Imaging was performed on a fluorescence microscope (DM IRE 2, Leica) with a cooled CoolSnap HQ digital camera (Photometrics). Dual emission intensity images were recorded simultaneously using the DualView beamsplitter with the OI-5-EM filterset (Optical Insights) and Metafluor 6.1r1 (Universal Imaging). Excitation light was attenuated to 40% of the Lambda DG4's output (Sutter Instruments). Images were acquired within the linear detection range of the camera. HANK's balanced salt solution was used to replace the growth medium before each measurement. During the measurement, cells were perfused with HANK's at 1 ml/min. Perfusion with HANK's containing the indicated concentrations was used to apply external glucose (grey bars).
Movies available in Quicktime format for viewing glucose dependent ratio changes
Cells expressing the nanosensors are pseudocolored to demonstrate glucose dependent ratio changes. Red color indicates high ratios representing low internal glucose levels, green color shows low ratios at high internal glucose concentrations. Integration over all cells was used to quantify the ratio, which is shown as a function of time. Red dots indicate progress of measurement.
Click on link below to view each movie:
COS-7 nuc 5 mM glc: COS-7 cells expressing FLIPglu-600µnucl in the nucleus.
HepG2 cyt 5 mM glc: HepG2 cells expressing FLIPglu-600µnucl in the cytosol.
HepG2 ER 10 mM glc: HepG2 cells expressing FLIPglu-600µER in the ER.