Supplementary Information

Microsecond Scale Vibrational Spectroscopic Imaging by Multiplex Stimulated Raman Scattering Microscopy

Chien-Sheng Liao et al.Supplementary results:

Characterization and optimization of 16-channel TAMP array

Our 16-channel TAMP array was assembled from single TAMPs. Each TAMP contained two stages. The first stage with dimensions of 1.2" × 1" × 0.25" included a LC circuit and a preamplifier. The LC circuit selectively amplified the SRL signal (the AC output) at the modulation frequency. After preamplification, the AC signal was sent to the second stage. The DC output of photodiode providing the intensity of the local oscillator was directly sent to the analog-to-digital converter (ADC) board. The second stage of TAMP was composed of an amplifier, a bandpass filter, and a rectifier. The amplifier provided four selectable amplifications, which enhanced the signal level to about 1.0 volt. The bandpass filter eliminated both low frequency and high frequency noise leaking through the resonant circuit. The filtered signal was rectified and then sent to the ADC for digitization. The first-stage TAMP array and the photodiode array were integrated into an 8" × 8" × 5" box that was smaller than a commercial lock-in amplifier. The second-stage TAMP array and the power supply, connecting to the first-stage TAMP array, were integrated into a 17" × 15" × 9" box.

Due to the tolerance (typically 1% to 10%) of electronic elements, such as capacitors and inductors in the LC circuit, the independent TAMPs may exhibit deviated resonant frequencies, which potentially can degrade the signal-to-noise ratio of the output. To validate our design, we measured the frequency response of each TAMP (Supplementary Fig. S2a) by sweeping the modulation frequency from 0 Hz to 5 MHz. The corresponding outputs from all TAMPs showed a resonant frequency centered at 2.1 MHz ± 38 kHz. The average bandwidth (at -3 dB) was 250 KHz, resulting in a Q factor of approx to 10. Because the standard deviation of the resonant frequency was smaller than the bandwidth by nearly one order of magnitude, our TAMPs were valid for parallel detection use.

The electronic tolerance also caused variation in the gain level between TAMPs. We measured the gains of all 16 TAMPs under four different gain settings. As Supplementary Fig. S2b shows, the four gain settings provided 78, 88, 98 and 108 dB amplifications, respectively, enhancing the signal level from several micro-volts to 1.0 volt. The deviation between channels was around 4 dB. To compensate for this gain deviation, we calibrated the AC output by the gain of each channel. After gain calibration, correct SRL spectra can be generated by the parallel detection scheme. To test linearity of the TAMP array, we measured

the laser power dependence of the SRL signal (i.e., AC output). We modulated the 800 nm beam and illuminated each channel separately in order to mimic the SRL signal. By tuning the laser power on each single channel, the linearity of the TAMP array at each gain setting was measured. Supplementary Fig. S2c shows the power dependence of channel 5. The AC output saturated at 1.0 volt, below which the output was linear with the local oscillator power in the range of 2 μW to 1.3 mW. All 16 channels have the same behavior of linear power dependence.

To confirm the source of noise in our measurement, we increased the local oscillator power and measured the noise level of each channel. The noise level of channel 5 as a function of local oscillator power is shown in Supplementary Fig. S2d. Below 0.2 mW, the noise was dominated by electronic noise which was close to 0.68 nV/Hz1/2. In the range from 0.1 to 1.0 mW, the noise gradually approached the theoretical shot noise level. The electronic noise intersected with the theoretical shot noise at 0.2 mW of local oscillator power, indicating that the shot noise is larger than the electronic noise when the local oscillator power was higher than 0.2 mW.

Supplementary Figures and Legends:

Supplementary Figure S1: A lab-built multiplex SRL microscope by broadband excitation and parallel detection. HW: half wave-plate, PBS: polarizing beam splitter, AOM: acousto-optic modulator, FG: function generator, L: lens, DM: dichroic mirror, G: grating, SL: slit, M: mirror, SU: scanning unit, OBJ: objective, MS: motorized stage, C: condenser, CL: cylindrical lens.

Supplementary Figure S2: Characterization of the TAMP array. (a) The frequency response of each channel. <fc> is the average central frequency. σfc is the standard deviation of central frequency. BW is the -3 dB bandwidth of the frequency response. (b) The actual gain levels of each channel at four different gain settings. The gain difference between channels was close to 4 dB. (c) The power dependence of channel 5. Our AC output was saturated at 1.0 volt. The four gain settings provided linear power dependence from 8×10 -7 to 2×10-4 dI/I. (d) The noise level of channel 5, which can be fitted based on the following equations: shot noise∝√ power andnoise∝√elec . noise2+shot noise2. The electronic noise was found to be 0.68 nV/Hz1/2 by fitting the experimental results. The electronic noise and the theoretical shot noise intersected at 0.2 mW LO power, indicating that the shot noise was larger than the electronic noise when the LO power was higher than 0.2 mW.

