— Supplementary Information—
Segmented Frequency-domain Fluorescence Lifetime Measurements: Minimizing the Effects of Photobleaching within a Multi-component System
Hadi M. Marwani1, Mark Lowry1, Patrick Keating1, Isiah M. Warner1, Robert L. Cook1,2*
1Department of Chemistry, LouisianaStateUniversity, Baton Rouge, LA70803
2 Department of Chemistry, Southern University, Baton Rouge, LA70813
*Correspondence to: Robert L. Cook
E-mail:
Tel: (225) 578-2980
Supplementary Information Table of Contents
PageContents
S-2Fig. 1S Data and fit of fluorescence emission spectra for Fl (0.02 µM), RhB (1 µM), and their mixture in 0.1 M NaOH excited at 490 nm
S-3Fig. 2S a Experimental frequency-domain data of pyrene (0.1 ppm) in argon degassed aqueous solution. b Normalized χ2 surfaces for NLLS analysis of the experimental data
S-4Fig. 3S a Experimental frequency-domain data of SRFAR (12 ppm) in argon degassed aqueous solution. b Normalized χ2 surfaces for NLLS analysis of the experimental data
S-5Fig. 4S Normalized χ2 surfaces for NLLS analysis of the pyrene and SRFAR mixture experimental data fora DDDD and b DfDfDfD models
S-6Fig. 5S Simulated frequency-domain LR data for pyrene and SRFAR mixture
S-7Fig. 6S Histograms of random errors used in the simulated data
S-8Fig. 7S Simulated frequency-domain LR and CR data after 0.5 degree and 0.005 random noise was added
S-9Fig. 8SThea lifetime, b fractional contribution, and cχ2for pyrene and SRFAR mixture of the simulated data after 0.5 degree and 0.005 random noise was added for the (D+D+D)FD model
S-10Fig. 9S The a lifetime, b fractional contribution, and cχ2for pyrene and SRFAR mixture of the simulated data after 0.5 degree and 0.005 random noise was added for the DDDD model
S-11Table 1S Fluorescence lifetimes and fractional contributions obtained from NLLS analyses for the (D+D+D)FD model of pyrene and SRFAR mixture after adding different random noises of equivalent magnitude
S-12Table 2SFluorescence lifetimes and fractional contributions obtained from NLLS analyses for the DDDD model of pyrene and SRFAR mixture after adding different random noises of equivalent magnitude
Fig. 1S Data and fit of fluorescence emission spectra for Fl (0.02 µM), RhB (1 µM), and their mixture in 0.1 M NaOH excited at 490 nm
a
b
Fig. 2S a Experimental frequency-domain data of pyrene (0.1 ppm) in argon degassed aqueous solution. b Normalized χ2 surface for NLLS analysis of the experimental data. Horizontal dashed line: one standard deviation from the minima of the χ2 surface. Corresponding fitting parameters are tabulated in Table 3
a
b
Fig. 3S a Experimental frequency-domain data of SRFAR (12 ppm) in argon degassed aqueous solution. b Normalized χ2 surfaces for NLLS analysis of the experimental data. Horizontal dashed lines: one standard deviation from the minima of the χ2 surface. Corresponding fitting parameters are tabulated in Table 3
a
b
