Supporting information

Low-frequency multiplex CARS microscopy with a high-repetition
near-infrared supercontinuum laser

Yusuke Arashida1, Atsushi Taninaka1,2, Takahiro Ochiai1,2, Hiroyuki Mogi1, Shoji Yoshida1,Masamichi Yoshimura3, Osamu Takeuchi1, and Hidemi Shigekawa1

1

Faculty of Pure and Applied Sciences, University of Tsukuba, Tsukuba 305-8573, Japan

2

TAKANO Co., LTD., Miyada-mura, Kamiina-gun, Nagano, 399-4301 Japan

3

Graduate School of Engineering, Toyota Technological Institute, Hisakata, Tempaku, Nagoya 468-8511, Japan1

1. Spectral shape caused by the interference between CARS and NRB

Fig. S1 Schematics of typical interference spectra with CARS and NRB. Red and bluecurves represent spectra for slightly different 𝜔! (± δω) values assuming (a) 𝐸"#$% ≪𝐸&$ and (b) 𝐸"#$% > 𝐸&$ . The blue and red lines are spectra for slightly different ωp,and when the difference between the spectra is considered, a peak appears at the vibration
frequency Ωi as shown by the green line. The green curves show the difference between
red and blue curves. When ECARS has a resonance peak shape and ENR is a non-phasesensitive background, the spectrum with the Fano shape appears when the NRB amplitude
is sufficiently larger than the CARS signal as shown in (a). On the other hand, when theeffect of interference by NRB is small, a spectrum with a peak appears as shown in (b)).
Then, when the difference between the two spectra obtained by changing ωp is considered,
the spectrum has a structure with the largest slope at the frequency Ωi. To obtain an
accurate Raman shift, it is necessary to consider fitting with the degree of interference.
Even if the effect of interference NRB is small, the background can be set to 0 by
considering the difference, which is useful for extracting CARS signals. However,depending on the degree of interference, a spectral shape between those in (a) and (b)may appear for the data.

22. Confirmation of wide bandwidthFig. S2 Broadband CARS spectrum of a polyimide film. The spectrum was obtained
as the difference between the two spectra measured for λp ± 0.2 nm (δλ = 0.4 nm), as
shown in Figs. 1 (d) and 1 (e). According to previous studies23-25, the peak at 1788 cm-1 isoriginated from the stretching vibration of the C-CO-C structure. The peak at 1394 cm-1and 1124 cm-1 are from the stretching and the bending vibration of the C-N-C structure,respectively. The multiple peaks observed in the region from 707 cm-1 to 776 cm-1 areconsidered to be oscillations due to the dianhydride structure. The peaks found at 865 cm1, 625 cm-1, and 586 cm-1 are vibrations of diamine ring breathing, C-O-C bonding, andmonosubstituted benzene deformation, respectively.

33. Chirp of the supercontinuum laser

Fig. S3 Wavelength dependence of group delay of ωs measured by frequency-resolved
optical gating (FROG). Spectra of the sum-frequency generation (SFG) between the ωpand ωs, which is generated by using a BBO crystal, were measured by changing the opticaldelay between the two beams of ωp and ωs. The wavelength λs of the vertical axis wascalculated by 1/λs = 1/λSFG -1/λp considering the wavelength of ωp for the wavelength ofsum-frequency light in the visible light region.

44. Spot size of the beams on the sample

Fig. S4, the spot size of the pump and the stokes beams on the focal plane measured with
optical microscope with the magnitude of x100 and using Si-based image sensor (ASI294MC, ZWO).

55. Spatial resolution of CARS imaging

Fig. S5 As an estimate of spatial resolution, the smallest spatial shape change of CARS
signal imaging in Fig. 4 was used for evaluation. (a) CARS image depicted from Fig. 4in the text. (b) The red dots show the cross section along the blue line in (a). The blue
curve shows the result of Gaussian fitting of the cross section. The spatial resolution ofour CARS is evaluated to 1. µm by using the upper limit of the fitting result of FWHM =1.78 ± 0.09 µm.

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6. Other advantages of the developed system

Fig. S6 High efficiency of the developed system and a damage induced by laser
illumination. (a) Raw spectrum of the sulfur crystal sample with an exposure time of 2ms, a repetition frequency of 40 MHz, and the power of 60 mW. Optical microscopy
images of sample obtained (b) before and (c) after the illumination with the IR laser. (d)Optical microscope image after illumination with a visible laser with a wavelength of 532
nm with the power of 60 mW. The black bar denotes 5 µm.

The sulfur crystal was used as the sample, and the laser repetition frequency was set to40 MHz. Figure 4S shows the results obtained with an exposure time of 2 ms. A Raman
signal with a 220 cm-1 peak clearly observed with an SN ratio of about 19. By IR
excitation multiplex CARS, we achieved the same high measurement efficiency as thatin previous research using visible light excitation, even on the low-wavenumber side of7the fingerprint region14. This microscopy is expected to enable the effective tracking ofmolecules containing heavy atoms and polymers.
Optical microscopy images of the sample obtained before and after themeasurement of the data shown in Fig. S5a are shown in Figs. S5b and S5c, respectively.
There are no major differences between the two images. Next, in the optical system shownin Fig. 1, the LPF was replaced with a BPF of 532 ± 10 nm. Then, the obtained visible
light was set to the same power as the IR (60 mW) and irradiated to the same part of thesample. As shown in Fig. S6d, the optical contrast changed. Since visible light has ahigher absorption rate than IR, the sample is considered to have been damaged. Namely,IR excitation could induce CARS while suppressing the sample damages caused by lightirradiation, as expected.

87. Optical microscopy images of the phase transition of S crystal.

Fig. S7 Pictures of the sulfur crystal sample sandwiched between a sapphire plate and agold mirror. Temperature dependence of the phase transition during heating and coolingare shown. The green arrows denote the points where phase transitions appeared.

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