Light-Sheet Fluorescence Imaging

CLAM illumination profiles in multiple views. Top right: 3D rendered image with three orthogonal standard-deviation-intensity projections of the tubular epithelial structure in the mouse kidney. Botton right: Sectional images of the mouse glomeruli captured by the CLAM microscope. (Source: Y.-X. Ren, J. Wu, Q. T. K. Lai, H. M. Lai, D. M. D. Siu, W. Wu, K. K. Y. Wong & K. K. Tsia)

An arsenal of advanced micro­scopy tools is now available to provide high-quality visualization of cells and organisms in 3D and has thus sub­stantiated our under­standing the complex biological systems and functions. Now, a research team led by the University of Hong Kong (HKU) developed a new form of imaging modality, coined coded light-sheet array micro­scopy (CLAM) that allows full 3D paralle­lized fluores­cence imaging without any scanning mechanism – a capability that is otherwise challenging in the existing techniques.

Established 3D biological micro­scopy techniques, notably confocal, multiphoton microscopy, and light-sheet fluores­cence micro­scopy (LSFM), pre­dominantly rely on laser-scanning for image capture. Yet, it comes at the expense of imaging speed because the entire volume has to be sequentially scanned point-by-point, line-by-line or plane-by-plane at a speed limited by the mechanical motions involving the imaging parts. Even worse, many serial scanning approaches repeatedly excite out-of-focus fluorescence, and thus accelerate photo­bleaching and photo­damage. They are thus not favorable for long-term, large-scale volumetric imaging critically required in applications as diverse as anatomical science, develop­mental biology and neuro­science.

3D parallelization in CLAM requires even gentler illu­mination to achieve a similar level of image sensitivity at the same volumetric frame rate. Hence, it further reduces the photobleaching rate and thus the risk of photo­damage. This is a critical attribute for preserving the biological specimen viability in long term monitoring studies. The heart of CLAM is the concept of infinity mirror (i.e., a pair of parallel mirrors), which is common in visual art and decoration, and has previously been adopted by the same team for enabling ultrafast opto­fluidic single-cell imaging. Here the team employed the infinity mirror together with simple beam shaping to transform a single laser beam into a high-density array of few tens of light-sheets for 3D paralle­lized fluores­cence excitation.

“One distinct feature of CLAM is its ability to flexibly reconfigure the spatial density and temporal coherence of the light sheet array, simply by tuning the mirror geometry, such as mirror separation and tilt angle,” explained Yuxuan Ren, a post­doctoral researcher. “This capa­bility has been challenging in the existing coherent wavefront shaping methods, yet could allow efficient parallelized 3D LSFM in scattered tissue imaging with minimal speckle artifact,” Ren added.

CLAM also adopts code division multi­plexing (CDM) (e.g., orthogonal frequency division multi­plexing demonstrated in this work), a technique widely used in telecommunication, to imprint the fluorescence signal from each image plane with a unique code. As a result, it allows paral­lelized 3D image capture with optical sectioning by using a 2D image sensor. “CLAM has no funda­mental limitation in scaling to higher volume rate as camera techno­logy conti­nually advances,” Kevin Tsia, Associate Professor in Department of Electrical and Electronic Engi­neering at HKU and the leading researcher of the team pointed out.

“Also, CLAM can be adapted to any existing LSFM systems with minimal hardware or software modification. Therefore, it is readily available for dissemination to the wider community of LSFM and related 3D imaging techniques,” added Tsia. (Source: CAS)

Reference: Y.-X. Ren et al.: Parallelized volumetric fluorescence microscopy with a reconfigurable coded incoherent light-sheet array, Light Sci. Appl. 9, 8 (2020); DOI: 10.1038/s41377-020-0245-8

Link: Dept. of Electrical and Electronic Engineering, University of Hong Kong, Hong Kong, China

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