Photoactivatable Fluorophores for Bioimaging Applications

Abstract

Photoactivatable fluorophores provide the opportunity to switch fluorescence on exclusively in a selected area within a sample of interest at a precise interval of time. Such a level of spatiotemporal fluorescence control enables the implementation of imaging schemes to monitor dynamic events in real time and visualize structural features with nanometer resolution. These transformative imaging methods are contributing fundamental insights on diverse cellular processes with profound implications in biology and medicine. Current photoactivatable fluorophores, however, become emissive only after the activation event, preventing the acquisition of fluorescence images and, hence, the visualization of the sample prior to activation. We developed a family of photoactivatable fluorophores capable of interconverting between emissive states with spectrally resolved fluorescence, instead of switching from a nonemissive state to an emissive one. We demonstrated that our compounds allow the real-time monitoring of molecules diffusing across the cellular blastoderm of developing embryos as well as of polymer beads translocating along the intestinal tract of live nematodes. Additionally, they also permit the tracking of single molecules in the lysosomal compartments of live cells and the visualization of these organelles with nanometer resolution. Indeed, our photoactivatable fluorophores may evolve into invaluable analytical tools for the investigation of the fundamental factors regulating the functions and structures of cells at the molecular level.

Keywords: fluorescence photoactivation and dissipation (FPD); photoactivatable fluorophores (PAFs); photoactivated localization microscopy (PALM); photochemical barcoding, single-molecule localization microscopy (SMLM); single-particle tracking photoactivated localization microscopy (spt-PALM).

Figure 1.

Irreversible and reversible fluorescence photoactivation in photocage−fluorophore and photochrome−fluorophore constructs respectively together with schematic representations of the absorption and emission spectra before (black) and after (green) switching for photoactivation mechanisms based on S₁ (a−d) and S₀ (e−h) control.

Figure 2.

Sequences of steps required for the implementation of FRAP (a−d), FPD (e−h), and our imaging protocol (i−m) to monitor diffusion on the basis of the bleaching of selected fluorophores (FRAP), switching of their emission from off to on (FPD) and shifting the spectral region of their fluorescence (our protocol).

Figure 3.

Structural design of our PAFs with substituents (R¹−R³) available for the regulation of their photochemical and photophysical properties.

Figure 4.

Normalized absorption and emission spectra of 1 (a and b) and 2 (c and d) in acetonitrile together with an overlap (e) of CLSM images, recorded in resolved spectral channels (green: ^Rλ_Ex = 514 nm, ^Rλ_Em = 525−600 nm; red: ^Pλ_Ex = 633 nm, ^Pλ_Em = 640−750 nm) after activation (λ_Ac = 405 nm, 50 mW cm⁻², 10 s) of the central region, of a Drosophila melanogaster embryo microinjected with 1 and images (f−k) captured sequentially in the red channel to monitor the gradual diffusion of 2.

Figure 5.

Normalized absorption and emission spectra of 3 (a and b), 4 (c and d), and 5 (e and f) in tetrahydrofuran together with overlaps of wide-field fluorescence images, recorded in resolved spectral channels (green: ^Rλ_Ex = 561 nm, ^Rλ_Em = 575−600 nm; red: ^Pλ_Ex = 633 nm, ^Pλ_Em = 645−660 nm blue: ^Pλ_Ex = 633 nm, ^Pλ_Em = 750−800 nm) after activation (λ_Ac = 405 nm, 50 mW cm⁻²) along the intestinal tract of the tail (g) and head (h) regions of a Caenorhabditis elegans nematode, fed with polystyrene beads containing 3, for 5 (i), 0 (j), 10 (k), 0 (l), and 20 min (m) and the corresponding relative emission intensities (bar chart).

Figure 6.

Sequence of steps (a−c) proposed for the photochemical barcoding and tracking of single cells on the basis of the photoinduced conversion of PAFs in selected cells under the influence of different doses of activating photons and the monitoring of their relative emission intensities.

Figure 7.

Schematic representations of a fluorescence microscope (a) and the diffraction pattern produced on the focal plane of the objective lens (b) together with the sequence of steps (c−k) required for the implementation of PALM on the basis of fluorescence activation and single-molecule localization.

Figure 8.

Evolution (a) of the single-molecule emission spectrum (^Pλ_Ex = 642 nm, 10 W cm⁻²) with the photoinduced conversion of 6 into 7 (λ_Ac = 405 nm, 10 W cm⁻²) and bleaching of the latter compound in poly(methyl methacrylate) together with diffraction-limited (b: ^Rλ_Ex = 532 nm, 10 W cm⁻²) and PALM (c: λ_Ac = 405 nm, 10 W cm⁻²; ^Pλ_Ex = 642 nm, 4 kW cm⁻²) images of COS-7 cells, labeled with 8, as well as magnifications (d and e) of the latter image with the corresponding spatial distributions (f) of the emission intensities across the fluorescent organelles in the field of view.

Figure 9.

Sequence of steps (a−i) required for the implementation of spt-PALM on the basis of fluorescence activation and single-molecule tracking.

Figure 10.

Single-molecule fluorescence images of COS-7 cells labeled with 6 (a−c) or 8 (d−f), recorded sequentially after activation (λ_Ac = 405 nm, 10 W cm⁻²; ^Pλ_Ex = 642 nm, 4 kW cm⁻²), and the single-molecule trajectories (g) of the corresponding photochemical products.