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Transcript of a b - Nature a b c 532 nm 307 pixels k = 1.733 nm/pixel ®»max = kd d Raw data Gaussian fit...

  • 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/pixel 532 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 16 0

    20

    40

    60

    80

    100

    λm ax

    sh ift

    (n m

    )

    Time (min)

    100 µM 50 µM 10 µM

    400 500 600 700 800 900 1000 0.0

    0.2

    0.4

    0.6

    0.8

    1.0 0 µM 10 µM 50 µM 100 µM

    E xt

    in ct

    io n

    Wavelength (nm)

    1.0 1.5 2.0 2.5 3.0 0 4 8

    12 16 20 24 28 32

    Fr eq

    ue nc

    y (%

    )

    Aspect Ratio c

    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

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    80

    -10 -8 -6 -4 -2 0 2 4 6 8 10 0.00

    0.05

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    0.20

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    0.30

    0.35

    0.40

    Δλmax (nm) F

    re qu

    en cy

    ( %

    )

    GSH & NaCl

    0 20 40 60 80 100 0.00

    0.05

    0.10

    0.15

    0.20

    0.25

    0.30

    0.35

    0.40

    F re

    qu en

    cy (

    % )

    Δλmax (nm)

    Na2S

    0 20 40 60 80 100 0.00

    0.05

    0.10

    0.15

    0.20

    0.25

    0.30

    0.35

    0.40

    F re

    qu en

    cy (

    % )

    Δλmax (nm)

    Cys & NaCl & Na2S

    0 20 40 60 80 100 0.00

    0.05

    0.10

    0.15

    0.20

    0.25

    0.30

    0.35

    0.40 F

    re qu

    en cy

    ( %

    )

    Δλmax (nm)

    CSH & NaCl & Na2S

    Time (min)

    Δ λm

    ax (

    nm )

    Cys & NaCl & Na2S GSH & NaCl & Na2S Na2S

    -10 -8 -6 -4 -2 0 2 4 6 8 10 0.00

    0.05

    0.10

    0.15

    0.20

    0.25

    0.30

    0.35

    0.40

    F re

    qu en

    cy (

    % )

    Δλmax (nm)

    Cys & NaCl a

    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 35 0

    20

    40

    60

    80

    100

    120

    140 λm

    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 40 590 600 610 620 630 640 650 660 670

    0 5 10 15 20 25 30 35 40 590 600 610 620 630 640 650 660 670

    Probe 1 Probe 2

    λm ax

    (n m

    )

    Time (min)

    b

    λm ax

    (n m

    )

    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)

    λm ax

    (n m

    )

    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 n