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SUPPORTING INFORMATION
Optical trapping and micro-Raman spectroscopy of functional red blood cells
using vortex beam for cell membrane studies
Ghanashyam C.
1, Sachin Shetty
3, Sanjay Bharati
3, Santhosh Chidangil
1,2, and Aseefhali
Bankapur
1,2*
1
Department of Atomic and Molecular Physics, Manipal Academy of Higher Education, Manipal,
576104, India
2
Centre of Excellence for Biophotonics, Manipal Academy of Higher Education, Manipal, 576104,
India
3
Department of Nuclear Medicine, Manipal College of Health Professions, Manipal Academy of
Higher Education, Manipal, 576104, India
*email:
[email protected]
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1. Raman experiment on Dye-Polystyrene system
The dye-polystyrene system was prepared by spreading polystyrene bead suspension on a fused
silica coverslip coated with Rhodamin-6G (Rh-6G) dye. Also, the size of the polystyrene bead
(~16 µm) was selected to just fit in the central null region of the donut beam, so that the donut
beam excites only the analyte (Rh-6G) surrounding the bead.
In the preliminary study, the micro-Raman spectra of a dye-polystyrene system were recorded
using both the Gaussian and vortex beam to test the advantage of using vortex beams for
membrane-targeted measurements. Figure 1S shows the comparison of Raman spectra of the
dye-polystyrene system recorded using both the beam profiles. As expected, the spectrum recorded
using the Gaussian beam is dominated by the peaks from polystyrene and that recorded using the
vortex beam has intense peaks from Rh-6G. This is because, while using the vortex beam, the
Raman signal is contributed from Rh-6G which forms the boundary of polystyrene bead, and with
the Gaussian beam the signal is arriving mostly from polystyrene bead. The experiment was
repeated to confirm the reliability and reproducibility of the obtained spectra.
600 800 1000 1200 1400 1600 0 500 1000 1500 Dye-polystyrene system Poly Poly Vortex Excitation
Int
ensi
ty
(a.u
)
Raman Shift (cm
-1)
Gaussian Beam Vortex Beam Gaussian Excitation Rh-6 G Rh-6 G Rh-6 G Rh-6 G Poly Poly Poly Rh-6 G Poly / Rh-6 G PolyFigure 1S. Comparison of Raman spectra of dye-polystyrene system recorded using
Gaussian and vortex beams. Laser power at 785 nm: 16 mW. Acquisition time for Gaussian
beam:10 s and vortex beam: 60 s
The vortex beam spectra of the dye-polystyrene system show three prominent peaks at 617, 996,
and 1026 cm
-1from polystyrene. The contribution of polystyrene is about 4.6% in the vortex beam
spectrum as compared to that in the Gaussian beam spectrum. The fractional contribution of
polystyrene is calculated by taking the peak intensity ratio at 996 cm
-1while normalizing the
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spectral intensities for 10 sec of acquisition. The results suggest that, while using the vortex beam
for excitation of the biological cell, about 5% contribution from the cytoplasm is inevitable.
2. Vortex Beam Trapping
Figure 2S: Schematic representation of the slipping of trapped RBC to an edge-on
orientation over cell transport. This escape from face-on orientation is probably because of an
imbalance in force due to the removal of one part of the cell from light while still keeping the
opposite part in the field when the cell experiences inertia after the transport.
3. Raman Frequency Assignments for single cell spectra of RBCs
Table 1S: Raman frequencies and corresponding band assignments for RBC trapped using
Gaussian and vortex beam at low power density (~ 3.6 ×10
4W/cm
2)
Sl.No. Vortex Gaussian Assignment
Sl.
No. Vortex Gaussian Assignment 1 562* --- ν(Fe–O
2)1 50 1179 --- Tyr, Arg 2
2 570 570 Thr 3 51 --- 1211# δ(C
mH) 1
3 588 587 δ(pyr deform)sym1 52 1216 --- ???
