Supplementary Figures

Transmission Grating

Dark-field Condenser

Sample

Objective

HQ2 CCD

Beam splitting lens

Color CCD

Transmission Grating

Dark-field Condenser

Sample

Objective

HQ2 CCD

Beam splitting lens

Color CCD

a b

c

532 nm

307 pixels

k = 1.733 nm/pixel λmax = k·d

d

Raw data Gaussian fitc

532 nm

307 pixels

k = 1.733 nm/pixel532 nm

307 pixels

k = 1.733 nm/pixel λmax = k·d

dd

Raw data Gaussian fit Raw data Gaussian fit

Supplementary Figure S1. Illustrations of single particle spectral measurement. (a) Device diagram

of the high-throughput single-particle dark-field spectral microscope. (b) Typical spectral image of PNP

probes absorbed on a glass slide surface. The scale bar is 10 µm. Notice that there is some overlap

between zero- and first-order images from different particles. However, since the first-order streaks are

always on the right side of the corresponding zero-order spots and have about the same peak-to-peak

distance, the spectrum of almost every particle is readily derived. (c) By calibrating using a 532 nm laser,

1 pixel is found to correspond to 1.733 nm. Gaussian fitting to the line graph of the first order image gives

the location of the spectral intensity maximum in pixels. The spectral maximum in nm can subsequently

be obtained.

0 2 4 6 8 10 12 14 160

20

40

60

80

100

λmax

shift

(nm

)

Time (min)

100 µM 50 µM 10 µM

400 500 600 700 800 900 10000.0

0.2

0.4

0.6

0.8

1.0 0 µM 10 µM 50 µM 100 µM

Ext

inct

ion

Wavelength (nm)

1.0 1.5 2.0 2.5 3.0048

121620242832

Freq

uenc

y (%

)

Aspect Ratioc

a b

d

Supplementary Figure S2. Characterization of the prepared 55×27 nm AuNR-Ag nanoprobes with

a 3 nm Ag shell optimized for dark-field imaging. (a) TEM image. (b) Aspect ratio distribution. Over

300 particles were counted. (c) Normalized extinction spectra of the PNP solution after adding 0, 10, 50

and 100 µM of Na2S. (d) Time courses of Δλmax with the addition of 10, 50 and 100 µM of Na2S obtained

via UV-Vis measurement.

0 2 4 6 8 10 12 14 16 18 20

0

10

20

30

40

50

60

70

80

-10 -8 -6 -4 -2 0 2 4 6 8 100.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

Δλmax (nm)F

requ

ency

(%

)

GSH & NaCl

0 20 40 60 80 1000.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

Fre

quen

cy (

%)

Δλmax (nm)

Na2S

0 20 40 60 80 1000.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

Fre

quen

cy (

%)

Δλmax (nm)

Cys & NaCl & Na2S

0 20 40 60 80 1000.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40F

requ

ency

(%

)

Δλmax (nm)

CSH & NaCl & Na2S

Time (min)

Δλm

ax (

nm)

Cys & NaCl & Na2S GSH & NaCl & Na2S Na2S

-10 -8 -6 -4 -2 0 2 4 6 8 100.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

Fre

quen

cy (

%)

Δλmax (nm)

Cys & NaCla

f

b c

d e

Supplementary Figure S3. Selectivity of AuNR-Ag nanoprobes in the presence of biothiols and

NaCl at single-particle level. Distribution of single PNP spectral shifts after adding (a) 5 mM cysteine

and 150 mM NaCl, (b) 5 mM glutathione and 150 mM NaCl, (c) 5 µM Na2S, (d) 5 mM cysteine, 150

mM NaCl and 5µM Na2S, and (e) 5 mM glutathione, 150 mM NaCl and 5 µM Na2S. The averaged single

PNP spectral shifts in the five cases are (a) 1.52 ± 2.22 nm, (b) 1.80 ± 2.46 nm, (c) 73.03 ± 10.85 nm, (d)

76.37 ± 9.99 nm, and (e) 77.89 ± 10.76 nm, respectively. (f) Typical time-dependent spectral shifts of

single PNPs with or without the addition of biothiols and NaCl. Thus the interference from biothiols and

NaCl is negligible.

.

