Photoactivatable fluorophores and techniques for biological imaging applications

Abstract

Photoactivatable fluorophores (PAFs) are powerful imaging probes for tracking molecular and cellular dynamics with high spatiotemporal resolution in biological systems. Recent developments in biological microscopy have raised new demands for engineering new PAFs with improved properties, such as high two photon excitation efficiency, reversibility, cellular delivery and targeting. Here we review the history and some of the recent developments in this area, emphasizing our efforts in developing a new class of caged coumarins and related imaging methods for studying dynamic cell-cell communication through gap junction channels, and in extending the application of these caged coumarins to new areas including spatiotemporal control of microRNA activity in vivo.

Figure 1

Examples of classical caged fluorophores based on NB or related caging groups.

Figure 2

Structures, photochemical and fluorescent properties of NPE-caged coumarins, NPE-HCCC and NPE-HCC. Parameters shown here are for NPE-HCCC. Parameters for NPE-HCC are shown in Table 1. Q_f, fluorescence quantum yield; Q_u, uncaging quantum yield; ε^365nm, extinction coefficient at 365 nm; δ_u^740nm, two photon uncaging cross section at 740 nm.

Figure 3

Absorption spectra of equmolar NPE-HCCC (a), 6-chloro-7-methoxy-coumarin 3-carboxamide (b), 1-(2-nitrophenyl)ethanol (c), and a 1:1 mixture of 6-chloro-7-methoxy-coumarin 3-carboxamide and 1-(2-nitrophenyl)ethanol (d). The spectra were taken in pH 7.3 Mops buffer (20 mM). Adapted from ref. with permission.

Figure 4

Structure and mode of action of a caged and cell membrane permeable coumarin, NPE-HCCC2/AM. Adapted from ref. with permission.

Figure 5

(A) Connexin, connexon and gap junction channel between a coupled cell pair. (B) Schematic of the LAMP technique for studying gap junction coupling. Black dots represent NPE-HCCC2. In step (1), cells were loaded with NPE-HCCC2/AM; Step (2), NPE-HCCC2 was photolyzed with a restricted light beam in a selected cell to generate HCCC2; Step (3), track HCCC2 diffusion to neighboring coupled cells by fluorescence microscopy. Steps (2) and (3) can be repeated multiple times, either by uncaging the same or a different donor cell, in order to track changes in cell coupling strength.

Figure 6

LAMP, a non-invasive and quantitative imaging assay of gap junctional communication. (A) Time courses of fluorescence intensities of HCCC2 in a pair of coupled human fibroblasts. Three episodes of UV flashes were applied to a cell (cell a, donor) at times indicated by the arrows at the top. Prior to the third uncaging, a GJ coupling blocker, α-Glycyrrhetinic acid (α-GA, 10 μM), was added. It stopped dye transfer. (B1–B5) Selected fluorescence images of coupled cells taken at times indicated by dashed arrows in (A). To aid visualization of both donor and recipient cells, they were briefly illuminated by UV light at the beginning (B1). (C1, C2) Quantification of HCCC2 transfer rates using data from (A) and Fick’s equation. The slopes of the fitted lines (linear least-square fits) gave rates of dye transfer after first (k₁) and second (k₂) uncagings (r² values of linear fittings in parentheses).

Figure 7

Two-photon uncaging spectra (top) of NPE-HCCC (triangle, X = Cl) and NPE-HCC (circle, X = H) and two photon excitation spectra of HCCC (triangle, X = Cl) and HCC (circle, X = H). Adapted from ref. with permission.

Figure 8

Structures of a caged FRET dye, caged coumarin-calcein (CCC-1), and its photolyzed product CC-1.

Figure 9

Two distinct types of bioconjugates of small synthetic PAFs. (A) In type 1 dextran conjugates of caged coumarin, fluorophore and dextran remain linked after photolysis. (B) In type 2 dextran conjugates of caged coumarin, fluorophore is separated from the dextran carrier upon uncaging.

Figure 10

Trojan-LAMP assay of cell junctional coupling in vivo. (A–D) Rapid dye transfer between pharyngeal muscle cells in a living C elegans larvae. A pulse of UV laser was delivered to a spot within the terminal bulb (indicated by the arrow in DIC image, A, scale bar = 10 μm) at time 0. Three additional pulses of UV laser were then delivered at 4, 11, and 16 sec. Coumarin fluorescence images at different time points were shown (B–D, image D was taken after moving the stage of the microscope to fit the entire pharynx into the viewing area of a CCD camera). 1, terminal bulb; 2, isthmus; 3, metacarpus; 4, procorpus. Adapted from ref. with permission. (E) Schematic of the anatomy of the pharynx of C. elegans. Eight groups of pharyngeal muscle cells, pm1 to pm8, are coupled by gap junction channels.

Figure 11

Structure, preparation, and mode of action of caged antimirs (cantimirs) for the spatial and temporal control of miRNA activity. Adapted from ref. with permission.