4 633 --- Glut 4 53 1221* 1222 δ(C
mH) 1
5 638 640 Tyr 5, p:C-S str 6 54 1227 --- Amide III (β- sheet) 7
6 652 --- Met 8 55 1232 1230 δ(C
mH) 1
7 672* 672 p:C-S str 9, ν7 (pyr
def)sym 1 56 1285 1285 Methylene wag
8 --- 676# ν
7 (pyr def)sym1 57 1291 1291 Lys, Arg 2
9 681 680 Cys 8 58 1303 1303 δ(C
aH=)2, PC, PI 10
10 685 --- Cys 8 59 1311 1309 Glut 4, p: Cα-H def 9,
Gal 11
11 702 701 Met 8 60 1329 1334 Trp 5, Gal 11, Fuc 12,
Glu 13
12 728 727 Amide V 6, PS 10 61 1338 1339 Phe, Tyr 5
13 743 743 Phe 5 62 1340 --- p: C
α-H def 9, Trp
14 749 749# ν15(pyr breathing),
γ1(pyr foldasym) 1
63 1343* 1345 ν
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15 752 754 νγ15(pyr breathing),1(pyr foldasym) 1 64 1358 --- Hist
14, Lys, Arg 2
16 756 --- Trp 5 65 1368 1367 δ(CH3) umbrella
mode 15
17 782 784 66 1373* 1373 Man11, ν4(pyr
half-ring)sym 1
18 787 --- PS 10 67 1377 1377 Gal, , Sia 16
19 792 794 68 1383 1384 Gal 11
20 800 801 69 1390 1390 Gal-Amine 12
21 806 --- Tyr 5 70 --- 1395 ν
20 (pyr half-ring)sym 1
22 825 823 71 1397* 1398 ν
40(pyr half-ring)sym 1
23 832 833 Tyr
5, O.P-O diester
antisym str, Gal, Man 11 72 1402 --- ν29(pyr half-ring)sym 1
24 853 852 Tyr 5, Lys 2, Sia 16 73 1417 --- Lys, Arg 2
25 --- 860 74 1427 1429 ν28(CαCm)sym 1
26 989 --- Pro 17 75 1436 Trp5, Sia 16
27 994 --- 76 1443 1442 Hist 14
28 999 999 Phe 5 77 1448 1446 Arg 2, PC, PE, PI 10
29 1017 1019 78 1453 1451, 55 p: CH2 and CH3 asym. def. 30 1005 1006 Cβ-vinyl str 1 79 1459 1461 Glut 4, Man 11, Glu 13, Sia 16, Trp 5 31 --- 1024 Phe 5 80 1520 1522# ν 38 1
32 --- 1033 Lys 2, Sia 16, Man 11 81 --- 1537 ???
33 1042 1045 Tyr 5, p: C-N str 82 1543 --- ν 11(CβCβ) 1 34 1048 1048 ??? 83 1548 1548# ν 11 (CβCβ) 1 35 1050 1051 ??? 84 1553 --- Trp 5, Amide II, Ace-Glu 36 1068 1065 Lys 2, PC, PE, PI, SM 10 85 1559* 1561 ν
2 (CβCβ) 1 37 1072 1075 Glu 13 86 1564* --- ν 2 (CβCβ) 1 38 1103 1104 p: C-N 18, Man 11 87 --- 1574 Hist 14 39 1109 --- p: C-N 18, Fuc 12, Sia 16 88 1577 --- Trp 5 40 1114 1116# ??? 89 1582 1583 Trp 5, ν 371 41 1119 1119 Glu 13 90 1589 --- ??? 42 --- 1122 ν5(CβC1)sym 1 91 1598 1597 ??? 43 --- 1124 p: C-N 18, PE, PI 10, Gal-Amine12 92 1604 1602 Phe 5, ν19 1 44 1128 --- 93 --- 1607# Phe 5, ν 10 1
45 1133 1133 p: C-N 9, SM 10, Fuc12 94 1616* 1617 Tyr 5, ν(Ca=Cb)vinyl
46 1149 --- ν441, Glu 95 1628 1629 Ace-Glu
47 1157 1155 p: C-C,C-N str
18, Gal
11, Fuc, Gal-Amine 12 96 --- 1634 Amide I (β-sheet) 19
48 1167 1165 Arg 2 97 1637* 1637 ν
10 1
49 1170 1172 ???
Amino Acids: Trp-Tryptophan, Tyr-Tyrosine, Phe-Phenylalanine, Hist-Histidine, Arg-Arginine, Lys-Lysine, Cys- Cysteine, Pro-Proline, Met- Methionine, Glut-Glutamic Acid, and Thr-Threonine; Lipids: PC-phosphatidylcholine, PE-phosphatidylethanolamine, PS-phosphatidylserine, PI-phosphatidylinositol, and SM-Sphingomyelin; $Carbohydrates: Gal-galactose, Man-mannose, Glu-glucose, Fuc-fucose, and Sia-sialic,
(Ace-Glu)-(acid N-acetyl-D-glucosamine) and, (Ace-Gal)-(N-acetyl-D-galactosamine). Bold- relative intense peak, *-oxy-marker, #- deoxy-marker
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Table 2S: Raman frequencies and corresponding band assignments for RBC trapped using Gaussian and vortex beam at high power density (~
15.4 ×10
4W/cm2) and frequency assignment corresponding to Hb free ghost pellets
Sl. No.