0 5 10 15 20 25 30 350

20

40

60

80

100

120

140λm

ax sh

ift (n

m)

CS,NP ( nM)

Supplementary Figure S4. Calculated λmax shifts of a 55×27 nm AuNR-Ag nanoprobe as a function

of the amount of sulfide consumed. The effective volume is designated to be 2.34×10-5 µL, which is

equivalent to the mean volume of sulfide ion diffusion in 1 s.

Supplementary Figure S5. Image processing procedures for retrieving single PNP spectra inside

live-cells. Because the scattering intensities of the nanoprobe are much higher than that of cellular

organelles, the zero-order (position) and first-order (spectrum) PNP images can be readily identified even

in the raw image (a). Image qualities are obviously improved via background subtraction (b). The

enlarged image is shown in (c). Comparing with the colour CCD image (d) leads to discrimination of red-

orange PNPs from white large intracellular organelles. The scale bar is 10 µm.

Supplementary Figure S6. Time dependent λmax fluctuation of two PNPs in the cytoplasm of a live

HeLa cell in the absence of Na2S. The standard deviations are 2.0 nm and 2.2 nm, respectively.

Supplementary Figure S7. Dark-field images of PNPs in live-cells. Original (a) and the enlarged (b)

spectral images of PNPs inside live HeLa cells after background subtraction that correspond to the colour

image (c) containing the two PNPs in Figure 5. The scale bar is 10 µm.

0 5 10 15 20 25 30 35 40590600610620630640650660670

0 5 10 15 20 25 30 35 40590600610620630640650660670

Probe 1 Probe 2

λmax

(nm

)

Time (min)

b

λmax

(nm

)

Time (min)

a

Supplementary Figure S8. Measured and calculated time-dependent λmax shifts of the nanoprobes

inside the cell. (a) Observed (hollow dots) and fitted (solid lines) time-dependent λmax shifts of the two

PNPs, P1 and P2, in Figure 5. (b) Simulated time-dependent λmax shifts of P1 (red) and P2 (green) if they

were exposed to the same concentrations of sulfide. Solid circles, 100 nM; open triangles, 10 nM. Since

the slopes for P1 and P2 are very different in a, the local sulfide concentrations experienced by P1 and P2

must be different.

a

c

b

d

a

c

b

d

0 5 10 15 20 25 30

600

620

640

660

680

700

Time (min)

λmax

(nm

)

negative control probe 3 probe 4 probe 5 probe 6

e

Supplementary Figure S9. Real-time detection of endogenous H2S in live-cells. Dark-field colour

images (a, c) and the corresponding background-subtracted spectral images (b, d) for endogenous H2S

imaging in HepG2 cells without (a, b) and with (c, d) the pretreatment of human insulin. The scale bar is

10 µM. (e) The observed (hollow dots) and fitted (solid lines) time-dependent λmax shifts of PNPs in the

HepG2 cells without adding sulfide sources (negative control), without insulin pretreatment (probe 3 and

4) and with the pretreatment of human insulin (probe 5 and 6). The large difference in the initial λmax

values of probe 5 and 6 is attributed to the variation in aspect ratios or Ag shell thicknesses of the AuNRs,

but that has no effect on Δλmax measurements.

Supplementary Notes Supplementary Note 1: relationship between PNP λmax shift and amount of sulfide consumed

The general expression for the refractive index change induced LSPR shift can be obtained from the

literature,32,56

1 2max dm n exp d / lΔλ ≈ ⋅Δ ⋅ − −⎡ ⎤⎣ ⎦（ ） , (S1)

where m is the sensitivity factor (in nm per refractive index unit (RIU)), Δn is the reaction induced

refractive index change, d is the effective adsorbate layer thickness and ld is the characteristic EM-field-

decay length. The refractive index change resulting from the chemical reaction is determined by the molar

fraction of the produced Ag2S, f = CS,NP / (CAg.0 - CS,NP), where CS,NP and CAg,0 are the concentrations of

consumed sulfide species and total Ag atoms for each nanoprobe. Then the refractive index change can be

described by the follow equation,

2[ ]Ag Ag S Agn 1-f n f n nΔ = ⋅ + ⋅ −( )

2( )Ag S Agf n n= ⋅ −

2 0( ) ( )= − ⋅Ag S Ag S ,NP Ag , S ,NPn n C / C -C (S2) where nAg and nAg2S are the refractive index of bulk Ag and Ag2S, respectively. By substituting equation

