Protection from photobleaching for live-cell imaging

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One of the key pain points when labeling cells and tissues with fluorescence is photobleaching, the degradation of fluorescent signals as samples are exposed to light. Photobleaching is a complex photodynamic process whereby a photoexcited fluorophore interacts with molecular oxygen, resulting in the destruction of the fluorophore and the production of highly reactive singlet oxygen (1O2) that can further degrade neighboring dye molecules. This loss of signal is particularly problematic when attempting to collect time-course data, image rare targets with low signal-to-noise ratios, or quantitatively compare fluorescently labeled samples. The recently introduced Invitrogen™ ProLong™ Live Antifade Reagent takes a novel approach to protect fluorescent proteins and dyes from photobleaching in live cells (Figure 1).

Multicolor live-cell imaging with ProLong Live Antifade Reagent

Figure 1. Multicolor live-cell imaging with ProLong Live Antifade Reagent. U2OS cells were transduced with CellLight™ Talin-GFP (green) and CellLight™ Golgi-RFP (orange) for 24 hr, labeled with MitoTracker™ Deep Red FM (purple) and NucBlue™ Live ReadyProbes™ Reagent (Hoechst™ 33342, blue), and incubated with ProLong™ Live Antifade Reagent for 90 min before imaging on a confocal microscope.

How ProLong Live Reagent protects live cells

While researchers using fixed-cell systems have long had the luxury of a number of commercial antifade mounting media to choose from— such as Invitrogen™ ProLong™ Diamond Antifade Mountant—these formulations are not compatible with live-cell systems. Moreover, the protection provided by homemade antifade formulations—such as Trolox™ antioxidant [1,2] or ascorbic acid—is not significant for most fluorescent dyes and virtually nonexistent for fluorescent proteins.

Based on Oxyrase™ antioxidant technology [3], ProLong Live Antifade Reagent contains enzymes isolated from E. coli plasma membranes that metabolize environmental components that exacerbate photobleaching. Furthermore, these enzymes are not cell permeant, so intracellular functions are minimally affected. ProLong Live Antifade Reagent is simply diluted into cell medium or a suitable imaging buffer, and then added directly to cells for a 15- to 120-minute incubation. After incubation, imaging can be performed for up to 24 hours with continuous protection from photobleaching. ProLong Live Antifade Reagent has been validated to provide protection for a wide range of organic dyes as well as fluorescent proteins (Figure 2). Best of all, ProLong Live Antifade Reagent has been rigorously tested and shows little to no measurable effect on cell vitality, proliferation, or incidence of apoptosis for at least 48 hours.

Figure 2. Quantitative analysis of protection from photobleaching by ProLong Live Antifade Reagent for key dyes and fluorescent proteins. HeLa or U2OS cells were stained with (A) Hoechst™ 33342, (B) CellLight™ Mitochondria-RFP, (C) CellLight™ Mitochondria-GFP, or (D) MitoTracker™ Green FM reagents. Cells were incubated for 2 hr in the dark either in complete medium (control) or in complete medium containing ProLong™ Live Antifade Reagent (Cat. No. P36974). After incubation, cells were imaged every 15 sec with the Thermo Scientific™ ArrayScan™ VTI HCS Reader, using optimal but consistent excitation/emission imaging conditions.

Protect your live cells from photobleaching

ProLong Live Antifade Reagent can help you maintain longer imaging times for scans or time-lapse experiments, and enable you to detect low-abundance targets without sacrificing cell health.

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