Supporting Information

© Wiley-VCH 2008

69451 Weinheim, Germany

S1

Supporting Information to Accompany “Two-Photon Fluorescent Probes for the

Acidic Vesicles in Live Cell and Tissue”

Hwan Myung Kim,[a] Myoung Jin An,[a] Jin Hee Hong,[b,c] Byeong Ha Jeong,[b,c] Ohyun Kwon,[e] Ju-Yong Hyon, [d] Seok-Cheol Hong, [c] Kyoung J. Lee,[b,c] and Bong Rae Cho*[a]

[a] Molecular Opto-Electronics Laboratory, Department of Chemistry, [b]National Creative Research Initiative Center for Neurodynamics, [c]Department of Physics, [d]Biomicrosystems Technology Program, Korea University, 1-Anamdong, Seoul, 136-701, Korea, [e] Samsung Advanced Institute of Technology, Korea.

Table of Contents

(Page) (S3) Synthesis and Material. (S4) Spectroscopic measurements. (S5) Water Solubility. (S6) pKa value. (S8) Computational Details. (S8) Measurement of Two-Photon Cross Section. (S9) Cell Culture. (S9) Two-Photon Fluorescence Microscopy. (S11) Preparation and Staining of acute rat Hippocampal slices. (S3) Scheme S1. Structures of the probes for acidic vesicles and synthesis of AH1, AH2, and AL1.

a) DCC, HOBt, CH2Cl2. b) 1. MeI, K2CO3, n-Bu4NI, acetone; 2. H2, Pd, EtOH. c) CF3CO2H. (S5) Figure S1. a, b, c) Normalized absorption and d, e, f) emission spectra of a, d) AH1, b, e) AH2

and c, f) AL1 in 1,4-dioxane, DMF, EtOH, and H2O. (S6) Figure S2. a, b, c) One-photon fluorescence spectra and d, e, f) plot of fluorescence intensity

against dye concentration for a, d) AH1, b, e) AH2 and c, f) AL1 in H2O. The excitation wavelength was 365 nm.

(S7) Figure S3. a, c) One-photon absorption and b, d) emission spectra of 1 μM a, b) AH1 and c, d) AH2 as a function of pH (3.2-10.5) in universal buffer. The excitation wavelength was 365 nm.

(S7) Figure S4. Two-photon emission spectra of AH1 as a function of pH (3.2-9.5) in universal buffer. The excitation wavelength was 780 nm.

(S8) Figure S5. a) One-photon absorption, b) emission and c) titration curves of AL1 as a function of pH (3.2-10.5) in universal buffer. The excitation wavelength was 365 nm.

(S8) Figure S6. HOMO energy level of Acedan, R1 and R2. These values were obtained from B3LYP/6-31G** calculations.

S2

(S9) Figure S7. Dependence of output fluorescence intensity (Iout) of 5 μM AH2 in buffer on the input laser power (Iin). The insert shows the linear dependence of Iout on Iin

2 (780 nm, 90 MHz, τ = 160 fs).

(S10) Figure S8. TPM images of (a-c) AH2- and (d-f) AL1-labeled (2 μM) macrophages collected at 500-620 nm with magnification 60× by excitation at 780 nm. Images were obtained after washing one (a,d), three (b,e), and five (c,f) times with phenol-red free DMEM. Scale bars are 30 μm. Cells shown are representative images from replicate experiments (n = 4). g) Normalized two-photon fluorescence intensities of AH2- and AL1-labeled (2 μM) macrophages collected after washing 1-5 times with phenol-red free DMEM. The fluorescence intensities are the average of four measurements.

(S11) Figure S9. a, b) Pseudo colored TPM images of AH1-labeled (2 μM) macrophages collected at a) 360-460 nm, and b) 500-620 nm, respectively. c) OPM image of LTR-labeled macrophages and d) co-localized image. The wavelengths for one- and two-photon excitation were 543 and 780 nm, respectively. Cells shown are representative images from replicate experiments. Scale bar is 30 μm.

(S11) Figure S10. a) Pseudo colored TPM images collected at 500-620 of AH2-labeled (4 μM) macrophages. The excitation wavelength was 780 nm. b) Two-photon excited fluorescence spectra from the intense spots (1) and bright domains (2) of AH2-labeled (4 μM) macrophages.

(S12) Figure S11. TPM images an acute rat hippocampal slice stained with 10 μM AL1. Images were taken at depth of ~100-250 μm with magnification 10× by excitation at 780 nm.

(S12) Figure S12. Images of an acute rat hippocampal slice stained with 15 μM AH2. a) Bright field image shows the CA1 and CA3 regions as well as the dentate gyrus (DG) upon magnification 10×. White dot lines indicate the pyramidal neuron layer. b) Accumulated 40 TPM images along the z-direction at the depth of ~100-250 μm with magnification 10× reveals the average distribution of the acidic vesicles in the same regions. c) TPM images were taken at depth of ~100-250 μm with magnification 10× by excitation at 780 nm. Scale bar is 300 μm.

(S13) Figure S13. TPM images an acute rat hippocampal slice stained with 10 μM a) AH1 and c) AL1. The excitation wavelength was 780 nm. a, c) Magnification 100× in CA3 regions at a depth of ~100 μm. b, d) Two-photon excited fluorescence spectra from the various spots of (a, c).

(S13) Figure S14. Plot of the TPEF intensity as a function of time. The data are taken from the real time image of the transportation of the acidic vesicles along the axon. Part of the image is shown in Figures 3c and 3d and movie S1. Intensities were recorded with 0.5 sec intervals for the duration of 1100 sec using xyt mode with 512 × 256 pixels at 800 Hz scan speed.

(S5) Table S1. Photophysical properties of AH1, AH2 and AL1 in various solvents.

Synthesis and Material. Synthesis of AH1, AH2 and AL1 is summarized in Scheme S1. 2-Hydroxy-4-nitrophenylcarbamic acid tert-butyl ester[1] (1) and 6-acyl-2-[N-methyl-N-(carboxymethyl)amino]naphthalene[2] (A) were prepared by the literature methods. Synthesis of other compounds is described below. LysoTracker Red was purchased from Molecular Probes (Eugene, OR).

O

NHN

OH2N

R

O

NHN

ON

R = H (AH1)R = OMe (AH2)

AL1

NB

N

NHNH

O

N

F F

LysoTracker Red

BocHN NO2

O

NHO

O A

a

NH2

H2N N

NH2

bA

a, cBocHN NH2

or

MeOHO

AH1or AL1

AH2

Scheme S1. Structures of the probes for acidic vesicles and synthesis of AH1, AH2, and AL1. a) DCC, HOBt, CH2Cl2. b) 1. MeI, K2CO3, n-Bu4NI, acetone; 2. H2, Pd, EtOH. c) CF3CO2H. 2-Methoxy-4-nitrophenylcarbamic acid tert-butyl ester (2). A mixture of 1 (3.0 g, 11.8 mmol), K2CO3 (2.5 g, 17.7 mmol), n-Bu4NI (0.87 g, 2.4 mmol), and MeI (1.8 g, 23.6 mmol) in dry acetone (50 mL) was refluxed for 20 h under N2. The cooled reaction mixture was poured into 100 mL of water, collected by filtration, and washed with water (100 mL) and hexane (100 mL). Yield 2.5 g (80 %); mp 117 °C; 1H NMR (300 MHz, CDCl3): δ 8.27 (d, 1H, J = 9 Hz), 7.91 (dd, 1H, J = 9, J = 2 Hz), 7.73 (d, 1H, J = 2 Hz) 7.36 (s, 1H), 3.98 (s, 3H), 1.54 (s, 9H); 13C NMR (100 MHz, CDCl3): δ 152.2, 147.0, 134.9, 118.3, 116.4, 105.5, 105.2, 81.9, 56.4, 28.6 ppm; Anal. Calcd for C12H16N2O5: C, 53.73; H, 6.01; N, 10.44. Found: C, 53.98; H, 5.89; N, 10.59. 4-Amino-2-methoxyphenylcarbamic acid tert-butyl ester (3). A mixture of 2 (2 g, 7.5 mmol) and 10 % Pd on carbon (0.16 g, 1.6 mmol) in ethanol (50 mL) was shaken under hydrogen for 5 h. The reaction mixture was filtered and washed with hot ethanol, and the solvent was removed in vacuo. Yield 1.7 g (95 %); 1H NMR (300 MHz, DMSO d6): δ 7.54 (s, 1H), 7.03 (d, J = 9 Hz, 1H), 6.23 (d, 1H, J = 2 Hz), 6.07 (dd, 1H, J = 9, J = 2 Hz), 4.93 (br s, 2H), 3.67 (s, 3H), 1.41 (s, 9H); 13C NMR (100 MHz, DMSO d6): δ 154.4, 147.3, 116.4, 106.0, 105.7, 98.6, 98.2, 78.7, 55.7, 28.7 ppm; Anal. Calcd for C12H18N2O3: C, 60.49; H, 7.61; N, 11.76. Found: C, 60.88; H, 7.46; N, 11.78. AH1. A mixture of A (0.20 g, 0.78 mmol), N,N’-dicyclohexylcarbodiimide (0.18 g, 0.86 mmol), and 1-hydroxybenzotriazole (0.13 g, 0.94 mmol) in CH2Cl2 (20 mL) was stirred for 30 min. To this mixture, p-phenylenediamine∙2HCl (0.50 g, 2.72 mmol) and Et3N (0.16 g 1.56 mmol) in CH2Cl2 (5 mL) were added and stirred for 3 h under N2. The resulting mixture was filtered and the filtrate was extracted with saturated NaHCO3 (aq), dried over Na2SO4, filtered and concentrated in vacuo. The crude product was purified by column chromatography using CHCl3/MeOH (10:1) as the eluent. Yield 0.13 g (47 %); mp 218 °C; 1H NMR (300 MHz, CDCl3): δ 8.37 (d, 1H, J = 2 Hz), 8.04 (s, 1H), 7.99 (dd, 1H, J = 9, J

S3

S4

= 2 Hz), 7.89 (d, 1H, J = 9 Hz), 7.72 (d, 1H, J = 9 Hz), 7.24 (d, 2H, J = 9 Hz), 7.17 (dd, 1H, J = 9, J = 2 Hz), 7.07 (d, 1H, J = 2 Hz), 6.64 (d, 2H, J = 9 Hz), 4.12 (s, 2H), 3.62 (br s, 2H), 3.25 (s, 3H), 2.69 (s, 3H); 13C NMR (100 MHz, DMSO d6): δ 197.7, 167.8, 150.2, 145.6, 137.8, 131.3, 131.1, 130.8, 128.6, 126.6, 125.3, 124.7, 121.7, 116.9, 114.8, 114.4, 105.5, 56.0, 27.1 ppm; Anal. Calcd for C21H21N3O2 : C, 72.60; H, 6.09; N, 12.10. Found: C, 72.78; H, 6.21; N, 12.39. AH2. The t-butoxycarbonyl-protected AH2 was obtained in 58 % overall yield from 3 according to above procedure of AH1. mp 192 °C; 1H NMR (300 MHz, CDCl3): δ 8.35 (d, 1H, J = 2 Hz), 8.23 (s, 1H), 7.97 (dd, 1H, J = 9, J = 2 Hz), 7.95 (d, 1H, J = 2 Hz), 7.87 (d, 1H, J = 9 Hz), 7.70 (d, 1H, J = 9 Hz), 7.57 (d, 1H, J = 2 Hz), 7.17 (dd, 1H, J = 9, J = 2 Hz), 7.07 (d, 1H, J = 2 Hz), 7.00 (s, 1H), 6.69 (dd, 1H, J = 9, J = 2 Hz), 4.11 (s, 2H), 3.87 (s, 3H), 3.24 (s, 3H), 2.68 (s, 3H), 1.50 (s, 9H); 13C NMR (100 MHz, CDCl3): δ 198.0, 168.1, 152.9, 149.3, 147.9, 137.3, 132.3, 132.1, 130.5, 130.1, 126.7, 125.2, 125.0, 116.6, 112.2, 111.9, 107.7, 103.4, 59.6, 55.9, 40.6, 40.3, 28.9, 28.4, 26.7 ppm; Anal. Calcd for C27H31N3O5: C, 67.91; H, 6.54; N, 8.80. Found: C, 67.78; H, 6.26; N, 8.51. This intermediate was dissolved in trifluoroacetic acid at 0 °C and the solution was stirred for 2 h.

