Fig. 4. FRET analysis of cells expressing Sla1p-YFP and Abp1p-CFP. FRET Images showing the interaction of Sla1p-YFP and Abp1p-CFP cortical patches. Fluorescence resonance energy transfer (FRET) between CFP and YFP was used to measure the relative proximity of Abp1 and Sla1p in KAY441 cells using the JP4 combination of excitation and emission filters sets and dichroic beamsplitter. (A) Deconvolved data from the FRET analysis showing (i) `YFP' fluorescence, (ii) `CFP' fluorescence, (iii) `FRET' fluorescence and (iv) `FRET control' fluorescence — zero in this case. Sla1p-YFP only populations (arrows), Abp1-CFP-only populations (arrowhead) and `FRET' populations can be observed. 3D histograms of the data are shown to the right indicating the relative levels of the `YFP', `CFP' and `FRET' signals in the raw data. The right hand panel shows a merged image showing the distinct overlapping populations. (B) The corrected FRET (FRET^C) image, after image arithmetic has been performed, is shown in quantitative pseudocolour representing arbitrary units of fluorescence intensity.

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