New Kind of Volumetric Fluorescence Microscopy

Yuxuan Ren, Queenie Lai and Kevin Tsia (from left) developed a new optical imaging technology to make 3D fluorescence microscopy more efficient and less damaging. (Source: HKU)

Scientists have been using fluores­cence micro­scopy to study the inner workings of biological cells and organisms for decades. However, many of these platforms are often too slow to follow the bio­logical action in 3D; and too damaging to the living biological specimens with strong light illu­mination. To address these challenges, a research team led by Kevin Tsia, Associate Professor of the Department of Electrical and Electronic Engi­neering of the University of Hong Kong (HKU), developed a new optical imaging tech­nology – Coded Light-sheet Array Micro­scopy (CLAM) – which can perform 3D imaging at high speed, and is power efficient and gentle to preserve the living specimens during scanning at a level that is not achieved by existing tech­nologies.

“CLAM allows 3D fluores­cence imaging at high frame rate comparable to state-of-the-art tech­nology (~10’s volumes per second). More importantly, it is much more power efficient, being over 1,000 times gentler than the standard 3D micro­scopes widely used in scientific labora­tories, which greatly reduces the damage done to living specimens during scanning,” explained Tsia. Existing 3D bio­logical microscopy platforms are slow because the entire volume of the specimen has to be sequentially scanned and imaged point-by-point, line-by-line or plane-by-plane. In these platforms, a single 3D snapshot requires repeated illu­mination on the specimen. The specimens are often illu­minated for thousands to million times more intense than the sunlight. It is likely to damage the specimen itself, thus is not favorable for long-term bio­logical imaging for diverse applications like ana­tomical science, develop­mental biology and neuro­science.

Moreover, these platforms often quickly exhaust the limited fluores­cence budget – a fundamental constraint that fluorescent light can only be generated upon illumination for a limited period before it permanently fades out in a photo-bleaching, which sets a limit to how many image acqui­sitions can be performed on a sample. “Repeated illu­mination on the specimen not only accelerates photo-bleaching, but also generates excessive fluores­cence light that does not eventually form the final image. Hence, the fluores­cence budget is largely wasted in these imaging platforms,” Tsia added.

The heart of CLAM is trans­forming a single laser beam into a high-density array of light-sheets with the use of a pair of parallel mirrors, to spread over a large area of the specimen as fluorescence excitation. “The image within the entire 3D volume is captured simul­taneously, without the need to scan the specimen point-by-point or line-by-line or plane-by-plane as required by other techniques. Such 3D paralleli­zation in CLAM leads to a very gentle and effi­cient 3D fluorescence imaging without sacrificing sensi­tivity and speed,” as pointed out by Yuxuan Ren, a postdoctoral researcher of the work. CLAM also out­performs the common 3D fluorescence imaging methods in reducing the effect of photo-bleaching.

To preserve the image reso­lution and quality in CLAM, the team turned to Code Division Multiplexing (CDM), an image encoding technique which is widely used in telecommunication for sending multiple signals simul­taneously. “This encoding technique allows us to use a 2D image sensor to capture and digitally reconstruct all image stacks in 3D simul­taneously. CDM has never been used in 3D imaging before. We adopted the tech­nology, which became a success,” explained by Queenie Lai, another postdoctoral researcher who developed the system.

As a proof-of-concept demons­tration, the team applied CLAM to capture 3D videos of fast microparticle flow in a microfluidic chip at a volume rate of over 10 volumes per second comparable to state-of-the-art technology. “CLAM has no funda­mental limitation in imaging speed. The only constraint is from the speed of the detector employed in the system, i.e. the camera for taking snapshots. As high-speed camera tech­nology continually advances, CLAM can always challenge its limit to attain an even higher speed in scanning,” highlighted by Jianglai Wu, the post­doctoral researcher who initiated the work.

The team has taken a step further to combine CLAM with HKU LKS Faculty of Medicine’s newly developed tissue clearing tech­nology to perform 3D visuali­zation of mouse glomeruli and intestine blood vasculature in high frame-rate. “We anti­cipate that this combined technique can be extended to large-scale 3D histo­pathological investigation of archival biological samples, like mapping the cellular organi­zation in brain for neuro­science research.” Tsia said.

“Since CLAM imaging is signi­ficantly gentler than all other methods, it uniquely favours long term and continuous ‘surveil­lance’ of biological specimen in their living form. This could poten­tially impact our funda­mental under­standing in many aspects of cell biology, e.g. to conti­nuously track how an animal embryo develops into its adult form; to monitor in real-time how the cells/organisms get infected by bacteria or viruses; to see how the cancer cells are killed by drugs, and other challenging tasks unachievable by existing tech­nologies today,” Tsia added. CLAM can be adapted to many current micro­scope systems with minimal hardware or software modi­fication. Taking advantage of this, the team is planning to further upgrade the current CLAM system for research in cell biology, animal and plant develop­mental biology. (Source: HKU)

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: Applied Life Photonics, Dept. of Electrical and Electronic Engineering, University of Hong Kong, Hong Kong

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