Fig. 4S Normalized χ2 surfaces for NLLS analysis of the pyrene and SRFAR mixture experimental data foraDDDD and bDfDfDfD models. Horizontal dashed lines: one standard deviation from the minima of the χ2 surface. Corresponding fitting parameters are tabulated in Table 3
Fig. 5S Simulated frequency-domain LR data for pyrene (= 181.89 ns,initial= 0.379) and SRFAR (initial=0.621,= 0.57 ns,1= 0.281, = 3.43 ns,2= 0.483, = 13.71 ns,3= 0.236) mixture. LR data were simulated assuming (0%, 0%), (20%, 0%), (50%, 0%), (0%, 20%), (0%, 50%) photobleaching for both pyrene and SRFAR respectively
Fig. 6S Histograms of random errors used in the simulated data
Fig. 7S Simulated frequency-domain LR and CR data after 0.5 degree and 0.005 random noise was added. Parameters: pyrene (= 181.89 ns,initial= 0.379) and SRFAR (initial=0.621,= 0.57 ns,1= 0.281, = 3.43 ns,2= 0.483, = 13.71 ns,3= 0.236) mixture. A 20% pyrene bleaching was assumed in the time the LR was exposed to radiation
a
b
c
Fig. 8SThea lifetime, b fractional contribution, and cχ2for pyrene and SRFAR mixture of the simulated data after 0.5 degree and 0.005 random noise was added for the (D+D+D)FD model. Horizontal dashed lines: ± one standard deviation from the average (horizontal solid lines). Corresponding fitting parameters are tabulated in Table 1S
a
b
c
Fig. 9S The a lifetime, b fractional contribution, and cχ2for pyrene and SRFAR mixture of the simulated data after 0.5 degree and 0.005 random noise was added for the DDDD model. Horizontal dashed lines: ± one standard deviation from the average (horizontal solid lines). Corresponding fitting parameters are tabulated in Table 2S
Table 1S Fluorescence lifetimes and fractional contributions obtained from NLLS analyses for the (D+D+D)FD model of pyrene and SRFAR mixture after adding different random noises of equivalent magnitude. UB denote the Unbleached run. N represents the added noise
Set / Model / αSRFAR / α1 / τ1 (ns) / α2 / τ2 (ns) / α3 / τ3 (ns) / α4 / τ4 (ns) / χ2UB N1 / (D+D+D)FD / 0.6208 / 0.28 / 0.57 / 0.48 / 3.43 / 0.24 / 13.71 / 0.3792 / 183.5811 / 1.3065
UB N2 / (D+D+D)FD / 0.6206 / 0.28 / 0.57 / 0.48 / 3.43 / 0.24 / 13.71 / 0.3794 / 182.7638 / 1.4342
UB N3 / (D+D+D)FD / 0.6206 / 0.28 / 0.57 / 0.48 / 3.43 / 0.24 / 13.71 / 0.3794 / 183.0103 / 1.3420
UB N4 / (D+D+D)FD / 0.6198 / 0.28 / 0.57 / 0.48 / 3.43 / 0.24 / 13.71 / 0.3802 / 181.0744 / 1.2725
UB N5 / (D+D+D)FD / 0.6208 / 0.28 / 0.57 / 0.48 / 3.43 / 0.24 / 13.71 / 0.3792 / 184.2694 / 1.2819
UB N6 / (D+D+D)FD / 0.6210 / 0.28 / 0.57 / 0.48 / 3.43 / 0.24 / 13.71 / 0.3790 / 183.5453 / 1.3919
UB N7 / (D+D+D)FD / 0.6196 / 0.28 / 0.57 / 0.48 / 3.43 / 0.24 / 13.71 / 0.3804 / 181.7271 / 1.3030
UB N8 / (D+D+D)FD / 0.6213 / 0.28 / 0.57 / 0.48 / 3.43 / 0.24 / 13.71 / 0.3787 / 183.6320 / 1.3385
UB N9 / (D+D+D)FD / 0.6191 / 0.28 / 0.57 / 0.48 / 3.43 / 0.24 / 13.71 / 0.3809 / 182.6028 / 1.3085
UB N10 / (D+D+D)FD / 0.6202 / 0.28 / 0.57 / 0.48 / 3.43 / 0.24 / 13.71 / 0.3798 / 182.7667 / 1.2440
