bio-molecules are conjugated to the carboxyl groups from PEG-acids. This two-step procedure helps to increase the hydrophilicity and to greatly reduce non-specific binding. c Simultaneous immunostaining of Rdots and fluorescence probes (shown in shaded antibodies). d Multiplexed imaging with stimulated Raman scattering (SRS) microscopy, thanks to the narrow peak width of Rdots.

Article Snippet: For the characterization of Rdots, spontaneous Raman spectra were acquired with a Horiba XploRA microspectrometer or a home-built Raman microspectrometer.

Techniques: Binding Assay, Immunostaining, Fluorescence, Imaging, Microscopy

Journal: Nature Communications

Article Title: Ultra-bright Raman dots for multiplexed optical imaging

doi: 10.1038/s41467-021-21570-0

Figure Lengend Snippet: a Spontaneous Raman spectra of six Rdots generated using Carbow dyes. arb. units: arbitrary units. b Raman spectra of Rdots and free Carbow dyes in DMSO, showing no significant peak shift or broadening. arb. units: arbitrary units. c RIE and Raman shift of Rdots made by various alkyne and nitrile containing probes. Rdots were made with a large variety of compounds that contain alkyne or nitrile molecules, and their RIE (relative Raman intensity versus EdU) values and Raman shifts were measured. RIE values were measured with Spontaneous Raman spectrometer with 532 nm excitation wavelength, except MARS dye, which were evaluated with SRS microscopy. Probes indicated with colored stars were used for immunostaining in this study. PDDA: poly(deca-4,6-diynedioic acid) , EdU: 5-ethynyl-2’-deoxyuridine. d Hydrodynamic diameter of PS nanoparticles before and after doping measured by DLS. The size distributions show no change when PS nanoparticles are doped with Raman dyes, and the resulting 21 nm Rdots are monodispersive. e Hydrodynamic diameter of Rdots after biofunctionalization.

Article Snippet: For the characterization of Rdots, spontaneous Raman spectra were acquired with a Horiba XploRA microspectrometer or a home-built Raman microspectrometer.

Techniques: Generated, Microscopy, Immunostaining

Journal: Nature Communications

Article Title: Ultra-bright Raman dots for multiplexed optical imaging

doi: 10.1038/s41467-021-21570-0

Figure Lengend Snippet: a Linear concentration dependence of Rdots with sub-nM SRS detection sensitivity. Red dashed line in the insert indicates shot-noise-limited SRS detection limit where SNR = 1. Error bars, mean ± s.d.; n = 3 measurements. Solid line shows a linear fitting ( R 2 = 0.999), shaded area indicates 95% confidence interval of the linear fitting. arb. units: arbitrary units. b A representative image of potential single-particle spots. Scale bar: 1 µm. c Raman spectra of ‘single’ Rdots2220 acquired by SRS microscopy by sweeping the pump wavelength. The original SRS images below at three marked pump wavelengths show the signal can be tuned off by only 1.5 nm. The spectra are in consistence with bulk measurements of Rdots by spontaneous Raman spectroscopy. Scale bar, 1 µm. arb. units: arbitrary units. d SRS intensity distribution of individual spots at pH 8.5. Red dash lines indicate fitted peak positions. The quantized distribution suggests spots corresponding to single-particle and double-particle aggregates. n = 289 from 7 replicates. e SRS intensity distribution of individual spots at pH 6.0. Red dash lines indicate fitted peak positions. The quantized distribution suggests spots corresponding to single-particles, double-particle aggregates, and triple-particle aggregates. n = 595 from 6 replicates. arb. units: arbitrary units. f A representative image of spots from single-particles and multi-particle aggregates. 1, 1’, and 1”: single-particle; 2: double-particle; 3: triple-particle; 4: quadruple-particle. Scale bar: 1 μm. g Line profiles across spots in f . Quantized signal intensity is evidently observed. arb. units: arbitrary units.

Article Snippet: For the characterization of Rdots, spontaneous Raman spectra were acquired with a Horiba XploRA microspectrometer or a home-built Raman microspectrometer.

Techniques: Concentration Assay, Single Particle, Microscopy, Raman Spectroscopy