三光子高速旋轉盤顯微術應用於腦成像

Abstract

在神經科學的研究中，徹底地了解大腦結構性與功能性的迴路是至關重要的目標。而高速深組織的活體腦成像是達到此目標的必要條件之一。近年來，隨著雷射技術的發展，結合高速成像技術 (如光片、旋轉盤或多焦點陣列掃描顯微術) 的雙光子激發螢光顯微鏡被研發出來，並應用在生物樣本上。這些技術使得對於小型模式動物的活體高速深組織光學腦成像變得逐漸可行。然而，儘管相比於雙光子激發，三光子激發在深組織有其優勢，但在目前的文獻中卻很少有高速三光子的系統被成功開發。

在本研究中，我們利用客製化的旋轉盤結合可調波長的高功率飛秒雷射系統，展示了首個三光子旋轉盤顯微鏡，我們達到了接近300 Hz的幀率進行三光子成像，為首個超過100 Hz 幀率的三光子顯微鏡。我們對不同的樣本進行成像，包含綠色螢光球、固定的小鼠腦片與活體的果蠅進行腦成像，以展示其能力。在實驗中我們實現了以3毫秒的曝光時間對微流道晶片中的流動螢光球進行高速成像。而在對老鼠與果蠅的腦成像實驗則顯示出，相比於雙光子旋轉盤成像，三光子旋轉盤成像具有更低的訊號衰減率。我們的研究實現了目前最高速的三光子成像，提供了一種可行的三光子高速成像系統。

Achieving a comprehensive understanding of the brain's structural and functional circuits is a crucial goal in neuroscience. To address this need, high-speed volumetric imaging of emergent properties within the connectomes of living animals is essential. Advances in laser techniques have enabled deep tissue imaging of the brain at high speed by combining two-photon excitation with rapid microscopy methods such as light-sheet, spinning disk, or multifoci array scanning microscopy. Despite the potential for improved imaging depth compared to two-photon excitation, high-speed three-photon imaging remains relatively scarce in the literature.

In this study, we utilize a customized spinning disk unit combined with a wavelength-tunable high-power femtosecond laser to showcase the three-photon microscopy at a frame rate of ~300 Hz, marking a significant milestone in achieving three-photon microscopy above the 100 Hz regime. We demonstrate its versatility by imaging various samples, including green fluorescent beads, in vivo Drosophila brains, and fixed mouse brains. Our experimental setup achieves three-photon high-speed imaging with an exposure time of only 3 ms for floating fluorescent beads inside a microfluidic chip. Furthermore, our brain imaging findings reveal that three-photon spinning disk imaging exhibits a lower attenuation rate compared to its two-photon spinning disk counterpart. Our work paves the way for utilizing three-photon excitation in high-speed deep tissue imaging applications.