Single-shot quantitative phase microscopy with color-multiplexed differential phase contrast (cDPC)

RESEARCH ARTICLE Single-shot quantitative phase microscopy with color-multiplexed differential phase contrast (cDPC) Zachary F. Phillips1☯*, Michael Chen2☯, Laura Waller1,2 1 Graduate Group in Applied Science and Technology, University of California, Berkeley, United States of America, 2 Dept. of Electrical Engineering and Computer Sciences, University of California, Berkeley, United States of America a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Phillips ZF, Chen M, Waller L (2017) Single-shot quantitative phase microscopy with color-multiplexed differential phase contrast (cDPC). PLoS ONE 12(2): e0171228. doi:10.1371/ journal.pone.0171228 Editor: Irene Georgakoudi, Tufts University, UNITED STATES ☯ These authors contributed equally to this work. * Abstract We present a new technique for quantitative phase and amplitude microscopy from a single color image with coded illumination. Our system consists of a commercial brightfield microscope with one hardware modification—an inexpensive 3D printed condenser insert. The method, color-multiplexed Differential Phase Contrast (cDPC), is a single-shot variant of Differential Phase Contrast (DPC), which recovers the phase of a sample from images with asymmetric illumination. We employ partially coherent illumination to achieve resolution corresponding to 2× the objective NA. Quantitative phase can then be used to synthesize DIC and phase contrast images or extract shape and density. We demonstrate amplitude and phase recovery at camera-limited frame rates (50 fps) for various in vitro cell samples and c. elegans in a micro-fluidic channel. Received: August 29, 2016 Accepted: January 17, 2017 Published: February 2, 2017 Copyright: © 2017 Phillips et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. In addition, we have included the CAD designs and example code for how to construct the device as well as process images using our method. Funding: This (research, publication, project, Web site, report, etc.) is funded by the Gordon and Betty Moore Foundation’s Data-Driven Discovery Initiative through Grant GBMF4562 to Laura Waller (UC Berkeley) and by the David & Lucille Packard Foundation Fellowship for Science & Engineering. Introduction Quantitative Phase Imaging (QPI) involves recovering the complex field of a sample—both amplitude and phase. This enables label-free and stain-free optical imaging of biological samples in vitro. In contrast to qualitative phase imaging methods, such as Zernike phase contrast (PhC) [1]) and Differential Interference Contrast (DIC), quantitative methods recover the phase delay caused by the sample, decoupled from absorption information. Modifications of PhC [2] and DIC [3] can make these setups quantitative, at a cost of requiring multiple images. More commonly, QPI methods use interferometry with coherent illumination and a reference beam [4–6], making them expensive and sensitive to misalignment and vibrations. Amongst the wide array of existing QPI methods, several are single-shot techniques. Offaxis holography interferes the sample beam with a tilted reference beam, then recovers phase by Fourier filtering [7]. Parallel phase-shifting can spatially multiplex several holograms within a single exposure via an array of polarizers [8]. Single-shot QPI add-ons based on amplitude gratings work with commercial microscopes, replacing the traditional camera module [9, 10]. Another add-on option uses two cameras to capture defocused images which can then be used PLOS ONE | DOI:10.1371/journal.pone.0171228 February 2, 2017 1 / 14 Single-shot phase microscopy with color illumination Competing Interests: This paper is related to the US provisional patent application for the method described in the manuscript, titled: OPTICAL PHASE RETRIEVAL SYSTEMS USING COLORMULTIPLEXED ILLUMINATION (Inventor (s): Laura Waller, Zachary Phillips, Michael Chen. Filing Date: April 15, 2016: Serial Number: 62/ 323,461) filed through UC Berkeley. This does not alter our adherence to PLOS ONE policies on sharing data and materials. to solve the Transport of Intensity Equation (TIE) [11]. Alternatively, if chromatic aberrations are large enough, they can enable single-shot color TIE [12] without any hardware changes. This concept of color multiplexing is similar to that used in photographic depth ranging [13]. All of these methods require some level of spatial or temporal coherence, limiting resolution. We seek here a single-shot QPI method that achieves the spatially incoherent resolution limit. Differential Phase Contrast (DPC) [14–17] is a partially coherent QPI technique that requires multiple images. Each is captured using a different asymmetric half-circle source pattern, which shifts the sample’s spectrum in Fourier space. Thus, a half circle source and its complement will cause the pupil function to crop opposite sides of the sample’s spectrum. Since imaginary information is encoded in Fourier asymmetry, these images can be used to recover phase. Assuming a linearized model for a weakly scattering sample, the inverse problem becomes a single-step deconvolution process [15, 17]. DPC recovers both amplitude and phase with resolution up to the incoherent resolution limit (2× better than coherent methods). Practically, the illumination switching can be done quickly and at low cost with an LED array [16–18]. At least two complementary source patterns are required, but generally 4 patterns (top, bottom, left, right half-circles) are used to avoid missing frequencies. The DPC method was recently extended to color multiplexing [19], where the 4 source patterns were encoded into two images by using a color camera in combination with a color LED array. Similarly, color photometric stereo has been used for retrographic surface profiling of large objects using off-axis color illumination in reflection mode [20]. Our method, termed color Differential Phase Contrast (cDPC), requires only a single color image for multiplexing source patterns. The three RGB source color channels are used to display three different half-circle source patterns. A 4th image is not needed, since it can be synthesized by taking the sum of two images acquired with opposite half-circle illuminations (a synthetic brightfield image) and subtracting that of a 90 degree rotated half-circle source. Thus we require only 3 illumination patterns and 3 measurements, which are collected in a single shot using a RGB Bayer filter sensor. We start by implementing the source pattern in an LED array microscope, which offers many imaging modalities in one platform [16–18, 21–24]. However, our configuration does not require a dynamic so (...truncated)