Lightfield

To determine the optical response of the SERS probes, a custom Raman microscope system was constructed. The system was retrofitted to a Nikon TE-2000-S inverted microscope base. A 785nm single-mode laser from Innovative Photonics Solutions (Monmouth, NJ) was coupled to a fiber optic cable with a numerical aperture of 0.39 and a core diameter of 400μm (Thorlabs Inc, Newton, NJ). The end of the fiber was attached to a laser entry port on the back of the microscope base to illuminate the sample on the microscope stage. Laser light was focused onto the sample and backscattered light was collected through the objective lens. A longpass filter removed elastic scattering and the Raman scattered light, which was passed to the spectrometer and dispersed using an IsoPlane 160 spectrometer (Princeton Instruments, Trenton, NJ) with a 1200 g/mm grating. The dispersed light was imaged with a Princeton Instruments Pixis 400 detector. All spectra were processed using LightField (Princeton Instruments) and Spekwin32 [75 ] spectral software. During Raman spectrum acquisition, 40mW of laser power was focused on the sample and Raman spectra were acquired using a 10-second acquisition time.

A sinusoid wave was generated by an OWON AG1022 waveform generator (Industry, CA, USA). The signal was passed through a Trek Model 2205 high-voltage amplifier (Lockport, NY, USA) and monitored using an EZ Digital OS-5030 oscilloscope (Gyeonggi-do, Korea). The applied AC field (350 V_RMS at 100 Hz) was delivered to the device using alligator clips. The sample flow was controlled by a New Era Pump Systems, Inc. NE-300 syringe pump (Farmingdale, NY, USA) operating at 5 μL/h during analysis.
Raman spectra were collected using an in-house built Raman microscope unit as described and used previously [18 (link),19 (link)]. The unit consists of an inverted Nikon Eclipse TE2000-S (Melville, NY, USA), a 785 nm single-mode laser (Innovative Photonic Solutions, Monmouth, NJ, USA), an IsoPlane 160 spectrometer equipped with a 1200 g/mm grating (Princeton Instruments, Trenton, NJ, USA), and a Pixis-400 CCD (Princeton Instruments). A 25 s integration time was used to acquire one spectrum. Spectra were processed using LightField (Princeton Instruments) and Renishaw Wire 4.1 (Gloucestershire, UK).

The raw imaging data were collected from LightField (Princeton Instruments), stored as a TIF stack and exported to Matlab (R2016b). The specific set of data mentioned in the paper is composed of a stack of 500 frames with a temporal acquisition of 0.6 s per frame. The Michaelson contrast of Fig. 1b was calculated for each SH image and plotted as a function of acquisition time. The Michelson contrast is defined as $C_{\mathrm{Michelson}}=\frac{\left(I_{\mathrm{SH},\max }-I_{\mathrm{SH},\min }\right)}{\left(I_{\mathrm{SH},\max }+I_{\mathrm{SH},\min }\right)}$ . With I_SH,max, the maximum SH intensity and I_SH,min the minimum SH intensity found in the image. Denoising was done by subtracting an offset, applying a Fast Fourier Transform filter along the temporal dimension, and applying thresholding and a median filter in the spatial dimension for each frame (kernel = 3).