SUPPLEMENTAL MATERIAL FOR
Label-free Spectroscopic Detection of Membrane Potential Using
Stimulated Raman Scattering
Bin Liu,1 Hyeon Jeong Lee,2 Delong Zhang,3 Chien-Sheng Liao,4 Na Ji,5
Yuanqin Xia,1,a) and Ji-Xin Cheng3,4,a)
1National
Key Laboratory of Science and Technology on Tunable Laser, Harbin Institute of Technology,
Harbin 150080, China
2Interdisciplinary
3Department
a)
Life Science Program, Purdue University, West Lafayette, Indiana 47907, United States
of Chemistry, Purdue University, West Lafayette, Indiana 47907, United States
4Weldon
School of Biomedical Engineering, Purdue University, West Lafayette, Indiana 47907, United States
5Janelia
Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147, United States
Authors to whom correspondence should be addressed. Electronic mails: xiayuanqin@hit.edu.cn and jcheng@purdue.edu.
1
Preparation of erythrocyte ghosts
The erythrocyte ghosts were prepared according to established protocols.1,2 In brief, the
blood of mouse was first diluted with the erythrocyte buffer solution1 at a ratio of 1:20. After
three times’ washing, the erythrocytes were then exposed to ice-cold 5 mM phosphate buffer (pH
8.0) which caused the erythrocytes to lyse. The samples were kept close to 0°C for 2 minutes,
after which the solution was centrifuged for 15 minutes at 18, 000 g and the supernatant was
removed from the pellet. Thereafter the resealing buffer (50 mM NaCl, 5 mM phosphate buffer,
pH 8.0) was added to allow for resealing of the membranes. The resealing process lasted 1 hour
at room temperature. In order to get rid of all hemoglobins from the cells, we repeated the lysis
and resealing procedures twice. When imaged by SRS microscopy, the formed erythrocyte ghost
appears as a spherical vesicle.
Intravesicular and extravesicular ionic compositions
The 3 different transmembrane potentials shown in Fig. 4 were generated by manipulating
the intravesicular and extravesicular ionic compositions. We first replaced resealing buffer with
the desired inside buffer, 65 mM NaCl/75 mM KCl/10 mM Hepes, pH 7.5/0.25 mM MgCl 2, and
equilibrated 16 hours at 4 ℃. Then the constituent of the outside buffer was varied to create 3
different transmembrane potentials according to ref. 3. Specifically, the outside buffer was 140
mM NaSCN, pH 7.5/0.25 mM MgCl2/10 mM Hepes for 10 mV, 65 mM NaCl/75 mM KCl/10
mM Hepes, pH 7.5/0.25 mM MgCl2 for 1 mV and 140 mM NaHepes, pH 7.5/0.25 mM MgSO4
for 10 mV. The inside and outside ionic compositions for the erythrocyte ghost shown in Fig. 3
were 65 mM NaCl/75 mM KCl/10 mM Hepes, pH 7.5/0.25 mM MgCl2 and 140 mM NaCl/10
mM Hepes, pH 7.5/0.25 mM MgCl2, respectively. All experiments shown in the manuscript were
done within 2 hours after establishing transmembrane gradients.
2
SRS Spectrum analysis
For SRS spectrum analysis, we first removed the background contribution from the
surrounding buffer. Then the resultant SRS spectrum was normalized with the two-photon
absorption signal of Rhodamine 6G. Thereafter the SRS spectrum was least-squares fitted as a
sum of seven Lorentzian bands.4,5
7
I SRS ( )  
i 1
2 Ai
i
 4   i  2  i2
,
where ω is the wavenumber, Ai is the area under the ith band, Γi is the width, and Ωi is the center
wavenumber of the ith band. Table SI lists the peak wavenumbers and widths for curve fitting.
Corresponding assignments of Raman peaks can be found in our previous paper.5 An example of
fitted curve parameters is shown in Table SII. The areas under Raman bands at 2930 cm1
(A2930) and 2850 cm1 (A2850) were used to characterize the transmembrane potential induced
SRS spectral profile change.
TABLE SI. Lorentzian fitting parameters.
Peak center
Width
(Ωi, cm)
(Γi, cm)
2845-2855
28.41
2856.5-2870
33.97
2875-2888
33.30
2885-2910
40.61
2930-2945
53.24
2958-2972
39.05
2980-3000
40.61
3
TABLE SII. Typical results of fitted curve parameters (peak center,
width and area under the peak). The numbers in parentheses are the
corresponding standard errors. Reduced Chi-Sqr: 0.01105; Adj. Rsquare: 0.94642.
Peak center
Width
Area under the
peak (a.u.)
(Ωi, cm)
(Γi, cm)
2850.46 (3.44)
28.41
24.45 (9.34)
2869.59 (10.39)
33.97
26.94 (15.62)
2887.48 (13.41)
33.30
23.15 (16.93)
2907.08 (16.46)
40.61
28.31 (18.95)
2931.74 (10.59)
53.24
72.94 (18.86)
2958.01 (11.00)
39.05
18.92 (13.93)
2989.90 (14.94)
40.61
8.77 (5.87)
References
1
J. Guck, R. Ananthakrishnan, H. Mahmood, T. J. Moon, C. C. Cunningham, and J. Kas, Biophys. J. 81 (2), 767
(2001).
2
E. O. Potma and X. S. Xie, J. Raman Spectrosc. 34 (9), 642 (2003).
3
R. B. Mikkelsen, S. P. Verma, and D. F. H. Wallach, Proc. Natl. Acad. Sci. USA 75 (11), 5478 (1978).
4
J.-x. Cheng, A. Volkmer, L. D. Book, and X. S. Xie, J. Phys. Chem. B 106 (34), 8493 (2002).
5
S. Yue, J. M. Cárdenas-Mora, L. S. Chaboub, S. A. Lelièvre, and J.-X. Cheng, Biophys. J. 102 (5), 1215 (2012).
4