Interference Microscopy and Mass Determination

Barer, R.

doi:10.1038/169366b0

Cited by 407 publications

(307 citation statements)

References 8 publications

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“…The potential of phase microscopy for the determination of cellular dry mass as a fundamental physical property was recognized several decades ago (4,8). However, practical realization of this potential has remained elusive for lack of appropriate optical methods.…”

Section: Discussionmentioning

confidence: 99%

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Optical imaging of cell mass and growth dynamics

Popescu¹,

Park²,

Lue³

et al. 2008

American Journal of Physiology-Cell Physiology

482

399

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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...

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Section: Discussionmentioning

confidence: 99%

“…In applying Eq. 15, we used ␣ ϭ 0.2 ml/g, which corresponds to an average of reported values (4). As shown, in addition to a measure of cell dry mass, the phase image also revealed the overall structure of the cell monolayer as in conventional optical microscopy.…”

Section: Quantitative Phase Imaging Of Live Cellsmentioning

confidence: 99%

Optical imaging of cell mass and growth dynamics

Popescu¹,

Park²,

Lue³

et al. 2008

American Journal of Physiology-Cell Physiology

482

399

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“…The introduction of the interference microscope to biological problems has made it possible to determine optical retardations of living cells (4,15). Unfortunately, to measure optical retardations of cellular inclusions such as nuclei, it is necessary to know the contributing optical retardation of the cytoplasm above and below it in the optical axis (5).…”

Section: Methodsmentioning

confidence: 99%

“…In this case a --k becomes 0.1 a and from Equations [4] and [8] In the determination of ~l~ot~ let us further assume that vertical thickness of the nucleus and nucleolus is equal to their shortest diameter in the optical plane. Then k --d can be replaced by 1 --d/k in which d and k can be measured in any similar units.…”

Section: Methodsmentioning

confidence: 99%

The Relative Concentration of Solids in the Nucleolus, Nucleus, and Cytoplasm of the Developing Nerve Cell of the Chick

Merriam

Koch

1960

The Journal of Cell Biology

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Growing and differentiating nerve cells of the fifth cranial ganglion of the chick embryo were studied by several means. During the period of 70 hours to 11 days of incubation (Hamburger-Hamilton stages 19 to 37) average cell mass increased more than 4.5 times while cells changed from relatively undifferentiated neuroblasts to morphologically characteristic nerve cells with long processes. By making simplifying assumptions about thickness of nucleus and nucleolus, relative to cytoplasmic thickness, it was possible to calculate solute concentration of nucleus and nucleolus relative to that of the cytoplasm from measurements of optical retardations through living cells. Differences in relative solute concentration were observed in nucleolus, cytoplasm, and nucleoplasm in the approximate ratio 1.2:1.0:0.8, respectively. The ratio remained essentially constant during the growth period examined despite the fact that the cell components grow at markedly different rates. This suggests that solid concentrations are physical characteristics of nucleus, nucleolus, and cytoplasm which are maintained even during rapid growth and differentiation.By cytochemical means it was demonstrated that mass increase in the nucleus is not associated with increase in deoxyrihonucleic acid. Both ribonucleic acid and protein are in greater concentration in nucleolus and cytoplasm than in the nucleoplasm. Electron microscopy shows interruptions in the nuclear envelope as well as an approximately even distribution of electron density in nucleus and cytoplasm. It is pointed out that consistent differences in solid concentration can exist on either side of the nuclear envelope even though it contains "pores." Implications of these data are discussed.

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“…Estimations of the dry mass of individual nuclei were calculated from data obtained using interferometry and using the equation of Barer (1952).…”

Section: Nuclear Dry Mass and Nuclear Volume Of Large And Small Genotmentioning

confidence: 99%