Figure 3. Imaging of cytosolic lactate.

(A) HEK293 cells expressing Laconic imaged at 440 excitation/535 emission). Scale bar is 20 µm. (B) Fluorescence ratio was measured at 0.01, 0.1, 1 and 10 mM extracellular lactate in HEK293 cells treated with metabolic inhibitors and permeabilized to H⁺ as described in Material and Methods. (C) The time course of mTFP and Venus fluorescences were measured in the presence of 2 mM glucose/1 mM lactate or 10 mM pyruvate, as indicated. A cell with low expression of the sensor requiring strong illumination was chosen to demonstrate the insensitivity of the fluorescence ratio to photobleaching. The fluorescence ratio was converted into lactate concentration using the ratio in pyruvate (zero lactate) as described in the text.