Research ArticleOPTICAL MICROSCOPY

Spectrometer-free vibrational imaging by retrieving stimulated Raman signal from highly scattered photons

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Science Advances  30 Oct 2015:
Vol. 1, no. 9, e1500738
DOI: 10.1126/sciadv.1500738
  • Fig. 1 Stimulated Raman spectroscopic imaging by spatial frequency multiplexing and single photodiode detection.

    (A) Principle. Every color of the pump laser was modulated at a specific megahertz frequency. Through the SRG process, the modulation frequency transferred to the Stokes beam. A time trace was recorded, from which fast Fourier transform was performed to retrieve an SRG spectrum. (B) A lab-built multiplex-modulation SRS microscope. D, dichroic mirror; G, grating; SU, scanning unit; M, mirror; OBJ, objective; P, polarizing beam splitter; PD, photodiode; PS, polygon scanner; Q, quarter waveplate; SL, slit. (C) Modulated pump laser intensity as a function of modulation frequency. (D) Linear relation between wavelength and modulation frequency. (E) SRG intensity as a function of modulation frequency. (F) Retrieved SRG spectrum (circles) and spontaneous Raman spectrum (solid line).

  • Fig. 2 Microsecond-scale acquisition of SRG spectra from completely diffused photons.

    (A) Experiment configuration. (B) SRG intensity of DMSO as a function of modulation frequency with 1.6- or 3.2-cm chicken breast tissues before photodiode. (C) Calibrated SRG spectra of DMSO. (D) SRG intensity of olive oil as a function of modulation frequency with 1.0-cm chicken breast tissues before photodiode. (E) Calibrated SRG spectra of olive oil. The integration time was 60 μs.

  • Fig. 3 In vivo monitoring of vitamin E distribution on mouse skin.

    (A) Experiment configuration. The nude mouse was anesthetized with isoflurane. (B)Spontaneous Raman spectra of vitamin E, olive oil (rich in CH2), and bovine serum albumin (rich in CH3). (C) MCR output spectra of three components assigned to lipid, protein, and vitamin E. (D) MCR concentration maps of lipid (in yellow) and protein (in green) before and after the treatment. (E) SRG images at 2911 cm−1. The pixel dwell time was 60 μs. Scale bar, 100 μm.

  • Fig. 4 In situ mapping of human patient breast cancer and stroma.

    (A) Digital picture of a 5-mm-thick human breast cancerous tissue. (B) SRG images of locations indicated in (A) at 2908 cm−1. (C) MCR output spectra of three components assigned to fat, fibrosis, and cell nuclei. (D) MCR concentration maps of fat (in yellow), fibrosis (in green), and cell nuclei (in pink). (E) Histological analysis (H&E staining) of the same tissue. The pixel dwell time was 600 μs. Scale bar, 100 μm.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/1/9/e1500738/DC1

    Vibrational spectroscopic imaging of live C. elegans

    Fig. S1. Comparison of photon collection efficiency by a spectrometer to that by a single detector.

    Fig. S2. Detection sensitivity of spatial frequency multiplexing SRS microscopy.

    Fig. S3. Spectroscopic imaging of a live wild-type C. elegans.

    References (4144)

  • Supplementary Materials

    This PDF file includes:

    • Vibrational spectroscopic imaging of live C. elegans
    • Fig. S1. Comparison of photon collection efficiency by a spectrometer to that by a single detector.
    • Fig. S2. Detection sensitivity of spatial frequency multiplexing SRS microscopy.
    • Fig. S3. Spectroscopic imaging of a live wild-type C. elegans.
    • References (41–44)

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