Significant progress has been made in the effort to overcome the technical challenges inherent in measuring biological phenomena in single cells. This progress has been made possible by applying approaches from very different fields of study.
The goal in any biological study is to develop a method that allows you to “see” what is going on under physiological conditions without perturbing the system by the use of large labels or probes. With that in mind, Manlio Tassieri, Ph.D., Royal Academy of Engineering research fellow at the University of Glasgow, has developed a simple and noninvasive experimental procedure to measure the linear viscoelastic properties of cells, which he described at Select BioSciences’ “Single Cell Analysis” conference.
How can rheology, the study of the flow of fluids, be used to develop models describing complex systems like cells? By applying rheology principles to the study of deformations occurring at micron length scales and with µL volumes, Dr. Tassieri and his group have contributed to the field of microrheology by moving their research focus from in vitro models to in vivo systems like living cells. In order to monitor the physiology in cells and get a sense of the underlying cytoskeleton, the team attached an antibody-coated 5 micron bead (anti-CD4) to a Jurkat cell resting on a coverslip.
The team then tracks the movement of the bead in relation to the larger lymphocyte by video monitoring. In the test system, movement of the bead was influenced by the combined action of both the cell’s cytoskeletal activity and the thermal fluctuations of the fluid molecules within the media around the cell. For example, the response to a hypo-osmotic shock could be followed by tracking the bead.
“Osmotic regulation is fundamental to homeostasis of the cell as documented in the literature. Using our microrheology method, we are able to show that when the cell is moved from iso- to hypo-osmotic conditions, the cell swells and then stiffens up to 300% of its normal rigidity,” explained Dr. Tassieri. “This rigidity is based on bundles of actin filaments realigning, leading to the re-organization of the cytoskeleton. Then over a period of minutes, the cell relaxes."
Similarly, the response to the addition of blebbistatin, a drug that blocks muscle contraction via interference with the actin/myosin interaction, can be monitored in the same way as shown for osmotic shock. The approach taken by the Tassieri lab is robust and reproducible, when compared to assays using techniques such as magnetic tweezers, optical tweezers, and atomic force microscopy. Further, the method has the advantage of revealing changes over a wide range of frequencies to a high level of accuracy.
Based on its simplicity and label-free nature, this application of microrheology could prove to be a valuable addition to studies that address cellular physiology under different pathological states.