A reporter of UV intensity delivered to the cytosol during photolytic uncaging

Abstract

Photolytic uncaging of biologically-active molecules within cells is a powerful technique. However, the delivery of uncaging light into the cytosol can vary between cell types, individual cells of the same type, and different loci within an individual cell because of optical differences in absorbance and light-scattering properties of the cytoplasm. Here, we demonstrate a simple technique for monitoring the magnitude of cytosolic ultraviolet delivery during uncaging, which also leaves a quantitative and persistent record of this within the cells. The simple method shown here provides a much needed universal monitor of the delivery of ultraviolet light to molecules within the cytosol, providing a much needed parameter for the correct interpretation of uncaging experiments.

Figure 1

Monitoring UV light delivery to the cytosol using hydroethidine. (a) The photo-induced reaction of hydroethidine to generate hydroxyethidium in the presence of molecular oxygen. (b) The different water solubilities of hydroethidine and its photo-induced product allows it to acts as a monitor for cytosolic UV exposure (c) Examples of the reaction in cells. The incubation medium for the cells contained hydroethidine (Sigma-Aldrich, Poole, UK, 20 μM) and UV illumination was for 10 s delivered through a Leica (Leica Microsystems, Milton Keynes, UK) confocal microscope (RS2) objective (x 63 oil) using a 50W Hg arc lamp with filters (330/80 nm input filter: 430 DCLPO2 dichroic; Omega Optics, Brattleboro, VT). The fluorescent signal was detected using 543 nm excitation HeNe laser line and emission at 600–700 nm.

Figure 2

Use of hydoethidine during uncaging (a) The graph shows the relationship between nuclear fluorescence (ex. 543 nm: em 600–700 nm) resulting from HE oxidation and the accumulated photons delivered (intensity × time) for the cell types shown in Fig.1C (HL60-blue, 3T3 - red, neutrophil - black) where the bars show the data range for each cell type. (b) A typical two-step exposure experiment demonstrating the repeatability and linearity of HE oxidation within an individual HL60 cell. Each pulse of UV light produced a similar magnitude of nuclear fluorescence at a constant rate of rise. (c) Uncaging of cytosolic IP₃ in a neutrophil loaded with both fluo4 (Invitrogen, Paisley, UK) as a Ca²⁺ probe and caged-IP₃ from its IP₃-PM ester (Alexis-Biochem, Nottingham, UK). The HE oxidation response begins at the start of UV light illumination, but the Ca²⁺ signal (in response to cytosolic IP₃) begins several seconds later. The data shows the phase contrast image of the cell (P/C), the fluo4 signal (Ca²⁺) and the fluorescence from photo-oxidation of HE (HEox). (d) The uniformity of UV exposure of cytosol in neutrophils within a cell population. HE oxidation in all neutrophils exposed to UV respond synchronously.