Real-time Raman microspectroscopy scanning of the single live sperm bound to human zona pellucida  Feng Liu, B.Sc., Yong Zhu, Ph.D., Yufei Liu, Xiaobo Wang, Ping Ping, M.D., Ph.D., Xinyuan Zhu, Ph.D., Hongliang Hu, Ph.D., Zheng Li, M.D., Ph.D., Lin He, Ph.D.  Fertility and Sterility  Volume 99, Issue 3, Pages 684-689.e4 (March 2013) DOI: 10.1016/j.fertnstert.2012.10.035 Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Mapping of Raman spectral peaks to different regions of sperm, including (A) acrosome, (B) nucleus, (C) equatorial, (D) neck, (E) midpiece, and (F) tail. (G) The combined spectral map. The x-axis presents the continuous wave lengths (cm−1) of the spectra, and the y-axis presents the intensity of the spectra, including three grades of intensity peaks: low intensity (50–100 a.u.), medium intensity (100–200 a.u.), and high intensity (>200 a.u.). Fertility and Sterility 2013 99, 684-689.e4DOI: (10.1016/j.fertnstert.2012.10.035) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 2 The grade of intensity shifts at (A) the acrosome region and (B) the equatorial region when treated by ZP-binding assay. The red arrow indicates the sperm treated by ZP binding, and the blue arrow indicates the sperm without ZP-binding treatment. Fertility and Sterility 2013 99, 684-689.e4DOI: (10.1016/j.fertnstert.2012.10.035) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Statistical analysis (P<.01; unpaired two-tailed Student t test) results of the two groups (ZP binding in red and ZP nonbinding in blue) showed that the mean spectral intensity of the 13 typical Raman peaks were differentiated at the acrosome, nucleus, and equatorial regions, at which the spectral lengths of (A) 789 cm−1, (B) 810 cm−1, (C) 838 cm−1, (D) 1,092 cm−1, (E) 1,100 cm−1, (F) 1,357 cm−1, (G) 1,421 cm−1, (H) 1,441 cm−1, (I) 1,482 cm−1, (J) 1,505 cm−1, (K) 1,575 cm−1, (L) 1,582 cm−1, and (M) 3,240 cm−1, as picked up by Opus 6.0 software. Fertility and Sterility 2013 99, 684-689.e4DOI: (10.1016/j.fertnstert.2012.10.035) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Principal component (PC) analysis shows the different spectral data between ZP binding (red) and ZP nonbinding (blue) and distinguished by these methods in three-dimensional coordinates. Fertility and Sterility 2013 99, 684-689.e4DOI: (10.1016/j.fertnstert.2012.10.035) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 The acrosome reaction was assessed by Pisum sativum agglutinin labeled with fluorescein isothiocyanate: (A) all sperm with intact acrosome in motile sperm isolated by gradient centrifugation; (B) the acrosome-reacted sperm bound to the ZP showing a single fluorescent band at the equatorial segment region. Fertility and Sterility 2013 99, 684-689.e4DOI: (10.1016/j.fertnstert.2012.10.035) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 (A) The marks of Raman scanning corresponding to sperm structure indicated by red Xs. (B) Schematic map of human sperm structure, including acrosome, nucleus, equatorial, neck, midpiece, and tail. Fertility and Sterility 2013 99, 684-689.e4DOI: (10.1016/j.fertnstert.2012.10.035) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions