We present a new type of surface-enhanced Raman scattering (SERS) substrate that exhibits extremely large and uniform cross-section enhancements over a macroscopic (greater than 25 mm2) area. The substrates are fabricated using a femtosecond laser nanostructuring process, followed by thermal deposition of silver. SERS signals from adsorbed molecules show a spatially uniform enhancement factor of approximately 10(7). Spectroscopic characterization of these substrates suggests their potential for use in few or single-molecule Raman spectroscopy.
Fast and selective isolation of single cells with unique spatial and morphological traits remains a technical challenge. Here, we address this by establishing high-speed image-enabled cell sorting (ICS), which records multicolor fluorescence images and sorts cells based on measurements from image data at speeds up to 15,000 events per second. We show that ICS quantifies cell morphology and localization of labeled proteins and increases the resolution of cell cycle analyses by separating mitotic stages. We combine ICS with CRISPR-pooled screens to identify regulators of the nuclear factor κB (NF-κB) pathway, enabling the completion of genome-wide image-based screens in about 9 hours of run time. By assessing complex cellular phenotypes, ICS substantially expands the phenotypic space accessible to cell-sorting applications and pooled genetic screening.
Fluorescence imaging is the most widely used method for unveiling the molecular composition of biological specimens. However, the weak optical emission of fluorescent probes and the tradeoff between imaging speed and sensitivity 1 is problematic for acquiring blur-free images of fast phenomena, such as sub-millisecond biochemical dynamics in live cells and tissues 2 , and cells flowing at high speed 3 . We report a solution that achieves real-time pixel readout rates one order of magnitude faster than a modern electron multiplier charge coupled device (EMCCD) -the gold standard in high-speed fluorescence imaging technology 4 . Deemed fluorescence imaging using radiofrequency-multiplexed excitation (FIRE), this approach maps the image into the radiofrequency spectrum using the beating of digitally synthesized optical fields. We demonstrate diffraction-limited confocal fluorescence imaging of stationary cells at a frame rate of 4.4 kHz, as well as fluorescence microscopy in flow at a throughput of approximately 50,000 cells per second.
The absolute Raman scattering cross section (σ RS ) for the 1584-cm −1 band of benzenethiol at 897 nm (1.383 eV) has been measured to be 8.9 ± 1.8 × 10 −30 cm 2 using a 785-nm pump laser. A temperature-controlled, small-cavity blackbody source was used to calibrate the signal output of the Raman spectrometer. We also measured the absolute surface-enhanced Raman scattering cross section (σ SERS ) of benzenethiol adsorbed onto a silver-coated, femtosecond laser-nanostructured substrate. Using the measured values of 8.9 ± 1.8 × 10 −30 and 6.6 ± 1.3 × 10 −24 cm 2 for σ RS and σ SERS respectively, we calculate an average cross-section enhancement factor (EF) of 0.8 ± 0.3 × 10 6 .
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