香港大學生物醫學工程研發
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過去數十年，科學家利用螢光顯微鏡，研究生物和生物細胞的内部運作。現有的顯微鏡技術存在不少局限 - 成像速度不夠快，未能有效地捕捉樣本在三維空間的生物動態；而且成像過程需要利用強光不斷照射生物活體，對樣本可能造成不可挽回的損害。 香港大學電機電子工程系副教授及生物醫學工程課程總監謝堅文博士領導的研究團隊，研發一種嶄新的三維螢光成像技術 - 編碼光片陣列顯微鏡（Coded Light-sheet Array Microscopy CLAM)，掃描速度媲美目前最前沿的三維掃描成像技術（每秒擷取超過10張立體影像的速率）。更重要的是，其對生物細胞所造成的光損害，比現時實驗室廣泛採用的標準三維技術，減低最少1,000倍，目前沒有任何技術能及。 此項嶄新技術已於期刊《光：科學與應用》上發表。發明正在申請一項美國應用專利。 CLAM的核心技術，是利用一組平行鏡內多重反射的原理，將掃描的單一激光光束，散換成高密度的薄片狀陣列（high density array of "light-sheets"），大範圍散落在成像的細胞樣本上，用作螢光激活。這不但能大幅改善目前生物應用的三維螢光成像顯微鏡的效率，更有助開拓前沿的科學研究領域，應用在人體解剖科學、發育生物學和神經科學等涉及精準細胞活動分析和追蹤等研究，例如追蹤動物胚胎如何演化到成年個體、實時監測細胞或器官如何受細菌或者病毒感染，或藥物如何破壞癌細胞等。 目前的三維生物顯微鏡的成像技術，是利用強光，逐點、逐行或逐個平面一步步地掃描整個樣本，成像速度很慢，強光也會對生物樣本造成破壞，並不適合應用作長時間的生物成像分析。掃描過程中樣本發出的螢光訊號很快被消耗盡，因爲當樣本被強光照射一定時間後，會出現光漂白現象，即當中用作顯影的螢光分子會永久失活不能再發光，樣本在顯微鏡下於是變得暗淡，無法繼續被偵測，這也是現有技術一個根本的局限。 謝堅文博士解釋說：「用現時的方法拍攝一張三維影像，需要不斷重複照射樣本，當中產生不少多餘而最終不會成爲影像一部分的螢光訊號，這不單無謂地浪費了本身已經非常有限的螢光訊號資源，加快光漂白現象，更重要的是，這些樣本經比太陽光強勁數千甚至數百萬倍的光源照射後，樣本本身也可能受到破壞。｣ ｢如何能在三維成像顯微鏡下，長時間拍攝一些活組織（不論是細胞或微生物），而要這些組織存活不死，一直都是生物醫學上的一大難題。實際上，要成功拍攝幾分鐘也非常艱難。｣謝博士說。 團隊成員博士後研究員任煜軒博士指出：「CLAM的3D平行化照射技術，在不影響成像的敏感度和速度的情況下，能達至非常柔和且高效的3D螢光成像，在減低光漂白的影響上比一般三維螢光成像方法優勝。」 為保持影像的解析度和質素，研究團隊採用了普遍用於電子通訊中同步傳播多頻道信號的「編碼劃分多工（Code Division Multiplexing CDM）」技術對影像編碼。 「我們利用二維圖像感測器來採集經過CDM技術編碼的螢光數據，透過電腦演算，把同一時間採集得的所有影像數據重組成3D影像。今次是首次把CDM編碼技術應用於三維掃描成像，並且取得成功，我感到非常鼓舞。」團隊成員黎芷君博士說。 研究團隊已成功利用CLAM以極快速每秒超過10張立體影像的速率，捕捉在微流體晶片中快速流動的微粒子的動態，成像速度與目前最前沿的三維掃描成像技術相約。 「CLAM的成像技術，速度上基本上並沒有上限，目前的主要限制在偵測儀器 -相機拍照的速度，是否夠快配合成像，而由於高速相機的技術日新月異，CLAM的成像速度基本上可不斷加快。」團隊另一位成員博士後研究員吳江來博士補充說。 在前沿研究領域上，研究團隊把CLAM與港大李嘉誠醫學院早前研發的「組織透明技術」結合，對老鼠腎小球及腸道血管進行高速3D結構成像。研究團隊期望這項技術可以延伸到各種對生物樣本的大範圍三維組織病理研究中，如神經科學研究腦組織中的細胞分佈架構。 ｢相比任何一個解決方案，CLAM獨特之處，是它產生的柔和光束，尤其適合一些持續長時間的生物監測研究，例如追蹤胚胎的成長，數天甚至數星期；監察細胞對一些治療藥物例如癌細胞對標靶藥的反應；以及細胞和器官受病毒或細菌感染的過程等。｣謝博士說。 CLAM的另一個優點是，它能夠結合多種現有的顯微鏡系統，不需要改動太多硬件或者 軟件，便可以轉化應用。團隊目前正計劃升級現有的CLAM系統，首先應用在細胞生物學、動物和植物生長生物學中的動態過程研究。 這項研究是香港大學工程學院和李嘉誠醫學院的跨學科合作，並得到研究資助局、創新科技支持專案、香港大學研發基金以及國家自然科學基金等資助。 請按此下載圖片及影片。
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HKU Biomedical Engineering develops novel 3D imaging technology to
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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 Dr Kevin Tsia, Associate Professor of the Department of Electrical and Electronic Engineering and Programme Director of Bachelor of Engineering in Biomedical 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. This advanced imaging technology was recently published in Light: Science & Applications. An US patent application has been filed for the innovation. "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 Dr 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 process called "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," Dr 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 (i.e. parallelized), 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 Dr 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 Dr 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 Dr Jianglai Wu, the postdoctoral research 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." Dr 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," Dr 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. This project is an interdisciplinary collaboration between HKU Faculty of Engineering and LKS Faculty of Medicine. It was funded by HKSAR Research Grants Council, Innovation and Technology Support Program, the University Development Funds of the University of Hong Kong and the Natural Science Foundation of China. For images and videos download please click here.
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