Supporting Information. Fine spatiotemporal control of nitric oxide release by infrared pulse-laser irradiation of a photo-labile donor

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1 Supporting Information Fine spatiotemporal control of nitric oxide release by infrared pulse-laser irradiation of a photo-labile donor Hidehiko Nakagawa, Kazuhiro Hishikawa, Kei Eto, Naoya Ieda, Tomotaka Namikawa, Kenji Kamada, Takayoshi Suzuki, Naoki Miyata, Jun-ichi Nabekura Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1, Tanabe-dori, Mizuho-ku, Nagoya, Aichi , Japan National Institute of Physiological Sciences, 38, Nishigonaka, Myodaiji, Okazaki, Aichi , Japan Kyoto Prefectural University of Medicine, 13, Taishogun, Nishitakatsukasa-cho, Kita-ku, Kyoto , Japan Japan Science and Technology Agency, PRESTO, Honcho, Kawaguchi, Saitama , Japan Advanced Institute of Sceince and Technology (Osaka branch), Japan Research Institute for Ubiquitous Energy Devices, National Institute of Advanced Industrial Science and Technology (AIST), Ikeda, Osaka , Japan Department of Chemistry, School of Science and Technology, Kwansei Gakuin University, Sanda, Hyogo , Japan Contents 1. Cell-loading efficiency and cellular distribution of Flu-DNB (Figure S1) 2. Near-infrared (NIR) pulse laser-induced NO release in Flu-DNB-treated cultured cells (Figure S2) 3. NIR pulse laser-induced NO release in Flu-DNB-treated mouse brain: vascular study (Figures S3, S4, and Videos S1, S2) 4. NIR pulse laser-induced NO release in Flu-DNB-treated mouse brain: microglial study (Figures S5, S6 and Videos S3, S4) 5. Quantum yield and two photon absorption cross section of Flu-DNB 6. Supplementary methods 1. Cell-loading efficiency and cellular distribution of Flu-DNB First, we examined whether or not Flu-DNB is available as an intracellular photocontrollable NO donor. When Flu-DNB was simply added to the culture medium, fluorescence due to its fluorescein moiety was observed in the intracellular region, even though it took for 24 hr to reach the maximum, confirming that Flu-DNB is taken up into the cells (Figure S1). Judging from the results in in vivo experiments (see main text), treatment with Flu-DNB for several hours was considered to be enough to conduct biological 1

2 experiments with controlled NO release. Based on these results and considerations, Flu-DNB was loaded into cells for 24 hr in the case of cultured cell experiments, and administered for 1 to 1.5 hr at higher concentration in in vivo experiments. In the cellular experiments, HCT cells were treated with Flu-DNB in culture medium for 24 h to load the compound into the cells. After 24 h incubation, the treated cells were observed with a confocal fluorescence microscope to confirm the cellular distribution of Flu-DNB by detecting the green fluorescence of Flu-DNB. As shown in Figure S1, Flu-DNB fluorescence was observed within the treated cells, and so Flu-DNB was confirmed to be loaded into the cells under these experimental conditions. Figure S1 Confocal fluorescence image of Flu-DNB-treated cells. 2. Near-infrared (NIR) pulse laser-induced NO release in Flu-DNB-treated cultured cells As described in the main text, we found that the fluorescence signal of DAR-4M T was increased only at the site irradiated with the laser. In addition to Figure 2, other representative images of the cells are presented in Figures S2. 2

3 Figure S2 Confocal fluorescence images of HCT116 cells irradiated with the 735 nm pulse laser in the presence of Flu-DNB, a photocontrollable NO donor, and DAF-4M AM, a fluorogenic NO probe: a) fluorescence image before pulse laser irradiation at the point indicated by the arrow; b) image of the same field after photoirradiation. 3. NIR pulse laser-induced NO release in Flu-DNB-treated mouse brain: vascular study As described in the main text, NIR pulse laser irradiation of an ROI on a blood vessel wall was found to induce a small but significant transient increase of its diameter only during the irradiation. In addition to Figures 3 and 4, other representative two-photon images are presented in Figures S3 and S4. Time-lapse images of Figure 4a-b and 4c-d are also shown as Videos S1 and S2 in separate files. Figure S3 shows the vasodilation effect in response to NIR pulse laser irradiation in Flu-DNB- or fluorescein (control)-treated mouse brain. Figure S4 shows that ODQ treatment attenuated the vasodilation induced by pulse laser irradiation in Flu-DNB-treated mouse brain. As can be seen in Figure S4e-h, this vessel showed a very small response to irradiation, so that the effect of ODQ seems to be limited. 3

