Apoptosis, programmed cell death, is necessary for maintaining tissue homeostasis. We addressed the issue of how intracellular transport changes during this process, applying fluorescence correlation spectroscopy (FCS). We used fluorescently labeled tracers with different sizes (radii from 1.3 to 20 nm) to measure changes in their intracellular mobility until the single cell died (based on a morphological assessment). Changes in the diffusion coefficient gave us information about the apoptotic cells’ nanoviscosity at multiple length scales. We showed that when apoptosis occurred, nanoviscosity suddenly increased by around 2–4 times. We connected this increase mainly to water efflux and volume loss during apoptosis, with a minor influence from caspase activity and cytoskeletal remodeling. A high value of nanoviscosity does not lead to apoptosis; however, a sudden rise in nanoviscosity invariably accompanies apoptosis. Our results show that abrupt slowing down of transport at different length scales marks cell death.

Most experimental procedures applied in modern biology involve cargo delivering into cells. One of the ways to cargo introduction is osmotic-mediated intracellular vesicle swelling. However, its widespread use was hindered due to cargo size (<10 nm) and cell-type-related restrictions. We addressed the issue of the composition of colloidal loading solution to enhance the efficiency of cellular delivery.

Quantitative description of biochemical processes inside living cells and at single-molecule levels remains a challenge at the forefront of modern instrumentation and spectroscopy. This paper demonstrates such single-cell, single-molecule analyses performed to study the mechanism of action of olaparib – an up-to-date, FDA-approved drug for germline-BRCA mutated metastatic breast cancer.