Abstract
Fourier ptychography microscopy (FPM) is a recently developed microscopic imaging method that allows the recovery of a high-resolution complex image by combining a sequence of bright and darkfield images acquired under inclined illumination. The capacity of FPM for high resolution imaging at low magnification makes it particularly attractive for applications in digital pathology which require imaging of large specimens such as tissue sections and blood films. To date most applications of FPM have been limited to imaging thin samples, simplifying both image reconstruction and analysis. In this work we show that, for samples of intermediate thickness (defined here as less than the depth of field of a raw captured image), numerical propagation of the reconstructed complex field allows effective digital refocusing of FPM images. The results are validated by comparison against images obtained with an equivalent high numerical aperture objective lens. We find that post reconstruction refocusing (PRR) yields images comparable in quality to adding a defocus term to the pupil function within the reconstruction algorithm, while reducing computing time by several orders of magnitude. We apply PRR to visualize FPM images of Giemsa-stained peripheral blood films and present a novel image processing pipeline to construct an effective extended depth of field image which optimally displays the 3D sample structure in a 2D image. We also show how digital refocusing allows effective correction of the chromatic focus shifts inherent to the low magnification objective lenses used in FPM setups, improving the overall quality of color FPM images.
Highlights
Fourier ptychography microscopy (FPM) is a computational imaging technique in which a series of low resolution brightfield and darkfield images, captured by illuminating the sample at different angles, are combined through an iterative phase retrieval algorithm to generate a single high resolution complex image with a large field of view (FoV) [1]
We explored the limits of post reconstruction refocusing (PRR) by imaging a 3D test sample fabricated using two chrome on glass calibration targets separated by microscope cover glasses with a 1.25x/0.04 objective lens (PlanApo N, Olympus) with a nominal depth of field (DoF) (Eq 1) of ∼ 470 μm at 632 nm
We applied digital refocusing using both initial pupil modification’ (IPM) and PRR methods and compared the results for four specific sample features (a 65 μm diameter disc, vertical and horizontal line pairs and the numeral ‘7’) to corresponding FPM reference images captured with the two calibration targets in focus
Summary
Fourier ptychography microscopy (FPM) is a computational imaging technique in which a series of low resolution brightfield and darkfield images, captured by illuminating the sample at different angles, are combined through an iterative phase retrieval algorithm to generate a single high resolution complex image with a large field of view (FoV) [1]. By relaxing the traditional trade-off between numerical aperture (NA) and magnification, FPM greatly increases the information gathering power of a light microscope and has significant potential for applications requiring both a large FoV and high spatial resolution such as imaging fixed and live cells [2,3,4] and tissue sections [5,6] as well as non-biological samples [7]. Combining the information contained within a series of such shifted passbands results in an image with an extended spatial frequency support, corresponding to an increase in the effective NA of the microscope from NAobj to NAsyn = NAobj + NAillu [8], where NAillu is the NA corresponding to the largest illumination angle. FPM images of even relatively thin samples typically contain out of focus information and it is not possible to visualise the full thickness of the sample in a single reconstructed 2D FPM image
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