4 Figure S3 Vasodilation effect in response to NIR pulse laser irradiation in Flu-DNB or fluorescein (control) treated mouse brain: a) two-photon fluorescence image of a vessel in Flu-DNB-treated mouse brain immediately before pulse laser irradiation; b) image at the same field as panel a, but during the uncaging pulse (735 nm) irradiation; c) image of another vessel in the Flu-DNB-treated brain immediately before irradiation; d) image at the same field as panel c, but during uncaging pulse irradiation; e) image of a vessel in fluorescein (control)-treated mouse brain; f) image at the same field as panel e but during uncaging pulse irradiation. The noise observed in the images during uncaging pulse irradiation is due to interaction of the uncaging pulse and acquisition pulse lasers during simultaneous scanning. The irradiated areas are indicated by red lines, and the vessel diameter before irradiation is shown by a yellow scale in each pair of images. 4

5 Figure S4 Vasodilation effect in response to NIR pulse laser irradiation in Flu-DNB- and ODQ-treated mouse brain: a) two-photon fluorescence image of a vessel before ODQ treatment in Flu-DNB-treated mouse brain immediately before the pulse laser irradiation; b) image of the same field as panel a, but during uncaging pulse (735 nm) irradiation; c) image of the same field as panel a, after ODQ treatment and immediately before irradiation; d) image of the same field as panel c, but during uncaging pulse irradiation; e) two-photon fluorescence image of another vessel before ODQ treatment in Flu-DNB treated mouse brain immediately before pulse laser irradiation; f) image of the same field as panel e, but during uncaging pulse (735 nm) irradiation; g) image of the same field as panel e, after ODQ treatment 5

6 and immediately before irradiation; h) image of the same field as panel g, but during uncaging pulse irradiation. The noise observed in the images during uncaging pulse irradiation is due to interaction of the uncaging pulse and acquisition pulse lasers during simultaneous scanning. The irradiated areas are indicated by red lines, and the vessel diameter before irradiation is shown by a yellow scale in each pair of images. Video S1 (separate file) Time-lapse images of Figure 3a-b. Animation was made by sampling every other frame at frame rate of 30 ms, showing an actual time range of 110s. Video S2 (separate file) Time-lapse images of 3c-d. Animation was made by sampling every other frame at frame rate of 30 ms, showing an actual time range of 110s. 4. NIR pulse laser-induced NO release in Flu-DNB-treated mouse brain: microglial study As described in the main text, dendrites of microglial cells were found to be attracted to the site of NIR pulse laser irradiation, depending on Flu-DNB treatment. In addition to Figure 5, other representative two-photon images of the brain are presented in Figures S5 and S6. Time-lapse images of Figure 5a-b and 5c-d are also shown as Videos S3 and S4 in separate files. These results also support the conclusion that Flu-DNB released NO upon pulse laser irradiation, and the released NO chemoattracted microglial dendrites to the irradiated site. 6

7 Figure S5 Attraction of microglial dendrites depending on photoirradiation in living mice. Iba1 mouse brain expressing GFP in microglia was treated with Flu-DNB and subjected to photoirradiation in the same manner as described in the Method section in the main text. Areas within broken circles were irradiated, and the same sites are indicated by arrows in the After images. Figure S6 No attraction of microglial dendrites to photoirradiated region in living mouse brain without Flu-DNB treatment. Mouse brain expressing GFP in microglia was treated with DMSO containing ACSF (vehicle) and subjected to photoirradiation in the same manner as described in the Method section in the main text. The area within the broken circle was irradiated, and the same site is indicated by an arrow in the After image. Video S3 (separate file) Time-lapse images of Figures 5a-b. Animation was done by sampling every third frame at frame rate of 60 ms, showing an actual time range of 490s. Video S4 (separate file) Time-lapse images of Figures 5c-d. Animation was done by sampling every third frame at frame rate of 60 ms, showing an actual time range of 490s. 5. Quantum yield and two-photon cross section of Flu-DNB Fluorescence quantum yield for Flu-DNB was determined by using a conventional fluorescence spectrometer, and two-photon cross section was determined by the two-photon-induced fluorescence (TFIP) method (ref. 1, 2). Table S1 Fluorescence quantum yields and two-photon cross section Two-photon cross section (GM) Compounds Quantum yield 780 nm 800 nm 7

