Abstract
Parallelized fluorescence imaging has been a long-standing pursuit that can address the unmet need for a comprehensive three-dimensional (3D) visualization of dynamical biological processes with minimal photodamage. However, the available approaches are limited to incomplete parallelization in only two dimensions or sparse sampling in three dimensions. We hereby develop a novel fluorescence imaging approach, called coded light-sheet array microscopy (CLAM), which allows complete parallelized 3D imaging without mechanical scanning. Harnessing the concept of an “infinity mirror”, CLAM generates a light-sheet array with controllable sheet density and degree of coherence. Thus, CLAM circumvents the common complications of multiple coherent light-sheet generation in terms of dedicated wavefront engineering and mechanical dithering/scanning. Moreover, the encoding of multiplexed optical sections in CLAM allows the synchronous capture of all sectioned images within the imaged volume. We demonstrate the utility of CLAM in different imaging scenarios, including a light-scattering medium, an optically cleared tissue, and microparticles in fluidic flow. CLAM can maximize the signal-to-noise ratio and the spatial duty cycle, and also provides a further reduction in photobleaching compared to the major scanning-based 3D imaging systems. The flexible implementation of CLAM regarding both hardware and software ensures compatibility with any light-sheet imaging modality and could thus be instrumental in a multitude of areas in biological research.
Highlights
The grand challenge of understanding the animate processes of complex biological systems in three dimensions lies in the lack of imaging modalities that monitor the dynamics at a sufficiently high spatiotemporal resolution and with a low level of photodamage
The unique feature that enables parallelized 3D imaging in coded light-sheet array microscopy (CLAM) is motivated by the concept of free-space angular-chirp-enhanced delay (FACED)[23,24,25], which creates an ultrafast laser linescanning action from multiple reflections of a pulsed laser beam between a pair of angle misaligned plane mirrors
In contrast to the existing light-sheet fluorescence microscopy (LSFM) modalities, the defining feature of CLAM is its generation of a dense light-sheet array (>30), which is reconfigurable in both temporal coherency and spatial density
Summary
The grand challenge of understanding the animate processes of complex biological systems in three dimensions lies in the lack of imaging modalities that monitor the dynamics at a sufficiently high spatiotemporal resolution and with a low level of photodamage. In addition to speeding up the imaging rate, parallelization in both the illumination and detection maximizes the spatial duty cycle and the photon budget This is a critical requirement that ensures high-degree of viability of a biological sample. In order to minimize the illumination artifacts originating from the interplay between a coherent beam and highly scattering tissue (e.g., speckle noise), these strategies are limited to sparsely sampling the imaged volume with a handful of light sheets. They necessitate serial beam scanning or dithering to effectively create a time-averaged superposition of incoherent light sheets[3]. The final 3D image is computationally reconstructed with algorithms that solve the inverse problem, leading to the limitations of a reduced lateral resolution and a high computational complexity
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