Encoded Planar Illumination Array Microscopy (EPAM) for High-Speed Parallelized Volumetric Imaging

Professor Tsia, Kevin Kin Man   (Principal Investigator (PI))

36

2017-01-01

675647

Encoded Planar Illumination Array Microscopy (EPAM) for High-Speed Parallelized Volumetric Imaging

biophotonics, high-speed optical imaging

Photonics,Others - Electrical and Electronic Engineering

Engineering (E)

17259316

General Research Fund (GRF)

2016

Completed

1) This project primarily aims to establish a new type of volumetric fluorescence imaging, called EPAM, by exploiting parallelized 3D encoded illumination and detection – a feature generally absent in the current imaging modalities. The rationale is that mechanical sample or light-beam scanning, and system synchronization are completely eliminated by parallelization in 3D. Not only can it guarantee high volumetric frame-rate and signal-to-noise ratio, but also minimize the exposure and thus photobleaching and phototoxicity. The encoding nature in EPAM also inherently provides optical sectioning and digital structured illumination for realizing 3D isotropic resolution without compromising the speed. Toward these goals, we specifically aim to: 2) Develop encoded planar-illumination array multiplexing – To realize 3D parallelization, we will develop a unique imaging strategy central to EPAM, featuring multiplexed planar-illumination array generation and encoding. The core element is an angle-misaligned mirror pair, which functions as an array of actively reconfigurable virtual sources that automatically generate a dense and incoherent 2D light-plane array – a unique and crucial element for high-quality illumination in EPAM. We will construct this planar-illumination array generator combined with an encoding module, which temporally modulates each of the 2D illumination planes with a unique code. In effect, the entire 3D image stack, which is captured simultaneously by a 2D image sensor, can be digitally demultiplexed (decoded) to reconstruct the 3D image. Such 3D multiplexing not only result in high volumetric frame rate with high SNR, it innately enables the optical sectioning capability. Particularly, we will adopt frequency-division multiplexing and coded-division multiplexing, which are routinely used in telecommunication, to the planar-illumination array, and investigate their compatibility to EPAM, in terms of imaging quality and speed; 3) Establish the EPAM prototype for high-speed volumetric imaging – We will integrate the encoded planar-illumination array generator in a microscope geometry resembling that typically adopted in light-sheet fluorescence microscopy (LSFM), i.e. one objective lens for excitation from one direction whereas another for fluorescence image capture from the orthogonal direction. This is motivated by the compatibility of this dual-objective-lens configuration with the working principle of EPAM. Also, EPAM in this configuration can inherit the key advantages (e.g. high speed, high spatial resolution), and thus the applications brought by LSFM. More importantly, the mechanical-scan-free nature of EPAM relaxes the physical limitation on the specimens/system. By incorporating the technique of extended depth-of-focus, which has broadly been applied in other microscope systems, we will establish the EPAM prototype that can record all optically-sectioned, in-focus 2D image planes simultaneously at a volumetric frame rate >10 Hz. We will characterize the performance of the EPAM prototype particularly in terms of the balance between spatial resolution, imaging speed, and detection sensitivity, and photodamage. Leveraging the encoded nature of 3D image stack, we will also implement structured-illumination digitally in the axial dimension without modifying the system and compromising the frame rate, favoring close-to-isotropic resolution; 4) Explore the potential applications of EPAM – Brought by the 3D multiplexing capability, which significantly minimizes the required exposure, another unique feature of EPAM is its unprecedentedly low photobleaching and phototoxicity. As a result, EPAM is thus well suited to address the unmet need in fundamental biology for long-term fast volumetric cellular/tissue/organism imaging at the sub-second temporal resolution. Built upon the prototype, we will, with the support from Faculty of Medicine at HKU, employ EPAM for two potential applications. First, we will use EPAM for large-scale monitoring dynamical changes in repair and regenerative processes of a live and freely-moving planarian – a unique regenerative worm model system used in stem cell biology due to its striking regenerative capability. Second, we will explore the use of EPAM for real-time 3D microparticle tracking, e.g. monitoring and understanding the spatiotemporal dynamics of the engineered bacteria (Salmonella) and the tumor vasculature system – a critical, yet missing, tool in the emerging field of bacterial cancer therapy.