Snapshot Quantitative Phase Imaging with Acousto-Optic Chromatic Aberration Control

Abstract

The transport of intensity equation enables quantitative phase imaging from only two axially displaced intensity images, facilitating the characterization of low-contrast samples like cells and microorganisms. However, the rapid selection of the correct defocused planes, crucial for real-time phase imaging of dynamic events, remains challenging. Additionally, the different images are normally acquired sequentially, further limiting phase-reconstruction speed. Here, we report on a system that addresses these issues and enables user-tuned defocusing with snapshot phase retrieval. Our approach is based on combining multi-color pulsed illumination with acousto-optic defocusing for microsecond-scale chromatic aberration control. By illuminating each plane with a different color and using a color camera, the information to reconstruct a phase map can be gathered in a single acquisition. We detail the fundamentals of our method, characterize its performance, and demonstrate live phase imaging of a freely moving microorganism at speeds of 150 phase reconstructions per second, limited only by the camera's frame rate.

Keywords: acousto-optics; illumination encoding; label-free microscopy; liquid lens; transport of intensity equation.

Figure 1

Principle of the TAG-enabled TIE microscope based on chromatic aberration control. (a) Schematic representation of the microscope and corresponding timing diagram. By controlling the TAG lens axial-scanning with pulsed multi-colored illumination, the focal plane corresponding to each color can be selected. (b) Plot of the microscope focal position versus different pulse delay values when illuminating with red, green, and blue light. (c) From left to right: Color-PSFs of the red, green, and blue channels at different time delays (see Figure 1b) measured using a 100 nm diameter microbead translated at different axial positions in steps of 2.5 µm. Scale bars 100 µm. (d) FRC measurements of the USAF target for red (left), green (middle), and blue (right) colors performed at 50 fps. The vertical solid black line indicates the cut-off frequency. The dashed lines indicate the 0.1 threshold criterion.

Figure 2

Quantitative phase imaging of a cheek cell. (a) Intensity images of a cheek cell captured with our chromatic aberration control system in a single snapshot, and the corresponding phase map retrieved by solving the TIE. The red, green, and blue channels, after color-splitting, correspond to axial planes z = +5 µm, z = 0 µm and z = −5 µm, respectively. (b) Intensity images of the same cheek cell and corresponding phase map captured sequentially using monochromatic illumination and mechanical translation of the sample at positions z = +5 µm, z = 0 µm and z = −5 µm. Scale bars 100 µm.

Figure 3

Rapid quantitative phase imaging of a moving Paramecium. (a) Phase maps of a Paramecium in water at different time instances were acquired at a rate of 150 phase maps/second. (b) Composite motion image of the Paramecium phase maps. (c) Plot of the Paramecium’s nucleus trajectory over time. Scale bars 100 µm.