Optical trap and digital holographic microscopy measure red blood cell biomechanics

Although more than 100 million units of blood are donated every year, chronic and acute blood shortages still limit the quality of medical care. And since red blood cells have a shelf life, constant replenishment is crucial. A reliable artificial blood replacement could moderate this problem, but so far that goal has proven elusive.

Red blood cells (RBCs) have unique mechanical properties that have proven difficult to replicate in artificial replacements. To characterize the problem, and perhaps lead to an evaluation and screening method to improve the artificial blood development process, a multidisciplinary team from Austria and Italy has measured RBC mechanical properties using both optical tweezers and digital holographic microscopy (DHM).¹

Measuring unique properties

Red blood cells transport oxygen and CO₂ through the body. They repeatedly force themselves through capillaries smaller than their diameter during their about-four-month lifetime by folding over to pass through, and recovering shape soon after. That squeezing and recovery is linked to the unmistakable biconcave disk shape, which is maintained by a cytoskeletal network of interconnected proteins. In vivo, immature reticulocytes experience numerous stimuli, perhaps both mechanical and chemical, triggering cytoskeletal network changes to create that shape. With ex vivo cultured RBCs, it’s proving difficult to replicate that maturation. Dan Cojoc, of the Institute of Materials at the National Research Council of Italy, Claudia Bernecker from the Medical University of Graz, and their colleagues realized that optical traps could provide a method to investigate RBC membrane mechanical properties.

The rapid change in intensity around a focused light beam results in forces pushing toward the focal point. These forces, although small, can manipulate microscopic entities and maintain them at the focal point, creating an optical trap. With deformable objects such as RBCs, the forces of the optical trap can compress the cell in the same way a capillary does. A native red blood cell (nRBC) would then fold in an optical trap the same way it does within a capillary (see figure).

Previous attempts to use optical traps with RBCs required silica microbeads to be attached to the membrane. That approach allows quantitative force measurement, but it also takes a significant amount of time. Cojoc and his colleagues traded off quantifiability for speed by using a single-beam optical trap to compress the RBCs. Their concept relies on moving a cell in and out of the trap while monitoring the light scattered through the cell. The measured intensity distribution for a completely deformable cell would only change with the cell’s position, while the distribution with a completely malleable membrane would vary widely as the cell changed shape. Thus, variation of intensity correlates with membrane stiffness.

To validate this procedure, the researchers created an optical trap from a 1064 nm beam focused through a 1.25 NA microscope objective. A focus tunable lens in the optical path shifts the focal point up to ±8 µm. The transmitted light is collected by a quadrant photodetector with a 5 kHz bandwidth. In parallel, a separate CMOS detector images the cell shape at 11 frames per second (fps). In practice, a cell was first identified, then held stationary in the trap for 10 seconds. The trap then oscillated at 0.75 Hz with a 5 µm amplitude for 10 seconds, then 7 µm for another 10 seconds, and then the cell was released from the trap and imaged as it recovered shape.

In a separate procedure, RBCs on glass slides were imaged with a DHM with a wavelength of 520 nm. The DHM separates the laser into a reference beam and sample beam, which create an interference pattern when they’re recombined. That provides an image of the variation in optical path length, which is related to cell thickness. By monitoring the image at 110 fps, the system quantifies cell membrane fluctuations.

Distinguishing native and cultured cells

For this proof-of-principle measurement, the quad-cell signal variance showed a slight but distinct difference between native RBCs and cultured RBCs matured in human platelet lysate, and a much greater difference with cultured RBCs matured in human plasma. That’s also consistent with observation, as the human plasma-treated RBCs remained spherical. Analysis of the DHM data confirmed that the signal variance is indeed correlated with membrane fluctuations. Although complete quantitative measurements were not possible, the measurements were consistent with accepted values for membrane bending modulus. More importantly, Cojoc says, “we’ve demonstrated better and more rapid biomechanical characterization of generated ex vivo-cultured RBCs versus native RBCs.”

“We plan to increase the throughput of the measurements by implementing multiple traps in parallel,” he says, “and also extend the technique to other cell types, such as native reticulocytes.”

REFERENCE

1. C. Bernecker et al., Cells, 10, 3, 552 (2021); doi:10.3390/cells10030552.