Controversy has long surrounded the question of whether spontaneous lateral demixing of membranes into coexisting liquid phases can organize proteins and lipids on micron scales within unperturbed, living cells. A clear answer hinges on observation of hallmarks of a reversible phase transition. Here, by directly imaging micron-scale membrane domains of yeast vacuoles both in vivo and cell free, we demonstrate that the domains arise through a phase separation mechanism. The domains are large, have smooth boundaries, and can merge quickly, consistent with fluid phases. Moreover, the domains disappear above a distinct miscibility transition temperature (T) and reappear below T, over multiple heating and cooling cycles. Hence, large-scale membrane organization in living cells under physiologically relevant conditions can be controlled by tuning a single thermodynamic parameter.

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We report here the first example of a new and novel method of determining the binary temperature-composition phase diagram of a chromonic material in water using its intrinsic fluorescence. Disodium cromoglycate, or cromolyn, is an anti-allergy medicine representative of a class of compounds known as the chromonics. We have discovered that cromolyn's fluorescence is very sensitive to the polarity, hence structure, of the phase it exhibits. The fluorescence signal shifts its wavelength maximum and its shape depending on whether the cromolyn is a single phase or in coexisting phases. Since the signal due to individual phases can be identified, the fluorescence signal can reveal the temperature-induced transitions between single phase and phase coexistence regions. By studying such fluorescence data for different compositions, an isobaric temperature-composition phase diagram may be constructed. We present here a phase diagram derived from fluorescence studies that is in agreement with previous determinations using other techniques. Our results suggest that the binary phase diagrams of other intrinsically fluorescent chromonic materials, such as perylene monoimide and bisimide derivatives used in organic optoelectronic devices, solar cells, and light-emitting diodes, can be studied in water using an analogous fluorescence approach.