Using novel interferometric quantitative phase microscopy methods, we demonstrate that the surface integral of the optical phase associated with live cells is invariant to cell water content. Thus, we provide an entirely noninvasive method to measure the nonaqueous content or "dry mass" of living cells. Given the extremely high stability of the interferometric microscope and the femtogram sensitivity of the method to changes in cellular dry mass, this new technique is not only ideal for quantifying cell growth but also reveals spatially resolved cellular and subcellular dynamics of living cells over many decades in a temporal scale. Specifically, we present quantitative histograms of individual cell mass characterizing the hypertrophic effect of high glucose in a mesangial cell model. In addition, we show that in an epithelial cell model observed for long periods of time, the mean squared displacement data reveal specific information about cellular and subcellular dynamics at various characteristic length and time scales. Overall, this study shows that interferometeric quantitative phase microscopy represents a noninvasive optical assay for monitoring cell growth, characterizing cellular motility, and investigating the subcellular motions of living cells. phase microscopy; interferometric microscopy; cell growth PHASE-CONTRAST (PC) and differential interference contrast (DIC) microscopy have been used extensively to study live cells without the need for exogenous contrast agents (32). The tremendous success of these methods is due to the fact that the optical phase shift through a given sample contains information about the refractive index (n) variations that directly result from structural features within the sample. Refractive index n can therefore be regarded as a powerful endogenous contrast agent for cellular structure (6). However, as the relationship between the irradiance and phase of the image field is generally nonlinear (30, 39), both PC and DIC are qualitative in nature and limited to morphological observations without specific structural data.Quantitative phase microscopy has received substantial interest in recent years, as quantifying optical phase shifts associated with cells provides structural and dynamical information at the nanometer scale without the need for any cell preparation or the use of exogenous contrast or labels. Existing methods for biological quantitative phase measurements can be divided into single-point and full-field techniques. Several point measurement techniques have been used for investigating the local structure and dynamics of live cells (1,7,10,14,29,36,37). In contrast, full-field phase measurement techniques provide simultaneous information from a large region of the sample, which offers the additional benefit of studying both the temporal and spatial behavior of the sample (2, 5, 9, 13, 18 -20, 40, 41).Over the past several years, our laboratory has developed new full-field phase imaging techniques that are suitable for spatially resolved investigation of live cell...

Microfluidic tools are providing many new insights into the chemical, physical and physicochemical responses of cells. Both suspension-level and single-cell measurements have been studied. We review our studies of these kinds of problems for red blood cells with particular focus on the shapes of individual cells in confined geometries, the development and use of a 'differential manometer' for evaluating the mechanical response of individual cells or other objects flowing in confined geometries, and the cross-streamline drift of cells that pass through a constriction. In particular, we show how fluid mechanical effects on suspended cells can be studied systematically in small devices, and how these features can be exploited to develop methods for characterizing physicochemical responses and possibly for the diagnosis of cellular-scale changes to environmental factors.

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 demonstrate a real-time blood testing system that can provide remote diagnosis with minimal human intervention in economically challenged areas. Our instrument combines novel advances in label-free optical imaging with parallel computing. Specifically, we use quantitative phase imaging for extracting red blood cell morphology with nanoscale sensitivity and NVIDIA’s CUDA programming language to perform real time cellular-level analysis. While the blood smear is translated through focus, our system is able to segment and analyze all the cells in the one megapixel field of view, at a rate of 40 frames/s. The variety of diagnostic parameters measured from each cell (e.g., surface area, sphericity, and minimum cylindrical diameter) are currently not available with current state of the art clinical instruments. In addition, we show that our instrument correctly recovers the red blood cell volume distribution, as evidenced by the excellent agreement with the cell counter results obtained on normal patients and those with microcytic and macrocytic anemia. The final data outputted by our instrument represent arrays of numbers associated with these morphological parameters and not images. Thus, the memory necessary to store these data is of the order of kilobytes, which allows for their remote transmission via, for example, the cellular network. We envision that such a system will dramatically increase access for blood testing and furthermore, may pave the way to digital hematology.

Multiple scattering and absorption limit the depth at which biological tissues can be imaged with light. In thick unlabeled specimens, multiple scattering randomizes the phase of the field and absorption attenuates light that travels long optical paths. These obstacles limit the performance of transmission imaging. To mitigate these challenges, we developed an epi-illumination gradient light interference microscope (epi-GLIM) as a label-free phase imaging modality applicable to bulk or opaque samples. Epi-GLIM enables studying turbid structures that are hundreds of microns thick and otherwise opaque to transmitted light. We demonstrate this approach with a variety of man-made and biological samples that are incompatible with imaging in a transmission geometry: semiconductors wafers, specimens on opaque and birefringent substrates, cells in microplates, and bulk tissues. We demonstrate that the epi-GLIM data can be used to solve the inverse scattering problem and reconstruct the tomography of single cells and model organisms.

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Phthalates are a family of synthetic chemicals that are used in producing a variety of consumer products. Di-(2-ethylhexyl) phthalate (DEHP) is an widely used phthalate and poses a public health concern. Prenatal exposure to DEHP has been shown to induce premature reproductive senescence in animal studies. In this study, we tested the hypothesis that prenatal exposure to DEHP impairs neurobehavior and recognition memory in her male offspring and we investigated one possible mechanism-oxidative damage in the hippocampus. Pregnant CD-1 female mice were orally administered 200 μg, 500 mg, or 750 mg/kg/day DEHP or vehicle from gestational day 11 until birth. The neurobehavioral impact of the prenatal DEHP exposure was assessed at the ages of 16-22 months. Elevated plus maze and open field tests were used to measure anxiety levels. Y-maze and novel object recognition tests were employed to measure memory function. The oxidative damage in the hippocampus was measured by the levels of oxidative DNA damage and by Spatial light interference microscopic counting of hippocampal neurons. Adult male mice that were prenatally exposed to DEHP exhibited anxious behaviors and impaired spatial and short-term recognition memory. The number of hippocampal pyramidal neurons was significantly decreased in the DEHP mice. Furthermore, DEHP mice expressed remarkably high levels of cyclooxygenase-2, 8-hydroxyguanine, and thymidine glycol in their hippocampal neurons. DEHP mice also had lower circulating testosterone concentrations and displayed a weaker immunoreactivity than the control mice to androgen receptor expression in the brain. This study found that prenatal exposure to DEHP caused elevated anxiety behavior and impaired recognition memory. These behavioral changes may originate from neurodegeneration caused by oxidative damage and inflammation in the hippocampus. Decreased circulating testosterone concentrations and decreased expression of androgen receptor in the brain also may be factors contributing to the impaired neurobehavior in the DEHP mice.

Here, we report the results of a study on the effects of ethanol exposure on human red blood cells (RBCs) using quantitative phase imaging techniques at the level of individual cells. Three-dimensional refractive index tomograms and dynamic membrane fluctuations of RBCs were measured using common-path diffraction optical tomography, from which morphological (volume, surface area, and sphericity); biochemical (hemoglobin (Hb) concentration and Hb content); and biomechanical (membrane fluctuation) parameters were retrieved at various concentrations of ethanol. RBCs exposed to the ethanol concentration of 0.1 and 0.3% v/v exhibited cell sphericities higher than those of normal cells. However, mean surface area and sphericity of RBCs in a lethal alcoholic condition (0.5% v/v) are not statistically different with those of healthy RBCs. Meanwhile, significant decreases of Hb content and concentration in RBC cytoplasm at the lethal condition were observed. Furthermore, dynamic fluctuation of RBC membranes increased significantly upon ethanol treatments, indicating ethanol-induced membrane fluidization.