Researchers have employed diffraction phase microscopy to gain more information about the morphology – form and structure – of human red blood cells. Their investigation could lead to advances in the treatment and screening of blood-cell morphology diseases such as malaria and sickle cell. The doughnut-shaped cells contain the molecule hemoglobin, which carries oxygen from the lungs throughout the body. They make up 40 to 45 percent of a person’s blood. The cells, which have indented centers, lack the internal structures found in other cells. Red blood cells have a membrane structure that enables them to be flexible and resilient, allowing them to squeeze through capillaries half their diameter. The cells transform from normal to spiculated to nearly spherical shape while accompanied by changes in cell mechanics. Little has been known about the mechanics of this membrane, a fluid lipid bilayer with an elastic 2-D network of spectrin, a cytoskeletal protein. Researchers used diffraction phase microscopy to study the mechanics of human red blood cell membranes. The cells are shown here. Courtesy of Gabriel Popescu. Diseases such as malaria, spherocytosis and sickle cell can cause changes in both the equilibrium shape and mechanics of the cells, which affect their transport function, according to the scientific report by YongKeun Park et al., published online in PNAS on March 29, 2010. The interdisciplinary team, led by professors Gabriel Popescu (electrical and computer engineering at the University of Illinois at Urbana-Champaign) and Alex Levine (chemistry and biochemistry at the University of California, Los Angeles), used diffraction phase microscopy to quantify the undulations of the membranes during the shape transition. The team was interested in learning how the deformability (alteration of form or shape) of the red blood cells related to their morphology. The deformability of the cells is considered to be their most important property. The highly sensitive measurement technique uses two beams of light – one going through the specimen and the other used as a reference. It enabled the scientists to see nanoscale membrane fluctuations in live cells and to measure them quantitatively. The researchers applied the noncontact optical interferometric technique to quantify the thermal fluctuations of the cell membranes with 3-nm accuracy over a range of spatial and temporal frequencies. By measuring “bumpy” red blood cells called echinocytes and round ones called spherocytes, they discovered that these deformed cells display less flexibility in their membranes – a finding that could provide insight into the mechanics and treatment of the diseases that affect the shape of red blood cells. The researchers used a new mathematical model of the red blood cell membrane fluctuations to extract from their fluctuation data the elastic properties of the red blood cell membranes. This mathematical model accounts for the curvature of the cells. Previous models had treated the membrane as a flat sheet. The dual optical method and theoretical model allowed them to find some new results where the shape and deformability are coupled. Microrheology Levine, who was involved in the theoretical aspects of the research, explained that the basic framework within which this sort of modeling falls is called microrheology. He said the basic idea is that in the macroscopic world it is fairly straightforward to push – or, more technically, shear – materials to measure how they deform or flow under stress, which is the study of rheology. “When you wish to study the rheology of microscopic and very fragile materials, it is obviously difficult to grab them and push on them. This is where the idea of studying the thermally driven fluctuations of soft microscopic materials comes in. By observing their fluctuations, you can work backwards mathematically to determine their elastic/mechanical properties,” Levine stated. “Popescu’s experiments provide a beautifully precise measure of the fluctuations of the membranes of these cells. That membrane is only nanometers thick and a few microns across. It is both extremely tiny and fragile on the scale of typical macroscopic objects. Our mathematical models allow us to analyze that fluctuation data and determine in detail the various elastic properties of the membrane. So this is an excellent example of microrheology being applied to cell membranes,” Levine said. The dual optical technique and mathematical model could have applications such as screening for certain blood diseases, and screening stored blood for membrane flexibility prior to transfusion – cellular changes in banked blood often occur. It may also allow scientists to study the effects of chemical agents, such as alcohol, on membranes, and to assess the effectiveness of medications in development for the treatment of blood cell morphology diseases. The investigation included collaborators from Massachusetts Institute of Technology in Cambridge, Mass.; from Harvard Medical School, Harvard School of Engineering and Applied Sciences, and Massachusetts General Hospital, all in Boston; and from the University of Colorado at Boulder.