The CHO cells were maintained in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. Two days prior to imaging, cells were seeded in 6-well plates and cultured overnight. The following day, plasmid DNA (2 µg per transfection reaction) and Lipofectamine 2000 were diluted in Opti-MEM medium (both from Life Technologies) and added to the cells. Analysis was performed 24 hr following transfection.

Using OptiMiS, spectrally resolved fluorescence information was obtained from CHO cells expressing either VCV or VCVV constructs. Examples of results obtained with both types of samples are displayed in Figure 2. The Cerulean and Venus emission intensity maps (FDA and FAD, respectively) are shown along with the corresponding FRET efficiency distributions. The histograms constructed using the FRET efficiency distributions of the two CHO cells are shown in Figure 2C. The addition of the third Venus fluorophore to the tetramer construct causes an increase in the FRET efficiency of the construct due to the presence of an additional acceptor (Venus molecule) in the vicinity of the Cerulean fluorophore. As can be seen from the graphs in Figure 2, this slight difference in FRET efficiency between the two FRET constructs is resolvable from a single scan of a cell using OptiMiS.

Figure 2. Two-dimensional fluorescence distribution maps for the donor (Cerulean, ‘C’) in the presence of the acceptor (FDA) and acceptor (Venus, ‘V’) in presence of the donor (FAD) along with the map of FRET efficiencies calculated from FDA and FAD for (A) a CHO cell expressing a VCVV construct, and (B) a CHO cell expressing a VCV construct. (Click to enlarge)

Probing calcium concentration changes in living cells

The Premo Cameleon FRET biosensor (Life Technologies) consists of two fluorophore subunits, eCFP and cpVenus, connected by a calmodulin-M13 moiety. When the calmodulin binds four Ca²⁺ molecules, the biosensor undergoes a conformational change which brings the fluorescent protein domains closer to one another. This configuration change causes an increase in FRET occurring between the eCFP (donor) and cpVenus (acceptor) molecules. Hence, by monitoring the FRET efficiency distribution over single cells or even subcellular regions, it is possible to detect changes in the calcium concentrations of said regions.

CHO cells expressing the rat M1 muscarinic acetylcholine receptor (CHO M1WT3, ATCC Inc) were plated on a glass bottom culture dish (MatTek Corporation) at a density of 250,000 cells per plate. The CHO cells were cultured in Ham's F12K medium with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and supplemented with 0.5 mg/ml G418 and 10% fetal bovine serum. The M1-expressing CHO cells were then transduced with the Premo Cameleon Calcium Sensor using the BacMAM transduction protocol provided by the manufacturer. Following the transduction procedure, the cells were incubated in their normal growth medium for 40 hours at 37°C and 5% CO₂. Individual cells expressing the Ca²⁺ biosensor were scanned with OptiMiS a number of times in order to establish a baseline FRET efficiency value for the sensor expressed in that particular cell. Then, 500 µM carbachol, a muscarinic receptor agonist, was added to the culture dish, and the cell was imaged repeatedly over an extended period of time at intervals of 15 seconds.

The FRET efficiency distribution of the CHO cell displayed in Figure 3 qualitatively illustrates a change in the configuration of a large number of the Cameleon sensors expressed in that cell. FRET efficiency distributions were constructed in this manner for each time point in the series of OptiMiS scans following carbachol stimulation of the cell. For each time point, a histogram of the FRET efficiencies was constructed; two such histograms are plotted in Figure 4A. An average FRET value over the entire cell was calculated from each histogram and also plotted as a function of time (Figure 4B). Plotted on the same graph in Figure 4B is the average FRET efficiency for a cell which was repeatedly scanned as a function of time, but at no point subjected to carbachol stimulation.

Figure 3. Imaging of a single CHO cell co-expressing the rat M1 muscarinic receptor and the Premo Cameleon FRET biosensor. (Click to enlarge)

Figure 4. Measurements of the FRET distributions in CHO cells expressing the rat M1 muscarinic receptor and the Premo Cameleon FRET Biosensor. (Click to enlarge)