Raman Frequencies Assignments
Gaussian Vortex Membrane Heme 1 Protein Lipids 10, 20 Carbohydrates
11-13, 16 Coordinates
1 532 533 532 p:S-S (t-g-t)
2 539 538 Glu, Sia δ(CCC, CCO, OCO)
3 545 544 543 p:S-S (t-g-t) 4 546, 549 546 Cholesterol 5 551 554 554 Ace-Glu 6 563 562 --- ν(Fe–O2) 7 569 568 567 Thr COO- rock 3
8 586 --- --- ν48 δ(pyr deform)sym
9 --- 631 --- Glut δ(COOH) 4
10 --- --- 643 Tyr, p:C-S str 6 C
γ-Cδ1-Cε15
11 --- 648 648 Sia δ(ring) + ρ(CH2)
12 655 656 Met Cγ-Sδ8
13 662 662 --- p:C-S str 9 Gal β(CCC), β(CCO), β(OCO)
14 669 669 --- p:C-S str 9 Fuc
15 673 674 674 ν7 p:C-S str 9 ν(pyr def)sym
16 678 680 682 Cys Cβ-Sγ8
17 --- 685 --- Cys Man Cβ-Sγ8
18 --- 692 --- ???
19 --- 695 696 ???
20 700 699 Met ν(C-S) 8
21 703 703 703 Cholesterol Gal β(CCC), β(CCO), β(OCO)
22 727 728 730 Amide V 6 PS Serine residue
23 734 --- --- s ???
24 --- 745 747 Phe ω(Cζ-H) 5
25 753 753 ν15, γ1 --- --- ν (pyr breathing), Pyr foldasym
26 --- --- 756 Trp Cε2-C2 5
27 785 784 785 Hist PS ω(Cε-H) 14
28 --- 806 806 Tyr ω(Cε1-H) 5
29 --- 813 --- Man
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31 --- 835 835 Tyr O.P-O diester antisym
str Gal, Man ω(Cε2-H)
5, ν(CC)
32 --- 843 841 Glu ν(CC)
33 --- 850 850 Tyr, Lys Sia ω(Cε2-H)
5, Cγ-twist 2, δ(CH,
CH2)
34 --- 855 855 Hist ω(Cε-H) 14
35 879 878 875 PC, SM Fuc
36 --- --- 871 PS Gal-Amine
37 --- 883 883 Trp Man, Sia Cε3-C ζ3-Cη5, ν(C–O–C)
38 885 886, 88 Gal-Amine
39 892 891 891 PC, PI, Palmitic acid Gal ν(CC)
40 --- 896 --- p: C-C skeletal 21 Sia δ(C–H) + τ(C–H2) 41 901 902 898 Lys Cγ-Cδ 2 42 --- 906 905 PC, PS 43 --- 915 --- Glu 44 925 925 926 p: N–Cα–C 9
45 930 929 931 p: N–Cα–C 9, Glut Man, Ace-Glu γ(OH) 4
46 940 --- --- ν32 δ(pyr deform)asym
47 --- 944 --- Sia ν(C–O–C) + ν(C–O)
48 953 --- 953 ???
49 972 --- --- ν(Cc-Cd)
50 977 975 --- ν(Cc-Cd) ν(Cc-Cd)
51 ---- 989 988 Pro Ring str 17
52 999 999 999 Phe Gal, Sia Cγ-Cδ1
5 β(CCH, CCO), ν(C–C)ring 53 1005 --- -- Cβ-vinyl str 54 1014 1011 1008 Trp C ζ3-Cη 5 55 1026 1027 1025 Phe Cε1-Cζ 5 56 1030 --- --- δ(=CbH2)4 δ(=CbH2)4
57 --- 1032 1032 Lys Sia, Man Cε-Nζ2, ν(O–C)
58 --- 1044 1042 Tyr, p: C-N str Cγ-Cδ2-Cε2 5
59 --- 1049 --- ???