Average / (D+D+D)FD / 0.6204 / 0.28 / 0.57 / 0.48 / 3.43 / 0.24 / 13.71 / 0.3796 / 182.8973 / 1.3223
LR N1 / (D+D+D)FD / 0.6427 / 0.28 / 0.57 / 0.48 / 3.43 / 0.24 / 13.71 / 0.3573 / 190.7667 / 2.1746
LR N2 / (D+D+D)FD / 0.6448 / 0.28 / 0.57 / 0.48 / 3.43 / 0.24 / 13.71 / 0.3552 / 192.9314 / 1.9826
LR N3 / (D+D+D)FD / 0.6448 / 0.28 / 0.57 / 0.48 / 3.43 / 0.24 / 13.71 / 0.3552 / 192.2744 / 2.1280
LR N4 / (D+D+D)FD / 0.6445 / 0.28 / 0.57 / 0.48 / 3.43 / 0.24 / 13.71 / 0.3555 / 193.2360 / 2.0078
LR N5 / (D+D+D)FD / 0.6432 / 0.28 / 0.57 / 0.48 / 3.43 / 0.24 / 13.71 / 0.3568 / 190.5144 / 1.9914
LR N6 / (D+D+D)FD / 0.6425 / 0.28 / 0.57 / 0.48 / 3.43 / 0.24 / 13.71 / 0.3575 / 187.7311 / 1.8975
LR N7 / (D+D+D)FD / 0.6454 / 0.28 / 0.57 / 0.48 / 3.43 / 0.24 / 13.71 / 0.3546 / 190.9960 / 1.6453
LR N8 / (D+D+D)FD / 0.6430 / 0.28 / 0.57 / 0.48 / 3.43 / 0.24 / 13.71 / 0.3570 / 189.3301 / 1.9278
LR N9 / (D+D+D)FD / 0.6436 / 0.28 / 0.57 / 0.48 / 3.43 / 0.24 / 13.71 / 0.3564 / 189.4139 / 2.1600
LR N10 / (D+D+D)FD / 0.6433 / 0.28 / 0.57 / 0.48 / 3.43 / 0.24 / 13.71 / 0.3567 / 187.5523 / 1.9304
Average / (D+D+D)FD / 0.6438 / 0.28 / 0.57 / 0.48 / 3.43 / 0.24 / 13.71 / 0.3562 / 190.4746 / 1.9845
CR N1 / (D+D+D)FD / 0.6272 / 0.28 / 0.57 / 0.48 / 3.43 / 0.24 / 13.71 / 0.3728 / 181.3705 / 1.4083
CR N2 / (D+D+D)FD / 0.6281 / 0.28 / 0.57 / 0.48 / 3.43 / 0.24 / 13.71 / 0.3719 / 184.7355 / 1.5217
CR N3 / (D+D+D)FD / 0.6286 / 0.28 / 0.57 / 0.48 / 3.43 / 0.24 / 13.71 / 0.3714 / 184.5163 / 1.5058
CR N4 / (D+D+D)FD / 0.6289 / 0.28 / 0.57 / 0.48 / 3.43 / 0.24 / 13.71 / 0.3711 / 186.1251 / 1.5661
CR N5 / (D+D+D)FD / 0.6293 / 0.28 / 0.57 / 0.48 / 3.43 / 0.24 / 13.71 / 0.3707 / 186.6040 / 1.3365
CR N6 / (D+D+D)FD / 0.6284 / 0.28 / 0.57 / 0.48 / 3.43 / 0.24 / 13.71 / 0.3716 / 182.8157 / 1.5438
CR N7 / (D+D+D)FD / 0.6285 / 0.28 / 0.57 / 0.48 / 3.43 / 0.24 / 13.71 / 0.3715 / 183.6359 / 1.5385
CR N8 / (D+D+D)FD / 0.6290 / 0.28 / 0.57 / 0.48 / 3.43 / 0.24 / 13.71 / 0.3710 / 184.9026 / 1.5374
CR N9 / (D+D+D)FD / 0.6283 / 0.28 / 0.57 / 0.48 / 3.43 / 0.24 / 13.71 / 0.3717 / 184.7760 / 1.5713
CR N10 / (D+D+D)FD / 0.6285 / 0.28 / 0.57 / 0.48 / 3.43 / 0.24 / 13.71 / 0.3715 / 184.6661 / 1.5493
Average / (D+D+D)FD / 0.6285 / 0.28 / 0.57 / 0.48 / 3.43 / 0.24 / 13.71 / 0.3715 / 184.4148 / 1.5079
Table 2SFluorescence lifetimes and fractional contributions obtained from NLLS analyses for the DDDD model of pyrene and SRFAR mixture after adding different random noises of equivalent magnitude. UB denote the Unbleached run. N represents the added noise
Set / Model / α1 / τ1 (ns) / α2 / τ2 (ns) / α3 / τ3 (ns) / α4 / τ4 (ns) / χ2UB N1 / DDDD / 0.2025 / 0.6942 / 0.2881 / 3.9065 / 0.1321 / 15.1694 / 0.3773 / 184.6364 / 1.3192
UB N2 / DDDD / 0.1813 / 0.5934 / 0.3297 / 3.7970 / 0.1200 / 19.9890 / 0.3690 / 188.4720 / 1.2483
UB N3 / DDDD / 0.1867 / 0.6312 / 0.2761 / 3.5157 / 0.1527 / 12.2418 / 0.3845 / 179.8925 / 1.3726
