Laura Waller

Multiplexed coded illumination for Fourier Ptychography with an LED array microscope

Fourier Ptychography is a new computational microscopy technique that achieves gigapixel images with both wide field of view and high resolution in both phase and amplitude. The hardware setup involves a simple replacement of the microscopes illumination unit with a programmable LED array, allowing one to flexibly pattern illumination angles without any moving parts. In previous work, a series of low-resolution images was taken by sequentially turning on each single LED in the array, and the data were then combined to recover a bandwidth much higher than the one allowed by the original imaging system. Here, we demonstrate a multiplexed illumination strategy in which multiple randomly selected LEDs are turned on for each image. Since each LED corresponds to a different area of Fourier space, the total number of images can be significantly reduced, without sacrificing image quality. We demonstrate this method experimentally in a modified commercial microscope. Compared to sequential scanning, our multiplexed strategy achieves similar results with approximately an order of magnitude reduction in both acquisition time and data capture requirements.

3D intensity and phase imaging from light field measurements in an LED array microscope

Realizing high resolution across large volumes is challenging for 3D imaging techniques with high-speed acquisition. Here, we describe a new method for 3D intensity and phase recovery from 4D light field measurements, achieving enhanced resolution via Fourier ptychography. Starting from geometric optics light field refocusing, we incorporate phase retrieval and correct diffraction artifacts. Further, we incorporate dark-field images to achieve lateral resolution beyond the diffraction limit of the objective (5× larger NA) and axial resolution better than the depth of field, using a low-magnification objective with a large field of view. Our iterative reconstruction algorithm uses a multislice coherent model to estimate the 3D complex transmittance function of the sample at multiple depths, without any weak or single-scattering approximations. Data are captured by an LED array microscope with computational illumination, which enables rapid scanning of angles for fast acquisition. We demonstrate the method with thick biological samples in a modified commercial microscope, indicating the technique’s versatility for a wide range of applications.

Transport-of-intensity approach to differential interference contrast (TI-DIC) microscopy for quantitative phase imaging

Differential interference contrast (DIC) microscopy is an inherently qualitative phase-imaging technique. What is obtained is an image with mixed phase-gradient and amplitude information rather than a true linear mapping of actual optical path length (OPL) differences. Here we investigate an approach that combines the transport-of-intensity equation (TIE) with DIC microscopy, thus improving direct visual observation. There is little hardware modification and the computation is noniterative. Numerically solving for the propagation of light in a series of through-focus DIC images allows linear phase information in a single slice to be completely determined and restored from DIC intensity values.

Transport of Intensity phase imaging by intensity spectrum fitting of exponentially spaced defocus planes

We propose an alternative method for solving the Transport of Intensity equation (TIE) from a stack of through-focus intensity images taken by a microscope or lensless imager. Our method enables quantitative phase and amplitude imaging with improved accuracy and reduced data capture, while also being computationally efficient and robust to noise. We use prior knowledge of how intensity varies with propagation in the spatial frequency domain in order to constrain a fitting algorithm [Gaussian process (GP) regression] for estimating the axial intensity derivative. Solving the problem in the frequency domain inspires an efficient measurement scheme which captures images at exponentially spaced focal steps, significantly reducing the number of images required. Low-frequency artifacts that plague traditional TIE methods can be suppressed without an excessive number of captured images. We validate our technique experimentally by recovering the phase of human cheek cells in a brightfield microscope.

Phase from chromatic aberrations

We show that phase objects may be computed accurately from a single color image in a brightfield microscope, with no hardware modification. Our technique uses the chromatic aberration that is inherent to every lens-based imaging system as a phase contrast mechanism. This leads to a simple and inexpensive way of achieving single-shot quantitative phase recovery by a modified Transport of Intensity Equation (TIE) solution, allowing real-time phase imaging in a traditional microscope.

