To the editor:

Our recent work demonstrated the advantages of a new imaging technique, local activation of fluorescent molecular probes (LAMP), which uses the cell-permeable caged dye NPE-HCCC2/AM to study cell gap junction coupling1. To extend this assay to cells in physiological preparations, we integrated two-photon uncaging and imaging (infrared-LAMP) to monitor intercellular dye diffusion in three dimensions (see Supplementary Methods online).

Activating caged dyes by two-photon excitation should offer superb resolution for tracking cellular and molecular movements2. This application requires caged probes with high two-photon uncaging cross-sections (δu). We measured the wavelength dependence of δu of our coumarin dyes, NPE-HCCC and NPE-HCC3 (Fig. 1a). Both caged probes were most sensitive to photolysis around 730 nm, and their δu values dropped to unmeasurable above 790 nm (Fig. 1b). The two-photon excitation cross-sections (σ2PE) of their parent fluorophores, HCCC and HCC (Fig. 1a), reached maxima of over 25 Goeppert-Mayer (GM; 1 GM = 10−50 cm4 × s / photon) at 790 nm (Fig. 1b), which is sufficient for live-cell two-photon imaging based on a comparison with the σ2PE of fluorescent proteins4.

Figure 1: Infrared-LAMP integrates two-photon uncaging and imaging to study junctional dye transfer in three dimensions.
figure 1

(a) Structures of coumarin dyes and NPE-caged forms. R, alkyl substitution. (b) Two-photon excitation spectra of HCCC and HCC, and wavelength dependence of two-photon uncaging efficiency of NPE-HCCC and NPE-HCC. (c–f) Volume-rendered three-dimensional images constructed from 43 slices (z-step, 1.25 μm) of 2PLSM images in the calcein channel (c) or coumarin channel (e and f; 30 s and 420 s after two-photon uncaging, respectively). The asterisk in c indicates the uncaged cell. Three spheroids in d outline the donor (1) and two recipient cells (2 and 3). (g) Time course of the average HCCC2 fluorescence intensities within the volume of each spheroid or the corresponding cells.

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Other caged dyes have δu well below 0.1 GM (refs. 3,5). Even at high laser power (>40 mW), the release of fluorophores by two-photon excitation remains challenging6. Although two-photon excitation limits photodamage to the focal plane, the possibility of bleaching and damage remains2,6. To minimize phototoxicity and diffusion of photolyzed products, it is important to be able to use a low laser power but still generate a sudden jump of effector mole-cules in a confined volume. The high spatial selectivity of two-photon excitation (<1 μm3) combined with a caged compound such as NPE-HCCC2/AM with a high δu value should be ideal for this.

To confirm the above measurements in cells, we loaded HeLa cells with NPE-HCCC2/AM and monitored the movement of released fluorophores by two-photon laser scanning microscopy (2PLSM). Before uncaging, cellular fluorescence was negligible; during continuous two-photon excitation at 790 nm, we detected no photolysis, but two-photon uncaging at 730 nm selectively activated HCCC2 (Supplementary Fig. 1 online). We used low laser power (<11 mW) to uncage and to image.

Finally, we used mouse pancreatic acini as a model system to test infrared-LAMP in tissue. The pancreatic acinar cell is the enzyme-secreting unit of the exocrine pancreas, where junctional communication is important for stimulation-secretion coupling. After loading dissected acini (Supplementary Fig. 2 online) with NPE-HCCC2/AM and calcein/AM, we acquired a stack of 2PLSM images (xy) of the calcein signal along the z axis (Fig. 1c). We achieved single-cell photoactivation by scanning the laser beam (730 nm) on a z plane across a region of interest encircled by the cell equator. We followed the diffusion of HCCC2 by acquiring 2PLSM z-stacks every 45 s. Post-acquisition analysis provided dynamic and quantitative information on cell-cell dye transfer in three dimensions (Fig. 1d–g and Supplementary Video 1 online). Experiments with mice were carried out according to a protocol approved by the Institutional Animal Care and Use Committee.

Note: Supplementary information is available on the Nature Methods website.