After the addition of toluene, the solution was evaporated to afford AH2. Yield 98 %; mp 157 °C; 1H NMR (400 MHz, CD3OD): δ 8.39 (d, 1H, J = 2 Hz), 7.86 (dd, 1H, J = 9, J = 2 Hz), 7.85 (d, 1H, J = 9 Hz), 7.65 (d, 1H, J = 2 Hz), 7.64 (d, 1H, J = 9 Hz), 7.28 (d, 1H, J = 9 Hz), 7.24 (dd, 1H, J = 9, J = 2 Hz), 7.22 (dd, 1H, J = 9, J = 2 Hz), 7.01 (d, 1H, J = 2 Hz), 4.34 (s, 2H), 3.92 (s, 3H), 3.32 (br s, 2H), 3.26 (s, 3H), 2.64 (s, 3H); 13C NMR (100 MHz, DMSO d6): δ 198.0, 168.8, 159.7, 159.5, 159.3, 159.0, 150.2, 137.8, 131.3, 131.1, 130.8, 126.6, 125.3, 124.6, 119.8, 116.8, 112.1, 105.5, 104.0, 56.7, 56.2, 27.0 ppm; Anal. Calcd for C22H23N3O3 : C, 70.01; H, 6.14; N, 11.13. Found: C, 70.18; H, 6.54; N, 11.03. AL1 was obtained in 77 % overall yield from N,N-dimethylethylenediamine according to above procedure of AH1. mp 69 °C; 1H NMR (400 MHz, CDCl3): δ 8.34 (d, 1H, J = 2 Hz), 7.96 (dd, 1H, J = 9, J = 2 Hz), 7.84 (d, 1H, J = 9 Hz), 7.68( d, 1H, J = 9 Hz), 7.11 (dd, 1H, J = 9, J = 2 Hz), 6.96 (d, 1H, J = 2 Hz), 6.92 (t, 1H, J = 7 Hz), 4.05 (s, 2H), 3.35 (q, 2H, J = 7 Hz), 3.18 (s, 3H), 2.68 (s, 3H), 2.34 (t, 2H, J = 7 Hz), 2.05 (s, 6H); 13C NMR (100 MHz, CDCl3): δ 197.8, 169.8, 148.9, 137.3, 131.6, 131.0, 130.2, 126.5, 124.8, 116.2, 106.6, 58.0, 57.8, 45.0, 39.9, 36.6, 33.98, 26.5 ppm; Anal. Calcd for C19H25N3O2: C, 69.70; H, 7.70; N, 12.83. Found: C, 69.78; H, 7.56; N, 12.77. Spectroscopic measurements. Absorption spectra were recorded on a Hewlett-Packard 8453 diode array spectrophotometer, and fluorescence spectra were obtained with Amico-Bowman series 2 luminescence spectrometer with a 1 cm standard quartz cell. The fluorescence quantum yield was determined by using Coumarin 307 and Rhodamine B as the reference by the literature method.[3]

S5

Figure S1. a, b, c) Normalized absorption and d, e, f) emission spectra of a, d) AH1, b, e) AH2 and c, f) AL1 in 1,4-dioxane, DMF, EtOH, and H2O. Table S1. Photophysical properties of AH1, AH2 and AL1 in various solvents.