8 Flu-DNB (in water containing 1% DMSO) Fluorescein 0.9 (ref. 3) 46 (ref. 4) 36 (ref. 4) References for this section 1. K. Kamada, L. Antonov, S. Yamada, K. Ohta, T. Yoshimura, K. Tahara, A. Inaba, M. Sonoda, Y. Tobe (2007), ChemPhysChem, 8, C. Xu, W. W. Webb (1996), J. Opt. Soc., Am. B, 13, J.M. Song, T. Inoue, H. Kawazumi, T Ogawa (1999), Anal. Sci., 15, N.S. Makarov, M. Drobizhev, A. Rebane (2008), Opt. Exp., 16, Supplementary Methods One-photon irradiation and confocal imaging of Flu-DNB-treated cultured cells HCT116 cells treated with 10 µm Flu-DNB and 10 µm DAF-FM-DA were irradiated with UVA light ( nm) from a photoilluminator MAX-302 (Asahi Spectra, Tokyo, Japan) equipped with a 300 W Xe lamp through a 25% ND filter. After irradiation, the cells were subjected to confocal fluorescence microscopy (LSM510, Carl Zeiss Japan Co. Ltd., Tokyo, Japan). Preparation of the treatment solution for in vivo studies A concentrated solution of Flu-DNB was prepared by dissolving the compound in DMSO on the day of the experiment. When mice were to be treated, the Flu-DNB solution in DMSO was diluted with artificial cerebrospinal fluid (a-csf) to the desired concentration (less than 1% DMSO in the final solution), and this solution was kept at room temperature in the dark. Details of TPLSM settings for the in vivo studies For vascular study Vessels were visualized using a TPLSM (Olympus, Tokyo, Japan) coupled with a mode-locked Ti-sapphire laser (pulse width of <100 fs, 80 MHz repetition frequency at wavelengths of 735 nm and 950 nm) (Mai Tai HP; Spectra-Physics, Mountain View, CA, USA). The excitation light was focused using a water-immersion objective lens (20x; 0.95 numerical aperture) (IR-LUMPlanFl, Olympus). The emitted fluorescence was divided into long (>570 nm) and short wavelengths using a dichroic mirror (570 nm; Olympus). 8

9 Time-lapse vascular imaging was performed with a 950 nm pulse laser. A hundred consecutive frames (512 x 512 pixels) were acquired at a rate of 1.1 s per frame. In the middle of scanning (frames 30 70), the 735 nm pulse laser was scanned over regions of interest (ROI) of arbitrary size. Vasodilation was evaluated by comparing the vessels at the sites just before irradiation and the 8th frame after starting irradiation. For microglia study Images of microglia expressing GFP were visualized using a TPLSM (Olympus, Tokyo, Japan) coupled with a mode-locked Ti-sapphire laser (pulse width of <100 fs, 80 MHz repetition frequency at wavelengths of 735 nm and 950 nm) (Mai Tai HP; Spectra-Physics, Mountain View, CA, USA). The excitation light was focused using a water-immersion objective lens (20x; 0.95 numerical aperture) (IR-LUMPlanFl, Olympus). The emitted fluorescence was divided into long (>570 nm) and short wavelengths using a dichroic mirror (570 nm; Olympus). Time-lapse imaging of GFP was performed with a 950 nm pulse laser. Three hundred consecutive frames (512 x 512 pixels) were acquired at a rate of 1.1 s per frame. In the first half of scanning (frames 1 150), the 735 nm pulse laser was scanned over regions of interest (ROI) of arbitrary size. Migration of dendrites was evaluated by comparing the fluorescence intensity at the ROI in the first frame and last frame with ImageJ software. 9