Photochemical Barcodes

Abstract

A photochemical strategy to encode fluorescence signals in vivo with spatial control was designed around the unique properties of a photoactivatable borondipyrromethene (BODIPY). The photoinduced disconnection of two oxazines, flanking a single BODIPY, in two consecutive steps produces a mixture of three emissive molecules with resolved fluorescence inside polymer beads. The relative amounts and emission intensities of the three fluorophores can be regulated precisely in each bead by adjusting the dose of activating photons to mark individual particles with distinct codes of fluorescence signals. The visible wavelengths and mild illumination sufficient to induce these transformations permit the photochemical barcoding of beads also in living nematodes. Different regions of the same animal can be labeled with distinct barcodes to allow the monitoring of their dynamics for long times with no toxic effects. Thus, our photochemical strategy for the generation of fluorescence barcodes can produce multiple and distinguishable labels in the same biological sample to enable the spatiotemporal tracking of, otherwise indistinguishable, targets.

Figure 1

Photoinduced transformation of 1 into 2, 3, and 4.

Figure 2

Normalized absorption and emission spectra of 1 (a,d) 2 (b,e), and 3 (c,f) in MeCN at 25 °C.

Figure 3

Brightfield image (a), overlap of three fluorescence channels (b), and relative emission intensities (c) of PS beads, doped with 1 (5% w/w), recorded after irradiation of individual beads for different times.

Figure 4

Overlaps of brightfield images and three fluorescence channels of C. elegans labeled with PS beads, doped with 1 (5% w/w), recorded before (a) and after (b) irradiation of the tail for 20 min; magnifications acquired before (c) and after (d–f) illumination for increasing times and relative emission intensities (g).

Figure 5

Overlaps (a,b) of brightfield images and three fluorescence channels of C. elegans labeled with PS beads, doped with 1 (5% w/w), recorded after irradiation of distinct regions for different times; magnifications of nonirradiated (d,f) and irradiated (c,e,g) areas and relative emission intensities (h).

Figure 6

Overlaps of brightfield images and three fluorescence channels (a–d) of C. elegans labeled with PS beads, doped with 1 (5% w/w), recorded sequentially over 77 s after irradiation of distinct regions for different times and storage for 50 min, and relative emission intensities (e) in the initial (a) and final (d) frames.