Solvent ( )[a] NTE (1)

maxλ [b] flmaxλ [b] Φ[c]

AH1 AH2 AL1 AH1 AH2 AL1 AH1 AH2 AL1 Dioxane (0.164) 345 345 346 414 417 417 0.38 0.12 0.79 DMF (0.386) 355 356 355 443 444 445 0.12 0.02 1.00 EtOH (0.654) 356 358 355 475 467 476 0.12 0.03 1.00 H2O (1.00) 364 362 362 495 496 497 0.03 0.01 0.76

[a] The numbers in the parenthesis are normalized empirical parameter of solvent polarity.[5] [b] λmax of the one-photon absorption and emission spectra in nm. [c] Fluorescence quantum yield, ± 15 %. Water solubility. Small amount of dye was dissolved in DMSO to prepare the stock solutions (1.0 × 10-3 M). The solution was diluted to (6.0 × 10-3 ~ 6.0 × 10-5) M and added to a cuvette containing 3.0 mL of H2O by using a micro syringe. In all cases, the concentration of DMSO in H2O was maintained to be 0.2 %.[4] The plots of fluorescence intensity against the dye concentration were linear at low concentration and showed downward curvature at higher concentration (Figure S2). The maximum concentration in the linear region was taken as the solubility. The solubilities of AH1, AH2, and AL1 in water are 5.0, 9.0, and 5.0 μM.

300 320 340 360 380 400 420 4400.0

0.5

1.0

Nor

mal

ized

Abs

orba

nce

Wavelength/ nm

Diox DMF EtOH H2O

c)

300 320 340 360 380 400 420 4400.0

0.5

1.0

Nor

mal

ized

Abs

orba

nce

Wavelength/ nm

Dioxane DMF EtOH H2O

a)

300 320 340 360 380 400 420 4400.0

0.5

1.0 Dioxane DMF

Nor

mal

ized

Abs

orba

nce

Wavelength/ nm

EtOH H2O

b)

380 400 420 440 460 480 500 520 540 5600.0

0.2

0.4

0.6

0.8

1.0

Nor

mal

ized

Flu

ores

cenc

e

Wavelength/ nm

Dioxane DMF EtOH H2O d)

380 400 420 440 460 480 500 520 540 5600.0

0.2

0.4

0.6

0.8

1.0

Nor

mal

ized

Flu

ores

cenc

e

Wavelength/ nm

Dioxane DMF EtOH H2O e)

380 400 420 440 460 480 500 520 540 5600.0

0.2

0.4

0.6

0.8

1.0

Nor

mal

ized

Flu

ores

cenc

e

Wavelength/ nm

Dioxane DMF EtOH H2O f)

S6

0.0 3.0x10-6 6.0x10-6 9.0x10-6 1.2x10-5 1.5x10-50

1x102

2x102

3x102

4x102

Figure S2. a, b, c) One-photon fluorescence spectra and d, e, f) plot of fluorescence intensity against dye concentration for a, d) AH1, b, e) AH2 and c, f) AL1 in H2O. The excitation wavelength was 365 nm. pKa value. A 3.0 μL of the stock solution of AH1 and AH2 in DMSO (1.0 × 10-3 M) was added to a cuvette containing 3.0 mL of universal buffer solution[6] (0.1 M citric acid, 0.1 M KH2PO4, 0.1 M Na2B4O7, 0.1 M Tris, 0.1 M KCl, pH 3.2-10.5) by using a microsyringe and the fluorescence intensity was measured as a function of the pH. The pKa values were estimated from the increase in the integrated area of the fluorescence spectra with pH 3.2-10.5 by using the relationship, log[(Imax - I)/(I - Imin)] = pH – pKa.[7] The calculated pKa values of AH1 and AH2 are 4.42 ± 0.03 and 4.48 ± 0.02, respectively. In order to determine the pKa

TP for the two-photon process, the TPEF spectra were obtained with a DM IRE2 Microscope (Leica) using the xyλ mode at 800 Hz scan speed. They were excited by a mode-locked titanium-sapphire laser source (Coherent Chameleon, 90 MHz, 200 fs) set at wavelength 780 nm and output power 1180 mW, which corresponded to approximately 10 mW average power in the focal plane. The calculated pKa

TP values of AH1 and AH2 are 4.46 ± 0.04 and 4.48 ± 0.04, respectively.

