以高效率的光參數放大器為光源使用於多光子顯微術提供高穿透深度影像

Abstract

西元1665年虎克利用光學顯微鏡觀察植物細胞，之後光學顯微技術被廣泛的使用於生醫影像中。傳統的光學顯微鏡並沒有光學切片能力，無法判定信號來自於哪個深度。Marvin Lee Minsky 在1957年提出了具有光學切片能力的共軛焦螢光顯微鏡。但根據散射理論，可知波長越短散射越強，光的穿透深度受光波長影響極大，共軛焦螢光顯微鏡使用可見光波段，穿透深度受到極大的限制，難以超過100微米。而多光子顯微術通常以紅外光為激發光，波長長散射弱，能有較好的穿透深度。另外由於為非線性效應，信號強度與入射光強度為非線性成長，只有聚焦點才有足夠的信號被偵測到，進而擁有與共軛焦顯微鏡相似的光學切片能力。那可否透過無限的拉長激發光波長，達到更深度的穿透深度呢？若拿生物樣本考慮，樣本內部以水含量最多，而水在對波長1.4 μm以上的光吸收變強，不適用於生醫影像。考慮波長盡量長且須避開水的吸收，1.3~1.4 μm的光將非常適用於高穿透深度生醫影像。 市面上常見的可產生1.3~1.4 μm的雷射光源有鈦：藍寶石雷射搭配光參數共振器(Ti: sapphire laser + optical parametric oscillator，Ti:sa + OPO)、超連續光譜雷射(Supercontinuum generation laser，SC)、鉻：鎂橄欖石雷射(Cr: forsterite)。鈦：藍寶石雷射搭配光參數共振器好處是脈衝非常短，可小於100 fs，調制波長快速，但輸出功率有限且光參數共振器架設較為複雜；超連續光譜一般常用光晶體光纖(Photonic crystal fiber，PCF)來產生，好處在於頻寬極寬，可以同時產生從紫外到紅外光的頻段，然而頻寬極寬也造成單位波長能量低，且光纖的色散會造成脈衝寬度過寬，不適合使用於多光子顯微術；鉻：鎂橄欖石雷射能達到高輸出功率，但在架設上也需要共振器。如何以簡單地單次通過(single pass)方式產生高功率的1.3~1.4 μm短脈衝光呢？上述的雷射技術都有部分缺失，無法達到我們設定的需求。 在實驗中成功締造光參數產生/放大(Optical parametric generation/amplification，OPG/OPA)的紀錄：在高重覆率條件下的輸出功率超過 1 W、量子轉換效率(輸入的光子數/信號光子數)達60%。且波段為1.3~1.4 μm、脈衝寬度約1 ps的短脈衝雷射光。另外，我們也以產生的1.36 μm波長的光作為多光子顯微術的激發光，並以第一型膠原蛋白作為生物樣本取得三度空間的二倍頻影像，並與1 μm激發光的影像比較，可明顯的看出1.36 μm能取得更深的影像，藉此證實我們所產生的光源非常適合使用在高穿透深度的多光子顯微術。

Since Hooke first used optical microscope to observe the plate cells in 1665, optical microscope has been widely applied to biomedical image. Convention optical microscope doesn’t provide optical sectioning which makes it fail to recognize signals from the depth of signals. Later in 1957, Marvin Lee Minsky displayed the confocal fluorescence microscope which has optical section. However, as we know from the theory of scattering, the shorter wavelength is, the stronger scattering will be, the penetration depth is highly related to wavelength. Generally, visible light is the source of confocal fluorescence microscopy, which restricts its penetration depth 100 μm. On the contrary, multiphoton microscopy usually uses infrared light as its source and thus has higher penetration depth because it has longer wavelength and lower scattering. Besides, the whole process is nonlinear, which means the signal power increases nonlinearly by source intensity. Only signal from focus spot is enough for detecting. Therefore, multiphoton microscopy also has optical section. From previous perspective, my core question is that whether we can get unlimited high penetration depth by lengthening the wavelength. Take biological sample into consideration, which mainly contains water. Water strongly absorb EM-wave of wavelength near 1.4 μm, which is unsuitable for biomedical imaging. In order to lengthen the wavelength as much as possible and avoid the absorption of water, light between 1.3~1.4 μm would be very suitable for deep-tissue imaging. People often use Ti: sapphire laser with optical parametric oscillator (Ti:sa + OPO), Supercontinuum generation laser (SC) and Cr: forsterite to generate 1.3~1.4 μm laser source. Ti:sa + OPO has ultrashort pulse which is often shorter than 100 fs and its peak wavelength can be fast tuned. But its output power is limited and oscillator is complex setup. SC usually uses photonic crystal fiber to generate. Its advantage is that spectrum is very broad, which is from ultraviolet to infrared. However, because it is broadband laser, its spectral power is low, usually ___. And the dispersion of fiber causes long pulse, which is not suitable for multiphoton microscopy. Cr:forsterite can achieve high output power, but its oscillator makes setup complex and less robust. How can we achieve easily single pass and high output power laser source with 1.3~1.4 μm wavelength? The aforementioned lasers all have some defects, which don’t fit our requirement. Our experiment succeeds in creating optical parametric generation/amplification record. We generate the pulse laser, whose output power is over 1 W, quantum efficiency (the number of the output photons/the number of the input photons) achieves 60% at high repetition rate, wavelength 1.3~1.4 μm and pulsewidth 1 ps. Besides, using 1.36 μm as source, which we generate, of multiphoton microscopy is success getting the second harmonic generation bulk image of the type 1 collagen. To compare the image with 1 μm as source obviously, 1.36 μm as source can get deeper image. It proved 1.36 μm is more suitable for deep-tissue multiphoton microscopy.