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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    ABSTRACT: In this work, we propose a dual-wavelength in-line digital holographic microscopy (DHM) configuration in order to eliminate the conjugate image and reach the maximum resolution of CCD. By using this configuration, two holograms are acquired in one shot. Our method is based not only on the recordings of two holograms at slightly different planes, but also on the diffraction patterns formed with two wavelengths. With this experimental setup, we are able to analyze fast dynamic processes at the microscopic scale in real time. Theoretical evaluation, computer simulations and experimental results validate our proposal. The experimental results are obtained using a phase-amplitude object and compared with those calculated from the well-established phase-shifting interferometry technique. As far as we know, in-axis configuration with a single exposure has not been used in DHM, as we present in this paper.
    Optics and Lasers in Engineering 07/2013; 51(7):883–889. DOI:10.1016/j.optlaseng.2013.02.005 · 2.24 Impact Factor
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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: Quantitative phase spectroscopy is presented as a novel method of measuring the wavelength-dependent refractive index of microscopic volumes. Light from a broadband source is filtered to an ~5 nm bandwidth and rapidly tuned across the visible spectrum in 1 nm increments by an acousto-optic tunable filter (AOTF). Quantitative phase images of semitransparent samples are recovered at each wavelength using off-axis interferometry and are processed to recover relative and absolute dispersion measurements. We demonstrate the utility of this approach by (i) spectrally averaging phase images to reduce coherent noise, (ii) measuring absorptive and dispersive features in microspheres, and (iii) quantifying bulk hemoglobin concentrations by absolute refractive index measurements. Considerations of using low coherence illumination and the extension of spectral techniques in quantitative phase measurements are discussed.
    Biomedical Optics Express 05/2012; 3(5):958-65. DOI:10.1364/BOE.3.000958 · 3.65 Impact Factor
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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.
    12/2010: pages 169-198;
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