A plan

The intrinsic fluorescence of the photoswitchable analogue GS-sw(FP), described previously^{77 (link)}, was used to study GS translocation into the cytoplasm. Approximately 1 ml of an E. faecalis cell suspension (OD₅₅₀ = 1.0) was co-incubated with 100 µg/ml GS-sw(FP) in order to observe cell uptake. Carboxyfluorescein succinimidyl ester (Sigma-Aldrich), which is unable to cross the plasma membrane, was applied for comparison to stain only the extracellular layers and outer  membrane leaflet. The bacterial suspension for the latter staining was resuspended in fresh 150 mM NaHCO₃ buffer (pH 8.3). At this increased pH, the amino groups of membrane proteins remain deprotonated and can react with the dye. The dye concentration was 1 µl/ml (stock solution 10 mg/ml in dimethyl sulfoxide). Both staining experiments were carried out at 37 °C for 30 min without agitation. Fluorescence was observed using a Axioskop 40 light microscope (Carl Zeiss) equipped with an “A-Plan” objective (100x/1.25 Ph3), a fluorescence filter (type 09, λ_ex = 450–490 nm, λ_em = 515 nm), and a digital camera (PowerShot G5, Canon). Due to the relatively low fluorescence intensity of GS-sw(FP), the exposure time was increased fourfold for the GS analogue compared to the control.