Merging of confocal and caging technologies: selective three-color communication with profluorescent reporters

Abstract

Falling apart, on cue: Signaling pathways often display a profound spatiotemporal component that is best studied using light-activatable reagents. Three separate photolabile moieties that can be distinguished based upon their response to three distinct wavelengths (360, 440, and 560 nm) have been synthesized and evaluated. This tri-color system is also applied to imaging in microwells and HeLa cells (see picture).

Figure 1

Structures of pro-fluorescent reporters of photolysis, where TAMRA in NB-1 and Cob-4 displays a λ_em = 580 nm, Rhodamine 110 in Coum-3 a _λem = 540 nm, and fluorescein in NB-2 displays a _λex = _λem = 520 nm. The structure of QSY7 is furnished in the Supporting Information.

Figure 2

(a) Fold increase of NB-1 fluorescence upon photolysis with a Hg arc lamp at 360 nm (●), 440 nm (◆) or 560 nm (▲). (b) Fold increase of Coum-3 fluorescence upon photolysis at 360 nm (●), 440 nm (◆) or 560 nm (▲). (c) Fold increase of Cob-4 fluorescence upon photolysis in a fluorimeter by excitation at 360 nm (●), 440 nm (◆) or 560 nm (▲). RF is an abbreviation for relative fluorescence.

Figure 3

Strategy for demonstrating the wavelength selective photolysis of Cob-4 in a NB-1/Coum-3/Cob-4 mixture. Cob-4 photolysis at 560 nm furnishes a mitochondria-free fluorescent response at 575 nm. Coum-3 photolysis at 440 nm provides a mitochondria-associated fluorescent response at 540 nm. Finally, NB-1 photolysis at 360 nm delivers a mitochondria-associated fluorescent response at 575 nm.

Figure 4

(a) Quantification of the fluorescent increase of NB-1 in HeLa cells before and after 25 frames of photolysis at 355 (●), 440 (■) or 559 nm (▲). (b) NB-1-loaded HeLa before and after photolysis at 355 nm. (c) Quantification of the fluorescent increase of Coum-3 in HeLa cells before and after 25 frames of photolysis at 355 nm (●), 440 nm (■) or 559 nm (▲). (d) Coum-3-loaded HeLa before and after photolysis at 44o nm. (e) Quantification of the fluorescent increase of Cob-4 in HeLa cells imaged from 1 – 50 frames at 559 nm. (f) Cob-4-loaded Hela before and after photolysis at 559 nm.