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

Ziji Liu,Lei Tian,Laura Waller,Sijia Liu

doi:10.1117/1.jbo.19.10.106002

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).

Highlights

Brightfield, darkfield, and phase contrast are the most common label-free contrast modes used in optical microscopy.[1]
We have demonstrated the ability to capture multicontrast microscopy in real time for dynamic biological samples
Our results enable one to simultaneously visualize and compare images in brightfield, darkfield, and two directions of phase contrast

Summary

Introduction

Brightfield, darkfield, and phase contrast are the most common label-free contrast modes used in optical microscopy.[1] Brightfield imaging is most suitable for observing samples with strong absorption. Darkfield imaging provides good contrast for subresolution features, since it only captures high-angle scattered light. Phase contrast is used for unstained and transparent biological samples, allowing visualization of shape and density variations. There are several commercial choices for phase contrast [e.g., Zernike phase contrast or differential interference contrast (DIC)2] and new methods which provide quantitative phase.[3,4,5,6] Since each of brightfield, darkfield, and phase imaging provides complementary information about a sample, it is often desired to use multiple methods at once. In a traditional microscope, each contrast mode relies on a different optical hardware configuration, requiring inserts at the condenser aperture, polarization components, and/or specialized objectives. We demonstrate a system which can achieve all three modes simultaneously by placing an LED array at the source plane and implementing simple postprocessing steps

Methods

Results

Conclusion