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Jesse B Schallek, Andres Guevara-Torres, David R Williams; Imaging the morphology, rheology and flux of single red blood cells in the living mouse eye without contrast agents . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3363. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Adaptive optics scanning light ophthalmoscopy (AOSLO) has been used to measure blood velocity in the living retina by tracking displacement of single blood cells. However, the complex morphology of blood cells has not yet been characterized due to insufficient cell boundary contrast. Here, we use differential imaging to resolve the shape of single red blood cells (RBCs) in the retinal circulation without using contrast agents.
Anesthetized C57BL/6J mice were imaged with an AOSLO using near infrared light. The confocal pinhole in the detection arm was replaced by a split-detector configuration (Scoles et al. 2014 IOVS) where the left and right half of the imaging point spread function was diverted into two, phase-locked photomultiplier tubes (PMT). Differencing the PMT signals provided differential contrast. 2-D point scanning at 25Hz was used to image slow moving RBCs. 1-D point scanning across a vessel at ~31 kHz provided high temporal resolution to image RBCs as they crossed the imaging beam.
We observed RBC deformation as cells 6.5 μm in diameter squeezed through the ~4 μm vessel lumen in the smallest capillaries (fig 1a). RBCs maintained a biconcave surface despite a high deformation index (length/diameter) that ranged from 1.55-2.42, similar to those reported in other tissues. 31 kHz 1-D scanning across a vessel imaged a train of erythrocytes that could be counted (RBC flux). Capillaries ranged from 40-161 cells/s (RBC volume of 1.9-7.7 picoliters/s). Capillaries showed robust modulations in RBC flux that corresponded to the heart rate of the anesthetized mouse (~300 beats/minute) demonstrating that pulsatile flow is pervasive in the smallest vessels (fig 1b). The leading and trailing edge of moving RBCs displayed classic “parachute” and “slipper” morphologies (fig 2a) revealing the microscopic rheology of RBC interactions with the vascular endothelium, plasma and glycocalyx. Capillaries showed heterogeneity in RBC packing density despite having similar velocity and size (fig 2bc).
This near infrared approach provides new hemodynamic information in capillaries while mitigating phototoxic exposure and obviating the need for blood contrast agents that may alter hemodynamics. Future studies analyzing the shape of moving RBCs have the potential to provide differential diagnosis in a variety of systemic diseases without requiring a blood draw.
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