Experimental robustness of Fourier ptychography phase retrieval algorithms

Fourier ptychography is a new computational microscopy technique that provides gigapixel-scale intensity and phase images with both wide field-of-view and high resolution. By capturing a stack of low-resolution images under different illumination angles, an inverse algorithm can be used to computationally reconstruct the high-resolution complex field. Here, we compare and classify multiple proposed inverse algorithms in terms of experimental robustness. We find that the main sources of error are noise, aberrations and mis-calibration (i.e. model mis-match). Using simulations and experiments, we demonstrate that the choice of cost function plays a critical role, with amplitude-based cost functions performing better than intensity-based ones. The reason for this is that Fourier ptychography datasets consist of images from both brightfield and darkfield illumination, representing a large range of measured intensities. Both noise (e.g. Poisson noise) and model mis-match errors are shown to scale with intensity. Hence, algorithms that use an appropriate cost function will be more tolerant to both noise and model mis-match. Given these insights, we propose a global Newtons method algorithm which is robust and accurate. Finally, we discuss the impact of procedures for algorithmic correction of aberrations and mis-calibration.

3D differential phase-contrast microscopy with computational illumination using an LED array

We demonstrate 3D differential phase-contrast (DPC) microscopy, based on computational illumination with a programmable LED array. By capturing intensity images with various illumination angles generated by sequentially patterning an LED array source, we digitally refocus images through various depths via light field processing. The intensity differences from images taken at complementary illumination angles are then used to generate DPC images, which are related to the gradient of phase. The proposed method achieves 3D DPC with simple, inexpensive optics and no moving parts. We experimentally demonstrate our method by imaging a camel hair sample in 3D.

Computational illumination for high-speed in vitro Fourier ptychographic microscopy

We demonstrate a new computational illumination technique that achieves large space-bandwidth-time product, for quantitative phase imaging of unstained live samples in vitro. Microscope lenses can have either large field of view (FOV) or high resolution, not both. Fourier ptychographic microscopy (FPM) is a new computational imaging technique that circumvents this limit by fusing information from multiple images taken with different illumination angles. The result is a gigapixel-scale image having both wide FOV and high resolution, i.e. large space-bandwidth product (SBP). FPM has enormous potential for revolutionizing microscopy and has already found application in digital pathology. However, it suffers from long acquisition times (on the order of minutes), limiting throughput. Faster capture times would not only improve imaging speed, but also allow studies of live samples, where motion artifacts degrade results. In contrast to fixed (e.g. pathology) slides, live samples are continuously evolving at various spatial and temporal scales. Here, we present a new source coding scheme, along with real-time hardware control, to achieve 0.8 NA resolution across a 4x FOV with sub-second capture times. We propose an improved algorithm and new initialization scheme, which allow robust phase reconstruction over long time-lapse experiments. We present the first FPM results for both growing and confluent in vitro cell cultures, capturing videos of subcellular dynamical phenomena in popular cell lines undergoing division and migration. Our method opens up FPM to applications with live samples, for observing rare events in both space and time.

Quantitative differential phase contrast imaging in an LED array microscope.

Illumination-based differential phase contrast (DPC) is a phase imaging method that uses a pair of images with asymmetric illumination patterns. Distinct from coherent techniques, DPC relies on spatially partially coherent light, providing 2× better lateral resolution, better optical sectioning and immunity to speckle noise. In this paper, we derive the 2D weak object transfer function (WOTF) and develop a quantitative phase reconstruction method that is robust to noise. The effect of spatial coherence is studied experimentally, and multiple-angle DPC is shown to provide improved frequency coverage for more stable phase recovery. Our method uses an LED array microscope to achieve real-time (10 Hz) quantitative phase imaging with in vitro live cell samples.

Real-time brightfield, darkfield, and phase contrast imaging in a light-emitting diode array microscope.

Abstract. We demonstrate a single-camera imaging system that can simultaneously acquire brightfield, darkfield, and phase contrast images in real time. Our method uses computational illumination via a programmable light-emitting diode (LED) array at the source plane, providing flexible patterning of illumination angles. Brightfield, darkfield, and differential phase contrast images are obtained by changing the LED patterns, without any moving parts. Previous work with LED array illumination was only valid for static samples because the hardware speed was not fast enough to meet real-time acquisition and processing requirements. Here, we time multiplex patterns for each of the three contrast modes in order to image dynamic biological processes in all three contrast modes simultaneously. We demonstrate multicontrast operation at the maximum frame rate of our camera (50 Hz with 2160×2560  pixels).