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In the course of the present work, we also considered relying on the o-aminocinnamic platform for uncaging applications as reported by Porter in ref 16. Indeed we expected that this series should be particularly favorable to get large k2 constants because of the lower electronegativity of the nitrogen atom with regard to the oxygen one. Thus we synthesized the 3-(6-amino-benzo(1,3)dioxo-5-yl)-acrylic acid ethyl ester in two steps by condensing the 1-carboxymethyliden triphenyl phosphorane on 6-amino-3,4-methylenedioxybenzaldehyde that was obtained from Fe/HCl reduction of 6-nitro-3,4-methylenedioxybenzaldehyde in a mixture of acetic acid, ethanol, and water. 3-(6-Amino-benzo(1,3)dioxo-5-yl)-acrylic acid ethyl ester exhibits satisfactory absorption properties: in Tris (pH, 7) 20 mM NaCl 100 mM buffer/acetonitrile 1/1 (v/v, εE(λ (1)E, 13 × 103 M-1 cm-1 at 385 nm
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1 is here typically two orders of magnitude smaller than in the o-hydroxycinnamate series; see Table 1S) forbidding to rely on the o-aminocinnamic platform to develop an efficient series of photolabile protecting groups for two-photon uncaging applications.
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To study uncaging with two-photon excitation for the whole series of cinnamates in a same solvent, we had to switch from the acetonitrile/buffer 1:1 (v/v) mixture to pure acetonitrile. Indeed, we observed in the former solvent that the fluorescence traces such as in Figure 3a periodically exhibited plateaus for the most hydrophobic derivatives (noticeably except for Ec which is fairly water-soluble).
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To study uncaging with two-photon excitation for the whole series of cinnamates in a same solvent, we had to switch from the acetonitrile/buffer 1:1 (v/v) mixture to pure acetonitrile. Indeed, we observed in the former solvent that the fluorescence traces such as in Figure 3a periodically exhibited plateaus for the most hydrophobic derivatives (noticeably except for Ec which is fairly water-soluble).
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In fact, one should also consider here that a caged compound might form a complex with some intracellular components (bilayer, proteins) not located at the laser focus. Then the apparent homogeneization time could be associated to the off-rate of the caged substrate from the complex
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In fact, one should also consider here that a caged compound might form a complex with some intracellular components (bilayer, proteins) not located at the laser focus. Then the apparent homogeneization time could be associated to the off-rate of the caged substrate from the complex.
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During the present series of experiments, we observed the formation of holes from focusing the laser beam in the zebrafish embryo at laser powers beyond 15 mW at 750 nm at the sample. We do not think that this detrimental effect results from heat associated with light absorption with residual one-photon excitation at 750 nm followed by nonradiative deexcitation. First, below 5 mW at 750 nm at the sample, water does not lead to any significant heating see: Schönle, A, Hell, S. W, Opt. Lett. 1998, 23, 325-327
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During the present series of experiments, we observed the formation of holes from focusing the laser beam in the zebrafish embryo at laser powers beyond 15 mW at 750 nm at the sample. We do not think that this detrimental effect results from heat associated with light absorption with residual one-photon excitation at 750 nm followed by nonradiative deexcitation. First, below 5 mW at 750 nm at the sample, water does not lead to any significant heating (see: Schönle, A.; Hell, S. W.; Opt. Lett. 1998, 23, 325-327).
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In addition, the zebrafish embryo does not contain any significant amount of interfering endogeneous chromophores at that step of development. In particular, we were never able to express GFP under control of a heat-shock promoter by simply focusing the IR laser beam in any targeted cell of an appropriate transgenic embryo. In fact, we estimate that our observation is more probably related to plasma formation that occurs at 10 mW at 750 nm even in pure solvents see: Sacchi, C. A. J. Opt. Soc. Am. B 1991, 8, 337-345
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In addition, the zebrafish embryo does not contain any significant amount of interfering endogeneous chromophores at that step of development. In particular, we were never able to express GFP under control of a heat-shock promoter by simply focusing the IR laser beam in any targeted cell of an appropriate transgenic embryo. In fact, we estimate that our observation is more probably related to plasma formation that occurs at 10 mW at 750 nm even in pure solvents (see: Sacchi, C. A. J. Opt. Soc. Am. B 1991, 8, 337-345.
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I, designate the brightnesses of Z and I, respectively.
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I, designate the brightnesses of Z and I, respectively.
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Although here less precise, a satisfactory order of magnitude of k1 can also be extracted from the FCS data. In the considered kinetic regime, E can be considered to directly afford F with k1 rate constant and the number of F molecules in the focal point, NF, increases linearly with time with a slope E0k1. Then the average number of molecules 1/G(0, < NF > [t1;t2] extracted from the FCS experiment between time t1 and t2 (0 and 1500 s, and then 4500 and 6000 s in the experiment shown in Figure 3b) is given by the expression: (Chemical Equation Presented) where N0F is the number of F molecules in the focal point a t, 0 s. Then the results from at least two FCS experiments provide NF0 and k1. In the case
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