This paper describes a method by which molecules that are impermeable to cells are encapsulated in dye-sensitized lipid nanocapsules for delivery into cells via endocytosis. Once inside the cells, the molecules are released from the lipid nanocapsules into the cytoplasm with a single nanosecond pulse from a laser in the far red (645nm). We demonstrate this method with the intracellular release of the second messenger IP 3 in CHO-M1 cells, and report that calcium responses from the cells changed from a sustained increase to a transient spike when the average number of IP 3 released is decreased below 50 molecules per nanocapsule. We also demonstrate the delivery of a 23 kDa AGT fusion protein into Ba/F3 cells to inhibit a key player BCR-ABL in the apoptotic pathway. We show that an average of ~ 8 molecules of the inhibitor is sufficient to induce apoptosis in the majority of Ba/F3 cells. KeywordsLipids; Dye-Sensitized; Nanocapsule; Intracellular; Photolysis To uncover the spatiotemporal dynamics of cellular function, we need techniques that allow us to perturb cells over space and time in a controlled fashion. The most common approach for the delivery of bioactive molecules to cells in a highly spatiotemporally-resolved fashion is the two-photon release of a chemically-caged compound. 1-5 Caged molecules, however, suffer from a number of drawbacks. The design and synthesis of a new caged compound can be tedious and the caging of large protein molecules can be difficult, if not impossible. For intracellular delivery, the caged molecules must be permeable to the cell; this requirement, in turn, makes it difficult to control the precise concentration of the caged molecules inside the cell because of their preferential partitioning into different cellular organelles and membranes. These drawbacks of caged compounds have prompted us to develop lightaddressable lipid nanocapsules as a general platform for caging a wide range of bioactive molecules. 6-12 Nanocapsules based on lipid vesicles represent an emerging class of physical cages,, some of which also have been used recently for intracellular release. [13][14][15][16][17][18] Our first nanocapsules were lipid vesicles which we manipulated with optical tweezers and photolyzed using a single nanosecond laser pulse in the UV. 6,8,10 This capability was sufficient for delivering molecules extracellularly in a cell-culture setting. Unfortunately, the lipid vesicles were fragile and could not be stored in solution for more than a few days. This * To whom correspondence should be addressed chiu@chem.washington.edu. Supporting Information