單一近紅外光激發波長達成即時多色多光子成像

Abstract

人類和其他靈長類一樣，是仰賴視覺的生物。我們藉由雙眼來理解這個世界。 因此，生命科學研究的一大核心在於發展組織成像的技術和工具。自從雷文霍克在西元1673年第一次由顯微鏡觀察到微生物後, 光學顯微鏡就一直是生物組織成像上的重要工具。尤其是在神經科學領域，神經的成像幫助我們了解大腦如何運作。 光學顯微鏡發展的一大突破是多光子顯微鏡的發明。多光子激發所具備的光學切片能力，讓我們能夠在不切片組織的條件下，觀察到大腦表層以下幾百甚至幾千個微米深的影像，而且影像不會因為大腦組織的散射特性而變得過於模糊，依然能夠維持亞細胞等級的分辨率。與此同時，螢光蛋白的開發，讓組織能夠在活體並且完整的情況下，藉由基因工程將標的細胞染上我們所想要的顏色。多光子顯微鏡和螢光蛋白科技的結合是當前最主流的生物組織成像工具，應用範圍包含活體和體外的功能性和結構性影像， 目前的螢光顯微鏡技術能夠清楚的追蹤單一標的細胞的型態和行為，但是大腦功能的運作通常是由好幾種不同細胞或分子所交互作用而成。即便最先進的螢光標記技術有能力將組織內各個標的物染上不同的顏色，因為各個顏色的螢光蛋白所需要的激發波長都不相同，為了達成即時的多色影像，目前的多光子顯微鏡主要仰賴用光參數震盪器來延展飛秒雷射的波長。這種雷射系統的價格非常昂貴，導致它很難普及於各個實驗室。我們希望能夠找到一個相對較為低價的解決辦法。 我們從文獻中發現許多螢光蛋白在紫外光的波長內都有一個共同的吸收高峰。這個吸收高峰已經被證明是來自於色胺酸，而這種胺基酸存在於任何一種螢光蛋白。因此，藉由激發色胺酸，我們期望能夠激發所有螢光蛋白。由於考量到紫外光對生物組織的光毒性質，我們巧妙地藉由一道740 nm的雷射，來三光子激發螢光蛋白內的色胺酸。 在這篇論文裡，我們成功的用這道雷射，在合理的雷射功率內，激發了六種不同顏色的螢光蛋白。並且在標有兩種顏色 －青色和黃色螢光蛋白－的老鼠腦片內，完成即時的多色多光子成像。這兩種顏色在過去從來沒有被單一一道740 nm的近紅外光同時激發且取像過。 因為740 nm 這個雷射波長可以由摻鈦藍寶石雷射所產生，而摻鈦藍寶石雷射又剛好是多光子顯微鏡中最常見的雷射源。因此，我們的發現為眾多使用多光子顯微鏡作為成像工具的實驗室，提供一個便宜而且方便的即時多色成像方法。

Humans, like all other primates, are highly visual creatures. We comprehend the world mainly through our eyes. Therefore, the essence of life science relies heavily on the construction of visualization techniques and apparatus. Not surprisingly, since van Leeuwenhoek’s observation of living animalcules in 1673, optical microscopy has played a crucial role in visualization in the field of bioimaging, especially neuroscience where imaging neuronal structure is the core of understanding how the brain operates. One of the major breakthroughs in optical imaging is multiphoton microscopy. The intrinsic optical sectioning ability of multiphoton excitation provides neuroscientists to see through the scattering brain tissue for hundred to thousands of micrometers deep with subcellular resolution. Furthermore, the introduction of fluorescent protein, a contrast agent that enables tissue labeling while keeping it intact via genetic fusion, greatly enhanced signal specificity amid non-fluorescing background. The incorporation of multiphoton microscopy and fluorescent protein technology is the mainstream for structural and functional imaging of brain tissues, fixed or alive. Although fluorescence imaging systems nowadays succeed in tracking the morphology and movement of individual neuron cells, the mysterious brain function often involves interactions between several components. State-of-the-art multicolor labeling strategies successfully labeled multiple targets with a large collection of fluorescent protein spectral variants. However, the discrete absorption peaks of these spectral variants pose a challenge to multiphoton microscopy. Current multiphoton microscopy usually depends on an excitation source that assembles a femtosecond second laser and an optical parametric oscillator for simultaneous multicolor imaging. These kinds of systems are expensive and prevent the widespread of multicolor multiphoton imaging. Hence, we aim to find a different strategy to reduce laser system complexity and expense. From various reports on fluorescent protein absorption spectrum measurement, an absorption peak in the deep ultraviolet region was found universal among spectral variants. This peak has been proven to originate from the absorption of Tryptophan, an autofluorescence amino acid that exists in every fluorescent protein. Therefore, we propose the possibility to excite all of the fluorescent protein spectral variants by a single wavelength. Since Tryptophan absorbs ultraviolet light, to avoid photo-toxicity, a 740 nm excitation wavelength is used for three-photon excitation. Based on our theory, in this study, we demonstrated simultaneous multicolor multiphoton imaging by a single 740 nm laser. We efficiently excite six different colors of fluorescent proteins separately within reasonable pulse energy and achieve simultaneous multicolor imaging of a cyan and yellow-colored mouse brain slice that was previously inaccessible with a 740 nm laser. Since the Ti:Sapphire laser is the most common laser source for two-photon microscopy, our technique provides a convenient way to accomplish simultaneous multicolor imaging without an additional wavelength conversion apparatus.