Fluo

resc

ence

Inte

nsity

(a.u

.)

Concentration

d)

~ 5 μM

0.0 3.0x10-6 6.0x10-6 9.0x10-6 1.2x10-5 1.5x10-50.0

2.0x101

4.0x101

6.0x101

8.0x101

1.0x102

1.2x102

1.4x102

~ 9 μM

Fluo

resc

ence

Inte

nsity

(a.u

.)

Concentration

e)

0.0 2.0x10-6 4.0x10-6 6.0x10-6 8.0x10-6 1.0x10-5 1.2x10-5 1.4x10-5 1.6x10-5

0

1x102

2x102

3x102

4x102

5x102

~ 5 μM

Fluo

resc

ence

Inte

nsity

(a.u

.)

Concentration

f)

400 450 500 550 600 650 7000

1

2

3

4

5

6 1.5E-5 1.2E-5 9.0E-6 7.5E-6 6.0E-6 5.0E-6 3.9E-6 3.0E-6 2.1E-6 1.2E-6 9.0E-7 6.0E-7 3.0E-7 1.2E-7Fl

uore

scec

e In

tens

tiy (a

.u.)

Wavelength/ nm

c)

400 450 500 550 600 650 7000.0

0.5

1.0

1.5 1.5E-5 1.2E-5 9.0E-6 7.5E-6 6.0E-6 5.0E-6 3.9E-6 3.0E-6 2.1E-6 1.2E-6 9.0E-7 6.0E-7 3.0E-7 1.2E-7

Fluo

resc

ece

Inte

nstiy

(a.u

.)

Wavelength/ nm

b)

400 450 500 550 600 650 7000

1

2

3

4

5

6

Fluo

resc

ence

Inte

nsity

(a.u

.)

Wavelength/ nm

1.5E-5 1.2E-5 9.0E-6 7.5E-6 6.0E-6 5.0E-6 3.9E-6 3.0E-6 2.1E-6 1.2E-6 9.0E-7 6.0E-7 3.0E-7 1.2E-7

a)

S7

400 450 500 550 600 6500

5

10

15

20

25

Figure S3. a, c) One-photon absorption and b, d) emission spectra of 1 μM a, b) AH1 and c, d) AH2 as a function of pH (3.2-10.5) in universal buffer. The excitation wavelength was 365 nm. Figure S4. Two-photon emission spectra of AH1 as a function of pH (3.2-9.5) in universal buffer. The excitation wavelength was 780 nm.

b)

Fluo

resc

ence

Inte

nsity

Wavelength/ nm

pH 3.2

pH 4.5

pH 10.5

300 350 400 4500.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035 a) 3.2 4.5 5.5 7.0 9.0

Abs

orba

nce

Wavelength/ nm

300 350 400 4500.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035 c)

Abs

orba

nce

Wavelength/ nm

3.2 4.5 5.5 7.0 9.0

400 450 500 550 600 6500

10

20

30

40

50

60

Fluo

resc

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Inte

nsity

Wavelength/ nm

d) pH 3.2

pH 4.5

pH 10.5

400 450 500 550 600 6500

5

10

15

20

25

Fluo

resc

ence

Inte

nstiy

Wavelength/ nm

pH 3.2

pH 4.5

pH 9.5

400 450 500 550 600 6500

10

20

30

40

50

60

70

80

S8

Figure S5. a) One-photon absorption, b) emission and c) titration curves of AL1 as a function of pH (3.2-10.5) in universal buffer. The excitation wavelength was 365 nm. Computational Details. Geometries of AH1, AH2, 4-acetamidoaniline (R1), and 4-acetamido-2-methoxyaniline (R2) were fully optimized by using B3LYP/6-31G** level[8a] in the density functional theory (DFT) formalism as implemented in Gaussian 98 program.[8b]

Figure S6. HOMO energy level of Acedan, R1 and R2. These values were obtained from B3LYP/6-31G** calculations.

