Research ArticleBIOPHYSICS

Polarization-sensitive stimulated Raman scattering imaging resolves amphotericin B orientation in Candida membrane

See allHide authors and affiliations

Science Advances  06 Jan 2021:
Vol. 7, no. 2, eabd5230
DOI: 10.1126/sciadv.abd5230
  • Fig. 1 Major models of AmB’s mechanism of action in a lipid membrane.

    (A and B) Chemical structures of AmB (A) and ergosterol (B), respectively. (C) Classic ion channel model. (D) Sterol sponge model where large extramembranous aggregates sequester ergosterol from lipid bilayers.

  • Fig. 2 Raman spectra of ergosterol, AmB, and AmB-treated single log-phase and stationary-phase CASC5314 cells.

    (A and B) Raman spectra of pure ergosterol (A) and AmB (B) dissolved in DMSO at a series of concentrations, respectively. a.u., arbitrary unit. (C) Raman spectra of AmB-treated single log-phase CASC5314 cells. (D) Raman spectra of AmB-treated single stationary-phase CASC5314 cells. Regions of interest are highlighted by green boxes.

  • Fig. 3 SRS microscope, performance characterization, and SRS imaging of untreated CASC5314 and CAC15 at 1556, 1602, and 1650 cm−1.

    (A) Schematic of the laboratory-built polarization-sensitive SRS microscope. Two half-wave plates (HWPs) were used to control the polarization of each beam. (B) SRS spectra of AmB (10 mg/ml) and pure ergosterol, with optimal Raman peaks highlighted. Data acquired under six-rod chirping. (C) SRS spectra of single AmB-treated CASC5314 cells. Data: means ± SD from at least 10 yeasts. AmB: 3.2 μg/ml for 1 hour. Data acquired under six-rod chirping condition. (D to F) SRS images of untreated CASC5314 cells at 1556, 1602, and 1650 cm−1, respectively. (G to I) SRS images of AmB-treated CASC5314 cells at 1556, 1602, and 1650 cm−1, respectively. (J to L) SRS images of untreated CAC15 cells at 1556, 1602, and 1650 cm−1, respectively. (M to O) SRS images of AmB-treated CAC15 cells at 1556, 1602, and 1650 cm−1, respectively. Pixel dwell time: 50 μs. Scale bar, 10 μm. Laser polarization direction is indicated by red arrows. Lipid droplet is indicated by white arrows.

  • Fig. 4 Polarization-sensitive SRS imaging of ghost RBCs at 2850 cm−1 and various AmB-treated fungal species at 1556 cm−1.

    (A1 and A2) SRS images of ghost RBCs at 2850 cm−1 in the horizontal and vertical polarization directions, respectively. Pump, 802 nm; Stokes, 1040 nm. Pixel dwell time: 50 μs. Scale bar, 2.5 μm. (B1 to J1) SRS images of AmB-treated S. cerevisiae AR-399, C. neoformans, C. duobushaemulonii AR-0394, C. glabrata ATCC2001, C. glabrata C1, C. glabrata C2, K. ohmeri AR-0396, C. albicans C14, and C. albicans SC5314 at 1556 cm−1 with laser polarization in the horizontal direction. Pump, 895 nm; Stokes, 1040 nm. Pixel dwell time: 50 μs. Scale bar, 10 μm. (B2 to J2) SRS images of the same strains but with laser polarization in the vertical direction. Pump, 895 nm; Stokes, 1040 nm. Pixel dwell time: 50 μs. Scale bar, 10 μm. (K) Zoom-in view of AmB-treated C. neoformans under two laser polarization directions. Scale bar, 2.5 μm. (L) Schematic of fungal cell membrane with angles defined. (M) Quantitative analysis of SRS signal intensity of single fungal cell at 1556 cm−1 under two laser polarization directions. AmB: 3.2 μg/ml, 1-hour treatment at 30°C. Laser orientation is indicated by red arrows.

  • Fig. 5 Polarization-sensitive SRS imaging of AmB-treated various C. auris strains at 1556 cm−1.

    (A1 to G1) SRS images of AmB-treated C. auris 1_MGH, C. auris 1_CDC, C. auris 2_CDC, C. auris 3_CDC, C. auris 4_CDC, C. auris 7_CDC, and C. auris 9_CDC at 1556 cm−1 with the laser polarization in the horizontal direction. (A2 to G2) SRS images of the same strains on the same field of view but with laser polarization in the vertical direction. (H) Orientation of ─CH2 in phospholipid backbone versus that of AmB. AmB: 3.2 μg/ml, 1-hour treatment at 30°C. Pixel dwell time: 50 μs. Scale bar, 10 um. Pump, 895 nm; Stokes, 1040 nm. Laser polarization direction is indicated by red arrows.

Supplementary Materials

  • Supplementary Materials

    Polarization-sensitive stimulated Raman scattering imaging resolves amphotericin B orientation in Candida membrane

    Pu-Ting Dong, Cheng Zong, Zeina Dagher, Jie Hui, Junjie Li, Yuewei Zhan, Meng Zhang, Michael K. Mansour, Ji-Xin Cheng

    Download Supplement

    This PDF file includes:

    • Figs. S1 to S10

    Files in this Data Supplement:

Stay Connected to Science Advances

Navigate This Article