60 --- 1054 1054 Arg Gal, Glu Cγ-twist
2
ν(CC), ν(CO), β(COH)
61 --- 1059 1060 Glut, Asp Gal-Amine ν(CN), ν(CC)
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63 1066 1066 1068 Lys PC, PE, PI, SM Cδ-Cε2
64 1071 1069 1073 Glut Gal, Sia ν(CN), ν(CC) ν(CC), ν(CO), β(COH), δ(C–H)
65 --- 1075 1077 Glu
66
67 1082 1081 --- δ(=CbH2)4 δ(=CbH2)4
68 1089 1088 1089 Hist, Arg PC, PS, PE Man Cδ-Nε 14, CζNη1H2 asym bend 2
69 1094 1094 1094 PI Ace-Glu,
Gal-Amine, Sia ν(C–C)ring
70 1098 1099 --- δ(=CbH2)as δ(=CbH2)as 71 1105 1105 --- p: C-N 18 Man 72 1111 1111 1111 p: C-N 18 Fuc, Sia ν(C–C) ring 73 --- 1116 1116 ??? 74 --- --- 1119 Glu 75 1121 --- --- ν5 ν(CβC1)sym 76 1124 1124 1124 p: C-N 18 PE, PI Gal-Amine C-C str 77 1131 --- 1131 p: C-N 9 SM Fuc
78 --- 1140 1139 Gal, Sia ν(CO),ν(CC), τ(CH2)
79 1146 1146 Gal-Amine
80 1152 1150 ν44 Glu ν(CβC1)sym
81 --- --- 1155 p: C-C,C-N str 18 Gal, Fuc,
Gal-Amine ν(CO),ν(CC)
82 1159, 61 1159 Hist, CH3 rock 6 Nε-Cε 14
83 1167 1166 1166 Arg Cβ-Cα-Hα2
84 1172 1172
85 --- --- 1177 Tyr, Arg Cδ1-Cε1-H, CζNη2H2 asym bend
86 1180 1183 1181 Lys, p: C-H bend 18 C
ε-rock 2
87 --- 1204 1201 Phe Cδ1 -Cε1 5
88 --- --- 1208 Phe, Tyr Cδ1 -Cε1, Cβ-twist5
89 1209 1212 ν5+ ν18 δ(CmH) 90 --- 1219 --- ??? 91 1221 1224 --- ν13 δ(CmH) 92 --- --- 1229 Amide III (β- sheet) 7 93 1231 1233 --- ν42 δ(CmH)
94 --- --- 1238 Hist Gal-Amine Cβ-twist 14
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(β- sheet) 7, 9 96 1249 1248 1247 Amide III (β- sheet) 9 Irregular/turns 7 Gal 40% ν(C–N)+30% δ(N–H) τ(CH2) 97 --- 1252 1251 Tyr Cζ-O-H 598 --- 1259 1257 Trp Fuc, Ace-Glu, Sia Cε2-Nε1 5
99 1265 1262 1265 Amide III (α-helix) 9, Tyr, Hist PC, PS, PI Man, Gal-Amine Cζ-O 5, Nδ-Cε-H 14
100 1267 --- --- 101 1274 1273 1273
Trp,
Amide III (α-helix)
9
Fuc Cε2-C ζ2-H 5
102 1284 1283 --- Methylene wag
103 --- 1287 --- Hist Cγ-Nδ 14
104 1294 1294 1293 Lys, Arg Cβ-twist, Cγ-rock 2
105 --- 1297 1296 PS, PE, SM Glu
106 1302 1302 -- δ(CaH=)2
107 1304 1305 1304 --- --- PC, PI
108 1307 --- --- ν21 δ(CmH)
109 1309, 14 1312 1315 Glut, p: Cα-H def 9 Gal ω(CH2)
110 1322 1320 1321 Hist, Arg Ace-Glu Cβ-rock 14, Cβ-twist 2
111 1328, 34 1332 1334 Trp Gal, Fuc, Glu Cβ-rock 5, ω(CH2)
112 1338 1339 1337 Phe, Tyr Cγ-Cδ1-H, Cγ-Cδ25
113 --- 1343 1341 p: Cα-H def 9, Trp Cδ2-Cγε2
114 1345 1343 --- ν41 ν(pyr half-ring)sym
115 1352 1351 1350 Trp Man Cδ2-Cγ 5
116 --- --- 1355 Hist, Lys, Arg Nδ-Cε 14, Cδ-twist 2
117 1365 --- --- ???
118 --- 1368 1368 ???