Measurement of Two-Photon Cross Section. The two-photon cross section (δ) was determined by using femto second (fs) fluorescence measurement technique as described.[9] AH1, AH2, AL1, and LysoTracker Red (DND-99) were dissolved in universal buffer (pH = 3.2) at concentrations of 5.0 × 10-6 M and then the two-photon induced fluorescence intensity was measured at 740−940 nm by using fluorescein (8.0 × 10-5 M, pH = 11) as the reference, whose two-photon property has been well characterized in the literature.[10] The intensities of the two-photon induced fluorescence spectra of the reference and sample emitted at the same excitation wavelength were determined. The TPA cross section was calculated according to Eq 1.

s r r rr

r s s s

S cS c

φδ δφ

Φ=

Φ (1)

Energy (HOMO)

-5.164 eV -5.044 eV

-4.827 eV

O

N

HN

OH2N

HN

OH2N

MeO

Energy (HOMO)

-5.164 eV -5.044 eV

-4.827 eV

O

N

HN

OH2N

HN

OH2N

MeO

Fluo

resc

ence

Inte

nsity

(a.u

.)

Wavelength/ nm

3.2 4.0 4.4 4.8 5.1 5.5 6.2 7.0 7.9 8.6 9.5 10.5

b)

3 4 5 6 7 8 9 10 110

10

20

30

40

50

60

70

80

90

Fluo

resc

ence

Inte

nsity

(a.u

.)

pH

c)

300 320 340 360 380 400 420 4400.000

0.005

0.010

0.015

0.020

3.2 4.4

Abs

orba

nce

Wavelength/ nm

6.2 7.9 9.5

a)

The plot of output fluorescence intensity (Iout) of 5 μM AH2 in buffer vs the input laser power (Iin) is shown in Figure S7. In all cases, the plot showed quadratic dependence of the TPEF signal on the excitation intensity, confirming the nonlinear absorption.

14

12 10

80

60

40

5 10 15 20 25 30 35 40 45 50

200

0

0

0

00

00

00

0 500 1000 1500 2000 2500

200

400

600

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1000

1200

1400

I20 (mW)

Fluo

resc

ence

Inte

nsity

(a.u

.)

I0(mW)

Fluo

resc

ence

Inte

nsity

(a.u

.)

Figure S7. Dependence of output fluorescence intensity (Iout) of 5 μM AH2 in buffer on the input laser power (Iin). The insert shows the linear dependence of Iout on Iin

2 (780 nm, 90 MHz, τ = 160 fs). Cell Culture. Macrophage (Raw 264.7) was cultured for 3 days in phenol-red free Dulbecco’s Modified Eagle’s Medium (DMEM; WelGene, Daegu, South Korea) supplemented with penicillin/streptomycin and 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA) in a CO2 incubator at 37 °C. The cells were loaded with 2-4 μM probes in phenol-red free DMEM for 20 min. After incubation, the cells were washed with phenol-red free DMEM. Two-Photon Fluorescence Microscopy. Two-photon fluorescence microscopy images of probe-labeled macrophages and tissues were obtained with spectral confocal and multiphoton microscopes (Leica TCS SP2) with a ×10 (NA = 0.30 DRY) and ×100 (NA = 1.30 OIL) objective lens. The two-photon fluorescence microscopy images were obtained with a DM IRE2 Microscope (Leica) by exciting the probes with a mode-locked titanium-sapphire laser source (Coherent Chameleon, 90 MHz, 200 fs) set at wavelength 780 nm and output power 1230 mW, which corresponded to approximately 10 mW average power in the focal plane. To obtain images at 360~460 nm and 500~620 nm range, internal PMTs were used to collect the signals in an 8 bit unsigned 512 × 512 pixels at 400 Hz scan speed. For the figures 3d, 3e and movie S1, images were recorded with 0.5 sec intervals for the duration of 1100 sec using xyt mode with 512 × 256 pixels at 800 Hz scan speed.

