December 2017
Spotlight Summary by Gabriel Popescu
Refractive index measurement of suspended cells using opposed-view digital holographic microscopy
The refractive index of cells can be used as an intrinsic contrast marker, thus revealing internal structure without the typical limitations associated with fluorescence imaging (e.g., photoxicity, photobleaching). The main challenge in extracting the refractive index of live cells is that this information is contained in the phase of the imaging field rather than its amplitude. In other words, a direct intensity-image of the specimen will not report on the refractive index. Over the past decade or so, a number of quantitative phase imaging (QPI) methods have been developed, which are capable of revealing the refractive index distribution. The QPI field is developing at a rapid pace, especially now that several technological approaches are robust enough to be commercialized.
The paper by Zheng et al., reports on applying opposed-view digital holographic microscopy (OV-DHM) to retrieve phase maps associated with live cells in suspension, and from those images inferring the cell refractive index. OV-DHM is a recently developed variant of digital holographic microscopy and employs a special optical geometry based on the Sagnac interferometer. As a result, the imaging is performed with two counter-propagating beams, each providing an off-axis interferogram, which are demodulated to extract two phase images. From the phase maps, the authors extract the refractive index value by assuming a spherical cell shape. This measurement can be obtained from a single measurement, but using two beams in a Sagnac geometry provides additional information that can be useful in determining the plane of focus, as well as in averaging the out-of-focus background.
In principle, in future experiments this refractive index measurement can be used to quantify cell growth during the cell cycle and to obtain correlations with disease states and various treatments. One wonders whether the Sagnac interferometer setup can potentially be used to extract the full 3D distribution of refractive index by solving a scattering inverse problem. Another interesting avenue to explore might be exploiting the sensitivity of this system to motion along the optical axis, which is otherwise difficult to assess. For example, internal vesicle transport within cells generates opposite-sign Doppler frequency shifts to the counter-propagating beams. Thus, with a differential measurement akin to the one presented here, one can potentially extract the Doppler shift and the velocity distribution along the optical axis, in a manner reminiscent of dynamic light scattering.
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The paper by Zheng et al., reports on applying opposed-view digital holographic microscopy (OV-DHM) to retrieve phase maps associated with live cells in suspension, and from those images inferring the cell refractive index. OV-DHM is a recently developed variant of digital holographic microscopy and employs a special optical geometry based on the Sagnac interferometer. As a result, the imaging is performed with two counter-propagating beams, each providing an off-axis interferogram, which are demodulated to extract two phase images. From the phase maps, the authors extract the refractive index value by assuming a spherical cell shape. This measurement can be obtained from a single measurement, but using two beams in a Sagnac geometry provides additional information that can be useful in determining the plane of focus, as well as in averaging the out-of-focus background.
In principle, in future experiments this refractive index measurement can be used to quantify cell growth during the cell cycle and to obtain correlations with disease states and various treatments. One wonders whether the Sagnac interferometer setup can potentially be used to extract the full 3D distribution of refractive index by solving a scattering inverse problem. Another interesting avenue to explore might be exploiting the sensitivity of this system to motion along the optical axis, which is otherwise difficult to assess. For example, internal vesicle transport within cells generates opposite-sign Doppler frequency shifts to the counter-propagating beams. Thus, with a differential measurement akin to the one presented here, one can potentially extract the Doppler shift and the velocity distribution along the optical axis, in a manner reminiscent of dynamic light scattering.
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Article Information
Refractive index measurement of suspended cells using opposed-view digital holographic microscopy
Juanjuan Zheng, Peng Gao, Xiaopeng Shao, and G. Ulrich Nienhaus
Appl. Opt. 56(32) 9000-9005 (2017) View: Abstract | HTML | PDF