S2 into equation S1, the Ag2S fraction dependent LSPR shift can be written as,

2 0[1 2 ]Δλ ≈ ⋅ − − ⋅ − ⋅max d Ag S Ag S,NP Ag , S ,NPm exp d / l n n C / C -C（ ）( ) ( ) (S3)

For the AuNR-Ag nanoprobes used in this study (L = 54.5 nm, D = 27 nm and Ag shell d thickness is

about 3 nm), the total amount of Ag atoms on the surface of an individual PNP, NAg,0, is about 1.4×10-18

mole. After designating an effective volume arbitrarily (e.g. the mean diffusion volume of sulfide ion in 1

s in this study), we can calculate the LSPR shift as a function of the amount of sulfide based on equation

S3 (Supplementary Fig. S3), where the sensitivity factor and characteristic EM-field-decay length were

3400 RIU and 320 nm according to a previous report.57

Supplementary Note 2: Kinetic spectral analysis for silver-sulfide reaction on the PNP surface

Because the concentration of dissolved oxygen in biological systems is generally in the sub-mM range

and is much higher than sulfide needed for Ag2S formation on the PNP surface, the reaction between

solid silver and sulfide in the presence of excessive oxygen can be reduced to a pseudo first-order

reaction,

22( )= ⋅

Ag SS ,eff Ag Sobs

dC k C -Cdt

(S4)

where kobs is the observed reaction rate constant, CAg2S is the concentration of consumed sulfide species

and Cs,eff is the effective concentration of sulfide species surrounding the nanoprobe. Cs,eff is determined

based on the Freundlich absorption relationship Cs,eff = KCS,0(1/p), where both K and p are temperature and

system related constants.47,58

Since the Ag2S formed on the surface of the Ag shell of the PNP might hinder the diffusion of the

sulfide species on the active Ag atom sites, the observed Ag2S formation reaction rate constant should be

corrected as

2⋅= ⋅

Ag Sobs

A-a Ck kA

(S5)

where A is a pre-exponential Arrhenius factor, a is the site hindrance factor related to Ag2S generated on

the PNP surface, and k is the actual reaction rate constant. So, equation S4 becomes

2 22

2 2

( )

(1 ) ( )

⋅= ⋅ ⋅

= ⋅ ⋅

Ag S Ag SS ,eff Ag S

Ag S S ,eff Ag S

dC A-a Ck C -Cdt A

ak - C C -CA

(S6)

Because at the end of the reaction, the reaction rate equals to zero, dCAg2S/dt = 0 and CAg2S = CAg,0/2, we

have A/a = CAg,0/2, so we can get

2 22

2 2

( )

2 ( ) ( )2

⋅= ⋅ ⋅

= ⋅ ⋅

Ag S Ag SS ,eff Ag S

Ag ,0Ag S S ,eff Ag S

Ag,0

dC A-a Ck C -Cdt A

Ck -C C -CC

(S7)

By integration with the initial condition that CAg2S = 0 when t = 0, equation S7 gives the following results,

2 2

2 2

2

( ) ( )2

= ⋅⋅

∫ ∫Ag S tAg S

0 0Ag ,0 Ag ,0Ag S S ,eff Ag S

C dC kdtC C-C C -C (S8)

22

2

2( ) ln( )2

⋅ =Ag ,0 Ag,0 Ag S S ,eff

Ag SAg,0 S ,eff Ag,0 S ,eff Ag S

C C - C Cf C = ktC - C C C -C

(S9)

By plotting f(CAg2S) versus time t according to the experimental data, the actual reaction rate constant k

can be estimated from the slope by least-squares regression. Subsequently, the theoretical kinetic spectral

shift curve can be calculated for each concentration of sulfide species.

Supplementary References

56. Willets, K.A. & Van Duyne, R.P. Localized surface plasmon resonance spectroscopy and

sensing. Annu. Rev. Phys. Chem. 58, 267-297 (2007).

57. Jung, L.S., Campbell, C.T., Chinowsky, T.M., Mar, M.N. & Yee, S.S. Quantitative interpretation

of the response of surface plasmon resonance sensors to adsorbed films. Langmuir 14, 5636-

5648 (1998).

58. Freundlich, H. Colloid and Capillary Chemistry. (Methuen, London, 1926).