119 1370 1370 -- ν4 Man ν(pyr half-ring)sym
120 1375 1375 1376 Gal, Sia ν(pyr half-ring)sym
ω(CH2), ν(C–C)
121 --- 1377 1377 Gal, Sia ω(CH2)
122 --- --- 1382 Gal
123 1387 1388 1387 ν12 Gal-Amine ν(pyr half-ring)sym
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125 --- 1394 --- ν20 ν(pyr quarter-ring) 126 1398 1400 --- ν40 ν(pyr quarter-ring) 127 1403 1405 --- ν29 ν(pyr quarter-ring) 128 1409 --- --- ν29 ν(pyr quarter-ring) 129 --- --- 1411 Hist Cβ-Cα-H 14 130 --- --- 1413 Phe Cβ-Cα-H 5131 1416 1416 1418 Lys, Arg Cδ-rock, Cβ-rock 2
132 1421 --- --- ???
133 --- 1425 --- ???
134 1430 1428 -- ν28 ν(CαCm)sym
135 --- 1433 1433 Trp Sia Cε2-Nε1-H 5, δ(C–H)
136 1440 --- 1438 Hist Cβ-rock 14
137 --- 1444 1445 Arg PC, PE, PI Nη1-Cζ-Nη2 Asym str 2
138 1448 1453 1450 p: CH2 and CH3
asym. def. PC, PS, PE, PI: CH def Fuc
139 1457 1458 1457 Glut Man, Glu, Sia δ(CH2), δ(CH2)
140 1461 --- 1463 Trp Cβ-bend 5
141 1472 1472 1468 Lys, Arg Ace-Glu Cε-bend, Cδ-bend 2
142 --- 1485 --- Gal δ(CH2) 143 1490 1490 --- ν3 ν(CαCm)sym 144 1498 1498 1497 Hist Cε- Nε14 145 --- 1500 1501 ??? 146 1507 1506 1505 ??? 147 1516 1516 1514 Tyr Cδ1-Cε1-H 5 148 1520 1518 --- ν38 ν(CβCβ) 149 1526 --- 1528 ??? 150 1543 1544 ---- ν11 --- --- ν(CβCβ)
151 --- 1551 1550 Trp, Amide II, Ace-Glu Cγ-Cδ15
152 1562 1561 1562 ν2 ν(CβCβ) 153 1568 --- --- ??? 154 --- --- 1571, 74 Hist Cγ-Cδ14 155 1578, 80 1579 1580 Trp Cδ2 -Cε3 5 156 1584 1584 --- ν37 --- --- ν(CαCm)asym 157 1600, 04 1603 --- ν19 Phe --- ν(CαCm)asym , Cδ2 -Cε2 5 158 --- --- 1606 Phe Cδ2 -Cε2 5 159 --- --- 1614 Tyr Cδ1-Cε1 5
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160 1616 1617 --- ν(Ca=Cb)vin yl Tyr Ca=Cb, Cδ1-Cε1 5 161 --- --- 1620 Trp Cε2 -Cζ2 5 162 1625 1627 Ace-Glu 163 1632 1632 --- ν(Ca=Cb)vin yl 164 --- --- 1635 Amide I (β-sheet) 19165 --- 1642 1642 Amide I (β-sheet) 19 80% ν(C=O)
166 1647 1648 1648 Amide I
(unordered) 19 80% ν(C=O)
167 1651 1652 1651, 53 ???
168 1658 1658 1661 Amide I (α-helix) 19 PC,PS,PI Sia 80% ν(C=O), ν(C=C), ν(C=O) 169 1673 --- 1665, 70, 75 Amide I (turn) 19 80% ν(C=O) 170 1681, 87 1682 1680 Amide I (turn) 19 [a] 80% ν(C=O)
*Amino Acids: Trp-Tryptophan, Tyr-Tyrosine, Phe-Phenylalanine, Hist-Histidine, Arg-Arginine, Lys-Lysine, Cys- Cysteine, Pro-Proline, Met- Methionine, Glut-Glutamic Acid, and Thr-Threonine; #Lipids: PC-phosphatidylcholine, PE-phosphatidylethanolamine, PS-phosphatidylserine, PI-phosphatidylinositol, and
SM-Sphingomyelin; $Carbohydrates: Gal-galactose, Man-mannose, Glu-glucose, Fuc-fucose, and Sia-sialic, (Ace-Glu)-(acid N-acetyl-D-glucosamine) and,
