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

Abstract

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 2π ambiguities. In addition, the phase noise contribution arising from spectral channel crosstalk on the color camera is quantified. © 2010 Optical Society of America OCIS codes: 090.5694, 090.4220, 100.5088, 180.3170. Transmission-geometry quantitative phase microscopy (QPM) has been developed for three-dimensional measurement and characterization of a wide variety of transparent samples, such as transparent optical elements (e.g., microlens arrays) [1], optical fibers [2], and living cells [3,4]. This interferometric measurement of optical path delays (OPDs) provides quantitative contrast arising from both the physical height of the sample and its refractive index changes. While QPM provides diffractionlimited lateral resolution and nanometer-scale axial resolution of OPDs, the axial range over which the phase can be unambiguously determined is limited to 2π, which corresponds to one full wavelength of the illumination light.To solve the 2π ambiguities in the acquired phase profiles and determine the correct relative OPDs across a field of view, two-dimensional unwrapping algorithms are typically employed. Classic unwrapping algorithms are based on gradient minimization, which adds multiples of 2π at specific points in the phase map [5]. While these algorithms can accurately recover a smooth and slowly varying phase map, they frequently fail to accurately reconstruct objects containing a phase difference greater than π between adjacent image pixels.An alternative approach to overcoming the limitations of phase unwrapping is the use of multiple illumination wavelengths. By processing the phase profiles obtained from different wavelengths, it becomes possible to synthesize a single phase profile that is unambiguous over the range of the "beat" wavelength [6,7], which is larger than the unambiguous range that results from using a single wavelength independently. Because this approach is based on an analytical solution and not an iterative numerical method, two-wavelength phase unwrapping is particularly useful when classic unwrapping algorithms fail to correctly unwrap across sharp phase discontinuities.Two-wavelength phase unwrapping has been previously employed in reflection-geometry QPM using both sequential [8] and simultaneous [9,10] illumination/ detection schemes to accurately reconstruct surface profiles of highly reflective structures. Two-wavelength phase unwrapping has also been used for transmissiongeometry phase microscopy [1,11]; however, these methods use sequential illumination and detection, which require...

Biomedical applications of holographic microspectroscopy [Invited]

“…They share the principle of interferometric microscopy and the concept of multispectral imaging. One direct method is to employ multiple lasers with different wavelengths in QPM [16,71,[81][82][83][84]. White light illumination equipped with various spectral filters [19,85] and second-harmonic generation (SHG) [75,86,87] also have been used.…”

“…White light illumination equipped with various spectral filters [19,85] and second-harmonic generation (SHG) [75,86,87] also have been used. A color image sensor has been used for multispectral imaging channels so that spectroscopic quantitative phase images can be simultaneously measured [71,84,88]. As a representative example, spectroscopic phase microscopy (SPM) [19] has been used for noninvasive label-free quantification of Hb protein in living human red blood cells (RBCs).…”

Diffraction phase microscopy: monitoring nanoscale dynamics in materials science [Invited]

“…Due to the modulo 2π nature of the phase, phase unwrapping errors can occur for sharp edges that undergo a greater than 2π phase shift (π in reflection mode) across adjacent pixels. Phase unwrapping errors can be eradicated using numerical methods [33] or measurements with 2 or more wavelengths [34,35]. Note also, that in pure reflection, the light travels only in the surrounding medium, so Δn must be replaced with the refractive index of the surrounding medium, which is typically 1 for air.…”

Real-time phase shift interference microscopy

“…In addition, in order to extend the imaging range beyond one fringe, different phase-unwrapping algorithms should be employed. Finally, for this system to be applicable also to discontinuous high-step samples, a two-wavelength phase shift [25][26][27][28][29] approach can be used. …”