RBC membrane fluctuations have been studied for nearly a century, as they offer a window into understanding the structure, dynamics, and function of this unique cell. Among the variety of experimental approaches available for study of cell mechanical properties [43
], optical microscopic techniques stand out as they enable collecting mechanical and dynamical data without physical contact with the sample. Moreover, since optical techniques can be extended into wide area imaging mode, these methods are ideally suited for high-throughput dynamical studies of live cells. Nevertheless, this has proven to be an experimentally challenging task, as most biologically relevant membrane motions occur at the nanometer and millisecond scales.
Over the past several decades, multiple optical methods have been used to study RBC mechanical properties. Phase contrast microscopy was used to study spatially-resolved RBC fluctuations [44
]. However, the phase contrast method is qualitative by nature, thus preventing full-field quantitative measurements. Reflection interference contrast (RIC) microscopy was developed to quantify the thermally-induced fluctuations of the erythrocyte membrane, but RIC is quantitative only in certain limiting situations [46
]. Fluorescence interference contrast (FLIC) microscopy relies on inferring the position of fluorescent dye molecules attached to the membrane from the absolute fluorescence intensity, and requires exogenous fluorescent markers by nature [48
]. Point dark field microscopy has been used to demonstrate the effect of ATP on RBC membrane dynamics [40
], but this technique only allows point measurements and is not suitable for studying spatial behavior of membrane fluctuations. In this report, we have presented several phase microscopy methods that enable full-field quantitative imaging of RBC dynamics with high sensitivity and at arbitrary time scales from milliseconds to hours.
Using FPM, which is a highly stable common-path interferometric method, we have measured spatially-resolved, low-frequency fluctuations across the RBC [31
]. These relatively slow motions, which appear to be confined to specific subcellular domains, have not been described before, and their physiological mechanism is unclear at this time. HPM, in contrast, is a single-shot full-field technique which provides spatially-resolved quantitative phase information at the millisecond scale [13
]. Using a stabilized HPM system, we have quantified the nanoscale thermal fluctuations of live RBCs [9
]. These measurements revealed a nonvanishing tension coefficient, which increases as cells transition from a normal discocytic shape to a spherical shape (). These findings are consistent with the common knowledge that discoid red cells have maximum mechanical flexibility, while more rigid forms such as spherocytes or elliptocytes exhibit less flexibility in microcirculation and are therefore more susceptible to mechanical damage. Furthermore, we have shown that the tension coefficient for RBC’s is significantly larger than giant unilamellar vesicles of comparable size, consistent with the cytoskeleton confinement model, in which the cytoskeleton hinders membrane fluctuations [9
DPM () can be essentially considered a hybrid instrument that combines the common-path geometry of FPM and the single shot capability of HPM, allowing fast and stable quantitative phase imaging [12
]. In addition, DPM can be readily combined with other microscopy methods, thus enabling multimodal microscopy [28
]. We have successfully used the DPM instrument to extract spatially resolved dynamical properties of live RBCs (–). The results reveal significant properties of temporal and spatial coherence associated with RBCs. Furthermore, we show that these correlations can be accounted for by the viscoelastic properties of the cell, and we extract the loss and storage moduli. Compared with other methods of extracting cell viscoelastic properties such as pipette aspiration [50
], electric field deformation [51
], optical tweezers [52
], dynamic scattering microscopy [34
], and magnetic beam excitation [53
], DPM measurements have the distinct advantage of being non-contact, non-invasive, and operate in the imaging mode.
Perhaps the most significant limitation of FPM, HPM, and DPM is the fact that they measure the line integral of the refractive index and the optical path length, thus effectively losing structural details in the z-direction. As discussed earlier, this is not a limitation for structurally homogenous samples such as RBCs, but it limits application on these techniques in more complicated cell types. Combination of DPM with light scattering in a method called dynamic scattering microscopy (DSM) enable measurement of cell membrane dynamical properties in all cell types, but unlike DPM, this method requires the use of exogenous microbeads [34
]. In order to fundamentally overcome this limitation, however, we have recently developed instrumentation and methods for tomographic phase microscopy or TPM [20
]. The TPM method enables disentangling the refractive index information from optical path length, thus providing detailed structural information without exogenous contrast (). Work in progress aims to extend dynamical studies of cell structures to 3-dimension using TPM and its future generations.
In summary, we believe that novel field-based microscopy techniques will soon replace conventional phase contrast methods for biological imaging. In particular, we have demonstrated the feasibility and power of these new methods in characterizing RBC dynamics. Given they are fast, non-contact, non-invasive and require no specific cell preparation, we believe that these methods will for the first time provide an opportunity for high-throughput analysis and study of RBC mechanical properties in health and disease.