UB N4 / DDDD / 0.1636 / 0.5155 / 0.2926 / 3.2746 / 0.1579 / 11.8165 / 0.3860 / 177.5789 / 1.2565
UB N5 / DDDD / 0.1480 / 0.3999 / 0.3141 / 3.1414 / 0.1559 / 12.7141 / 0.3819 / 182.2980 / 1.2009
UB N6 / DDDD / 0.1513 / 0.4218 / 0.3338 / 3.3480 / 0.1382 / 15.1204 / 0.3767 / 184.6898 / 1.2757
UB N7 / DDDD / 0.1747 / 0.5394 / 0.2926 / 3.4666 / 0.1494 / 12.8069 / 0.3833 / 179.8344 / 1.2513
UB N8 / DDDD / 0.1748 / 0.5523 / 0.3220 / 3.6541 / 0.1269 / 16.1118 / 0.3763 / 184.7844 / 1.3667
UB N9 / DDDD / 0.1791 / 0.5918 / 0.3061 / 3.5554 / 0.1343 / 15.1639 / 0.3805 / 182.4397 / 1.3300
UB N10 / DDDD / 0.1630 / 0.5149 / 0.3045 / 3.2856 / 0.1532 / 13.6740 / 0.3792 / 183.0951 / 1.3363
Average / DDDD / 0.1725 / 0.5454 / 0.3060 / 3.4945 / 0.1421 / 14.4808 / 0.3795 / 182.7721 / 1.2957
LR N1 / DDDD / 0.1997 / 0.6340 / 0.3224 / 3.8823 / 0.1090 / 14.0056 / 0.3689 / 182.1143 / 1.6169
LR N2 / DDDD / 0.1926 / 0.6166 / 0.3030 / 3.5145 / 0.1453 / 13.7026 / 0.3592 / 189.6099 / 1.9192
LR N3 / DDDD / 0.2029 / 0.6822 / 0.3005 / 3.7580 / 0.1292 / 12.3668 / 0.3674 / 183.6609 / 1.6769
LR N4 / DDDD / 0.1911 / 0.6342 / 0.3195 / 3.5720 / 0.1270 / 14.6895 / 0.3623 / 187.6884 / 1.5310
LR N5 / DDDD / 0.1911 / 0.6342 / 0.3195 / 3.5720 / 0.1270 / 14.6895 / 0.3623 / 187.6884 / 1.5310
LR N6 / DDDD / 0.2150 / 0.7177 / 0.3039 / 3.9875 / 0.1153 / 14.5331 / 0.3658 / 181.5801 / 1.4620
LR N7 / DDDD / 0.2150 / 0.7177 / 0.3039 / 3.9875 / 0.1153 / 14.5331 / 0.3658 / 181.5801 / 1.4620
LR N8 / DDDD / 0.2112 / 0.6891 / 0.2976 / 3.8981 / 0.1255 / 13.4764 / 0.3656 / 182.9953 / 1.6370
LR N9 / DDDD / 0.2112 / 0.6891 / 0.2976 / 3.8981 / 0.1255 / 13.4764 / 0.3656 / 182.9953 / 1.6370
LR N10 / DDDD / 0.2017 / 0.6734 / 0.3149 / 3.7704 / 0.1216 / 15.3545 / 0.3618 / 183.4274 / 1.6072
Average / DDDD / 0.2032 / 0.6688 / 0.3083 / 3.7840 / 0.1241 / 14.0827 / 0.3645 / 184.3340 / 1.6080
CR N1 / DDDD / 0.1645 / 0.5049 / 0.3258 / 3.4682 / 0.1425 / 15.5910 / 0.3671 / 185.0040 / 1.3842
CR N2 / DDDD / 0.1923 / 0.6352 / 0.3040 / 3.8047 / 0.1374 / 15.6384 / 0.3663 / 186.3729 / 1.6001
CR N3 / DDDD / 0.1911 / 0.6350 / 0.2978 / 3.7080 / 0.1410 / 14.5761 / 0.3701 / 185.9265 / 1.6254
CR N4 / DDDD / 0.1759 / 0.5685 / 0.3218 / 3.6190 / 0.1314 / 15.2066 / 0.3709 / 183.8512 / 1.5807
CR N5 / DDDD / 0.1872 / 0.5899 / 0.2952 / 3.6467 / 0.1468 / 13.7715 / 0.3707 / 185.0009 / 1.4833
CR N6 / DDDD / 0.2030 / 0.6663 / 0.3085 / 4.0759 / 0.1244 / 17.1768 / 0.3641 / 189.3314 / 1.5128
CR N7 / DDDD / 0.1506 / 0.4549 / 0.2892 / 2.8802 / 0.1874 / 11.6380 / 0.3729 / 185.1022 / 1.6199
CR N8 / DDDD / 0.1823 / 0.5917 / 0.3063 / 3.5718 / 0.1370 / 14.0630 / 0.3744 / 182.7348 / 1.6438
CR N9 / DDDD / 0.1838 / 0.5779 / 0.3237 / 3.8359 / 0.1244 / 15.9962 / 0.3681 / 188.1707 / 1.3179
CR N10 / DDDD / 0.1972 / 0.6501 / 0.3001 / 3.8340 / 0.1324 / 15.1573 / 0.3702 / 185.3044 / 1.6249
Average / DDDD / 0.1828 / 0.5874 / 0.3072 / 3.6444 / 0.1405 / 14.8815 / 0.3695 / 185.6799 / 1.5393
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