Supplementary Figure S3: SRL spectral recording of DMSO solution by lock-in free parallel detection. (a) DC signal. Upper row shows images of the DMSO/air interface acquired by each spectral channel. The wavelength at each channel was determined by fitting the 16-channel DC outputs to the spectrum measured by a spectrometer. (b) AC signal. Upper row shows the SRL spectral images of the DMSO/air interface acquired by each spectral channel. The AC signal of each channel was calibrated by the DC amplitude and the gain. Scale bar: 20 μm. The spectra in (a) and (b) were derived from a single pixel.

Supplementary Figure S4: Lipid droplet trafficking in single live PC3 cells. (a) SRL image at 2874 cm-1. Enlarged image shows the movement of lipid droplets. (b-c) Time-lapsed SRL spectra of lipid droplets 1 and 2 shown in (a). Offsets were added for comparison between time-lapsed spectra. The pixel dwell time was 32 μs. Scale bar: 10 μm

Supplementary Figure S5: Frame-by-frame acquisition results in spectral distortion. To simulate the condition of low temporal resolution, we acquired 16 sets of spectroscopic SRS images and constructed one set of hyperspectral image by selecting one spectral channel from each of the 16 spectroscopic images. Since each spectroscopic image took 3 seconds, the spectral acquisition time was 48 seconds in the frame-by-frame hyperspectral mode. (a) SRL images at 2930 cm-1 and 2860 cm-1 of living tumor cells. These two spectral channels were acquired at time 18 seconds and 33 seconds, respectively. (b) The spectral profiles of the lipid droplets as indicated in (a). The spectral profiles were distorted because of the intracellular motion of lipid droplets. The pixel dwell time was 32 μs. Scale bar: 10 μm

Supplementary Figure S6: Spontaneous Raman spectra of lipid droplets in normal and avasimibe-treated PC3 cells. (a) Lipid droplets in normal PC3 cells showed a distinct Raman band at 2860 cm-1, which arises from the C-H stretch vibration in the cholesterol ring and a distinct Raman peak at 702 cm-1, which arises from the cholesterol ring vibration. (b) Lipid droplets in avasimibe-treated PC3 cells showed a dip in the 2860 cm -1 region and a weak Raman peak at 702 cm-1. The acquisition time for each spectrum was 30 seconds. In order to confirm the SRL spectroscopic imaging result, the percentage of CE in the droplet was independently quantified by the ratio of Raman peaks at 702 and 1442 cm-1, according to a calibration curve reported by Yue et al. in Cell Metabolism, 2014, 19: 393-406.

Supplementary Figure S7: Spontaneous Raman spectra of triglyceride (TG) which is abundant in CH2 groups, and bovine serum albumin (BSA) which is abundant in CH3

groups.

Supplementary Figure S8: Discrimination of fat droplets from protein-rich organelles in live C. elegans. (a) SRL image at 1411 cm-1. (b) SRL image at 1444 cm-1. (c) SRL spectral profiles of compartment 1 and 2 indicated in (a) and (b). (d) MCR concentration map of fat droplets. (e) MCR concentration map of protein-rich organelles. (f) MCR output spectra of fat droplets and protein-rich organelles. The SRL spectroscopic images were averaged 5 times, which resulted in 160 μs pixel dwell time. Scale bar: 10 μm.

Supplementary Figure S9: SRL image of static PS and PMMA microsphere mixtures. (a) SRL image at 2914 cm-1 and (b) MCR outputs of concentration maps of PS and PMMA. (c) MCR output spectra and the corresponding Raman spectra of PS and PMMA. The pixel dwell time was 32 μs. Scale bar: 20 μm.

Supplementary Movies:Supplementary Movie 1: Time sequenced SRL imaging of a live PC3 cell at 2874 cm-1.

Supplementary Movie 2: Time sequenced SRL imaging of fast flowing mixture of PMMA and PS beads at a speed of 1.0 mm/s. The Raman shift was at 2914 cm-1.

Supplementary Movie 3: Time sequenced MCR output of fast flowing PMMA and PS beads.

Supplementary Movie 4: Time sequenced SRL imaging of fast flowing live PC3 cells at a speed of 0.5 mm/s. The Raman shift was at 2874 cm-1.