Abstract
We present a phase-imaging technique to quantitatively study the three-dimensional structure of cells. The method, based on the simultaneous dual-wavelength digital holography, allows for higher axial range at which the unambiguous phase imaging can be performed. The technique is capable of nanometer axial resolution. The noise level, which increases as a result of using two wavelengths, is then reduced to the level of a single wavelength. The method compares favorably to software unwrapping, as the technique does not produce non-existent phase steps. Curvature mismatch between the reference and object beams is numerically compensated. The 3D images of SKOV-3 ovarian cancer cells are presented.
Highlights
In holography, the interference between the coherent object and reference waves produces a holographic recording, which contains the information about the intensity of light, and its phase
Hologram is sampled by a high resolution CCD array [2,3,4] and the detected light intensity profile is transferred to a computer as an array of numbers
We present a simple ways of correcting a curvature mismatch between the reference and object beams, based on the phase correction within the angular spectrum algorithm
Summary
The interference between the coherent object and reference waves produces a holographic recording, which contains the information about the intensity of light (amplitude signal), and its phase. The optical path length can be converted to physical thickness, providing the sample height information This property of holograms offers a phase-contrast techniques, which can be used for quantitative 3D imaging. As their optical paths lengths differ and the beams interfere when they are recombined, it creates shadow effects, giving the appearance of a three-dimensional image Both ZPC and DIC phase contrast microscopy techniques cannot be utilized to extract the quantitative phase information. In certain cases, the software unwrapping algorithm can mistakenly identify low intensity areas as multiple phase steps, producing nonexistent height features This problem is not present in dual-wavelength optical unwrapping, as it does not rely on surrounding pixels to correct the phase discontinuities, but compares the two single wavelength phase images, taken simultaneously. We present a simple ways of correcting a curvature mismatch between the reference and object beams, based on the phase correction within the angular spectrum algorithm
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