We have developed diffraction phase microscopy as a new technique for quantitative phase imaging of biological structures. The method combines the principles of common path interferometry and single-shot phase imaging and is characterized by subnanometer path-length stability and millisecond-scale acquisition time. The potential of the technique for quantifying nanoscale motions in live cells is demonstrated by experiments on red blood cells.
We introduce Hilbert phase microscopy (HPM) as a novel optical technique for measuring high transverse resolution quantitative phase images associated with optically transparent objects. Because of its single-shot nature, HPM is suitable for investigating rapid phenomena that take place in transparent structures such as biological cells. The potential of this technique for studying biological systems is demonstrated with measurements of red blood cells, and its ability to quantify dynamic processes on a millisecond scale is exemplified with measurements of evaporating micrometer-sized water droplets.
Using a novel noncontact technique based on optical interferometry, we quantify the nanoscale thermal fluctuations of red blood cells (RBCs) and giant unilamellar vesicles (GUVs). The measurements reveal a nonvanishing tension coefficient for RBCs, which increases as cells transition from a discocytic shape to a spherical shape. The tension coefficient measured for GUVs is, however, a factor of 4-24 smaller. By contrast, the bending moduli for cells and vesicles have similar values. This is consistent with the cytoskeleton confinement model, in which the cytoskeleton inhibits membrane fluctuations [Gov et al., Phys. Rev. Lett. 90, 228101, (2003) The red blood cell (RBC) has a composite membrane consisting of a lipid bilayer coupled to a two-dimensional spectrin network, which grants the cell its characteristic properties of both softness and strong shear elasticity [1,2]. Because mature RBCs lack nuclei, they represent a convenient model for studying cell membranes, which have broad applications in both science and technology [3,4]. The lipid bilayer is 4 -5 nm thick, and exhibits fluidlike behavior, characterized by a finite bending modulus and a vanishing shear modulus, 0. The resistance to shear, crucial for RBC function, is provided by the spectrin network, which has a mesh size of 80 nm.Spontaneous membrane fluctuations, or ''flickering,'' have been modeled theoretically under both static and dynamic conditions [5][6][7][8][9]. These thermally induced membrane motions exhibit 100 nm scale amplitudes at frequencies of tens of Hz. In past studies, measurements of the membrane mean squared displacement versus spatial wave vector, u 2 q, revealed a q ÿ4 dependence predicted by the equipartition theorem, which is indicative of fluidlike behavior [6,10 -13]. These results conflict with the static deformation measurements provided by micropipette aspiration [14,15], high-frequency electric fields [16,17], and, more recently, optical tweezers [18], which indicate an average value for the shear elasticity of the order of 10 ÿ6 J=m 2 . Gov et al. predicted that the cytoskeleton pinning of the membrane has an overall effect of confining the fluctuations and, thus, gives rise to superficial tension much larger than in the case of free bilayers [5]. This confinement model may offer new insight into the cytoskeleton-bilayer interaction that determines the morphology and physiology of the cell [19].RBCs can be assumed optically homogeneous. Therefore, measurement of the cell optical path length via interferometric techniques can provide information about the physical topography of the membrane with subwavelength accuracy and without contact. However, existing optical methods, including phase contrast microscopy (PCM) [6], reflection interference contrast microscopy (RICM) [10], and fluorescence interference contrast (FLIC) [20], are limited in their ability to measure cell membrane displacements. PCM provides phase shifts quantitatively only for samples that are optically much thinner than the wavelength of light, which ...
We present a new quantitative method for investigating red blood cell morphology and dynamics. The instrument integrates quantitative phase microscopy with an inverted microscope, which makes it particularly suitable for the noninvasive assessment of live erythrocytes. In particular, we demonstrate the ability of this approach to quantify noninvasively cell volume and dynamic morphology. The subnanometer path-length sensitivity at the millisecond time scales is exemplified by measuring the hemoglobin flow out of the cell during hemolysis.
Using Hilbert phase microscopy for extracting quantitative phase images, we measured the average refractive index associated with live cells in culture. To decouple the contributions to the phase signal from the cell refractive index and thickness, we confined the cells in microchannels. The results are confirmed by comparison with measurements of spherical cells in suspension.
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