Simultaneous two-wavelength transmission quantitative phase microscopy with a color camera

Department of Biomedical Engineering, Fitzpatrick Institute for Photonics, Duke University, Durham, North Carolina 27708, USA.
Optics Letters (Impact Factor: 3.29). 08/2010; 35(15):2612-4. DOI: 10.1364/OL.35.002612
Source: PubMed


We present a quantitative phase microscopy method that uses a Bayer mosaic color camera to simultaneously acquire off-axis interferograms in transmission mode at two distinct wavelengths. Wrapped phase information is processed using a two-wavelength algorithm to extend the range of the optical path delay measurements that can be detected using a single temporal acquisition. We experimentally demonstrate this technique by acquiring the phase profiles of optically clear microstructures without 2pi ambiguities. In addition, the phase noise contribution arising from spectral channel crosstalk on the color camera is quantified.

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    • "It can be seen that the quantitative experimental result of the proposed method is in agreement with the standard four-step PSI result. The slight difference between profiles can be attributed to the phase noise due to the aforementioned intensity crosstalk between the red and green channels [26] "
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    • "Fu et al. used a two-wavelength QPM system to measure and map dispersion in live HeLa cells [3]. Multi-wavelength illumination has also been employed to aid phase unwrapping [4] and to decouple refractive index from cell thickness [5]. All of these methods exploit dispersive effects in QPM at small numbers of discrete spectral points. "
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    ABSTRACT: Interferometric phase measurements of wide-field images of biological cells provide a quantitative tool for cell biology, as well as for medical diagnosis and monitoring. Visualizing rapid dynamic cell phenomena by interferometric phase microscopy can be performed at very fast rates of up to several thousands of full frames per second, while retaining high resolution and contrast to enable measurements of fine cellular features. With this approach, no special sample preparation, staining, or fluorescent labeling is required, and the resulting phase profiles yield the optical path delay profile of the cell with sub-nanometer accuracy. In spite of these unique advantages, interferometric phase microscopy has not been widely applied for recording the dynamic behavior of live cells compared to other traditional phase microscopy methods such as phase contrast and differential interference contrast (DIC) microscopy, which are label free but inherently qualitative. Recent developments in the field of interferometric phase microscopy are likely to result in a change in this situation in the near future. Through careful consideration of the capabilities and limitations of interferometric phase microscopy, important new contributions in the fields of cell biology and biomedicine will be realized. This chapter presents the current state of the art of interferometric phase microscopy of biological cell dynamics, the open questions in this area, and specific solutions developed in our laboratory.
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