To determine the effects of washing of the probe-labeled cells with phenol-red free DMEM, two-photon fluorescence microscopy images of AH2- and AL1-labeled macrophages were obtained with upright two-photon microscopy (TPM; BX51WI, Olympus) with a ×60 (NA = 0.90 W) water

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immersion objective (LUMPlanFI/IR, Olympus). The cells were loaded with 2 μM AH2 and AL1 as described above, and the TPM images were obtained with a BX51WI Microscope (Olympus) by exciting the probes with a mode-locked titanium-sapphire laser source (Coherent Chameleon, 90 MHz, 200 fs) set at wavelength 780 nm. Then the imaging solution (1 mL) was discarded and replaced 3 times by 500 μL of phenol-red free DMEM, and the images were recorded. Further iterations were followed by the same procedures. The TPM images of AH2- and AL1-labeled (2 μM) macrophages after washing 1, 3, and 5 times with phenol-red free DMEM are shown in Figure S8.

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Figure S8. TPM images of (a-c) AH2- and (d-f) AL1-labeled (2 μM) macrophages collected at 500-620 nm with magnification 60× by excitation at 780 nm. Images were obtained after washing one (a,d), three (b,e), and five (c,f) times with phenol-red free DMEM. Scale bars are 30 μm. Cells shown are representative images from replicate experiments (n = 4). g) Normalized two-photon fluorescence intensities of AH2- and AL1-labeled (2 μM) macrophages collected after washing 1-5 times with phenol-red free DMEM. The fluorescence intensities are the average of four measurements.

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Figure S9. a, b) Pseudo colored TPM images of AH1-labeled (2 μM) macrophages collected at a) 360-460 nm, and b) 500-620 nm, respectively. c) OPM image of LTR-labeled macrophages and d) co-localized image. The wavelengths for one- and two-photon excitation were 543 and 780 nm, respectively. Cells shown are representative images from replicate experiments (n = 5). Scale bar is 30 μm.

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Figure S10. a) Pseudo colored TPM images collected at 500-620 of AH2-labeled (4 μM) macrophages. The excitation wavelength was 780 nm. b) Two-photon excited fluorescence spectra from the intense spots (1) and bright domains (2) of AH2-labeled (4 μM) macrophages. Preparation and Staining of acute rat Hippocampal slices. Slices were prepared from the hippocampi of 2-day-old rat (Sprague-Dawley; SD). Coronal slices were cut into 400 μm-thick using a vibrating-blade microtome in artificial cerebrospinal fluid (ACSF; composition in mM: 138.6 NaCl, 3.5 KCl, 21 NaHCO3, 0.6 NaH2PO4, 9.9 D-glucose, 1 CaCl2, and 3 MgCl2). Slices were incubated with 10-

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Figure S11. TPM images an acute rat hippocampal slice stained with 10 μM AL1. Images were taken at depth of ~100-250 μm with magnification 10× by excitation at 780 nm.

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Figure S12. Images of an acute rat hippocampal slice stained with 15 μM AH2. a) Bright field image shows the CA1 and CA3 regions as well as the dentate gyrus (DG) upon magnification 10×. White dot lines indicate the pyramidal neuron layer. b) 40 TPM images were accumulated along the z-direction at the depth of ~100-250 μm with magnification 10×. The accumulated image reveals the average distribution of the acidic vesicles in the same regions. c) TPM images were taken at depth of ~100-250 μm with magnification 10× by excitation at 780 nm. Scale bar is 300 μm.

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Figure S13. TPM images an acute rat hippocampal slice stained with 10 μM a) AH1 and c) AL1. The excitation wavelength was 780 nm. a, c) Magnification 100× in CA3 regions at a depth of ~100 μm. b, d) Two-photon excited fluorescence spectra from the various spots of (a, c).

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Figure S14. Plot of the TPEF intensity as a function of time. The data are taken from the real time image of the transportation of the acidic vesicles along the axon. Part of the image is shown in Figures 3c and 3d and movie S1. Intensities were recorded with 0.5 sec intervals for the duration of 1,100 sec using xyt mode with 512 × 256 pixels at 800 Hz scan speed.

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