1. Nonlinear Advanced Optical Microscopy (先進非線性光學顯微術)
。Fast optical sectioning images for real-time applications(快速光學切片影像技術)
Temporal focusing-based multiphoton excitation microscopy (TFMPEM) is a system composed of these key elements, a diffractive grating and a set of 4f system of a collimating lens and high numerical aperture objective lens. It makes all the frequency components of pulse laser overlap and in-phase to each other to achieve the narrowest pulse width only on the focal plane of the objective lens. It not only can provide axial resolved ability but a large area excitation without mechanical scanning, which will limit the temporal resolution of the image. Therefore, TFMPEM is suitable for realizing the high frame rate applications.
時域聚焦之多光子激發顯微術(temporal focusing-based multiphoton excitation microscopy,TFMPEM)藉由繞射光柵及一組由準直透鏡和高數值孔徑的物鏡所組成的4f架構,使脈衝雷射的所有頻率僅在物鏡的焦平面上重和並彼此同相位而形成最窄的脈衝,不僅擁有縱向解析照明能力同時也可大面積激發無須掃描,不受限機械的移動速度並可實現超高幀速度的應用。
In order to expand the application of temporal focusing-based multiphoton excitation microscopy, a digital micromirror device (DMD) is used to substitute the blazed grating as a diffraction component. By controlling the micromirror on DMD, the arbitrary excitation pattern can be produced, which also acts as an optical mask yet preventing the tilt aberration.
為了在時域聚焦之多光子激發顯微系統上產生任意的圖形照射以拓展更多應用,利用一組數位微型反射鏡元件(digital micromirror device,DMD)取代繞射光柵做為分光元件,並透過控制元件上各個像素的工作狀態,可同時產生任意圖形進而達到光罩的作用。
。Thick tissues imaging with adaptive optics (藉由適應性光學於厚組織影像)
Temporal profile distortions reduce excitation efficiency and degrade image quality in temporal focusing-based microscopy. In order to compensate for the distortions, a wavefront sensorless adaptive optics system (AOS) was integrated. The feedback control signal of the AOS was acquired from local image intensity maximization via a hill-climbing algorithm. The control signal was then utilized to drive a deformable mirror in such a way as to eliminate the distortions.
在時域聚焦之多光子激發顯微術中,系統的時間域干擾會降低其激發效率並且降低光學影像品質,為了補償此干擾,我們將系統整合無波前感測式的適應性光學系統,經由爬山演算法得到局部的光學影像最強化的回饋訊號,控制可調變聚焦鏡的以降低其時間域干擾。
For real-time adaptive optics applications, an easily implementable AOS based on a real-time FPGA platform with state-space multichannel control has been developed, and also integrated into a laser focusing system successfully. The overall system with a 32-channel driving signal for a deformable mirror as input and a Zernike polynomial via SHWS as output is optimally identified to construct a multichannel state-space model off-line. Then, a state-space multichannel controller designed by the identified model is implemented in the FPGA to drive the DM for dynamic phase distortion compensation.
在即時的適應性光學應用,我們進一步發展一可輕易整合式的適應性光學系統,在基於FPGA平台下發展即時狀態空間多通道控制器,並且成功整合至一雷射聚焦系統。此系統設定可調變聚焦鏡的32通道當作輸入,Shack-Hartmann波前感測器量得的Zernike多項式當作輸出來離線建立一個多通道狀態空間的系統模型。針對此系統模型做最佳化設計的狀態空間控制器將會驅動可調變聚焦鏡進行波前干擾的補償。
。Breaking diffraction limit for super-resolution imaging(超解析度影像技術)
By using the 2nd-order nonlinear structured-illumination microscopy to illuminate a periodically structured pattern on the sample, the spatial frequency of the sample higher than the system collection cutoff frequency will be modulated into a lower frequency region, where the spatial frequency can be collected by the system. Extract these higher frequency components by different pattern phase shifts and remapping them to their real position. Therefore, we can get a super-resolution image.
利用二階非線性結構性照射顯微術(nonlinear structured-illumination microscopy)將周期性的結構照射在樣品上,將原本無法被顯微系統接收到的高頻資訊透過週期性結構的調變至系統可接收的區間內,再透過不同結構之相位將調變的高頻資訊取出並重新組合,進而得到一超高空間解析度的影像。
We presented a method to overcome the limitation of the time resolution of the multiphoton imaging using fast frame rate of the temporal focusing multiphoton excitation microscopy (TFMPEM) and using an astigmatism method to provide 3D tracking ability. The sectioning zoom-in images show the pattern changes from a vertical ellipse to a horizontal ellipse by shifting 600 nm of z step between each other at the top of the figure. Based on the recorded images at each 20 nm location, the following left figure shows that the response of the 4 μm depth was reconstructed by fitting the equation. We demonstrated the dynamical 3D tracking measurement performance for the freely fluorescent beads as the bio-fluorescent material by this method. By 10 ms temporal resolution, the 3D trajectories of the 200nm spherical particle with diffusing in the DI water were recorded as shown in the following right figure.
利用廣視域搭配像散法(astigmatism)之多光子激發螢光顯微鏡,將原本的不完美影像中像散效應所產生的橢圓之長短比值進行分析處理,獲得螢光分子團在相對應縱軸上更精細的位置。進而提高次微米級單一特定螢光分子團的三維空間解析度,並搭配TFMPEM具有高速與大視野的優點,因而提供即時高速進行觀測螢光分子團在三維空間中位移。下左圖為放大光切片影像,其拍攝已固定螢光球於每600nm位移(Z軸位置)所得到之像散效應影像;每20nm的Z軸位置上記錄其影像分析可得4µm區間內X方向(紅色)與Y方向(藍色)的像散反應曲線。我們使用自由熒光珠作為該方法的生物熒光材料進行證明三維動態追踪測量性能。在10 ms的時間分辨率下,在200 nm的球形顆粒與擴散在DI水中的三維軌跡可被記錄,如下右圖所示。
。Multiphoton-induced laser ablation (多光子誘發雷射蝕除)
Plasma-induced ablation is beneficial for the removal of biotissue without photothermal damage. In order to induce ablation, the photon energy must be greater than the chemical bonding energy to directly break the chemical bonds of machined samples, thereby generating plasma. Herein, we adopt the temporal focusing technique featuring high-throughput multiphoton excitation to achieve rapid disruption of biotissues. Experimental results of machining chicken tendon show that large-area ablation machining without dramatic photothermal damage in non-machining regions can be efficiently achieved with a machining speed of approximately 1.6 × 106 μm3/s.
光蝕除是一種雷射加工現象,是利用高功率強度雷射直接破壞掉分子之間的鍵結,當光子能量大於分子間之鍵結能時,經過線性單光子或非線性多光子吸收,分子會躍升到激發態並且分解噴射出材料表面,而造成蝕除的現象。此種加工方式非常精確,並且在加工處不會出現熱效應的影響。研究使用了時間聚焦的高通量(high-throughput)之多光子激發技術來完成生物組織加工,目前初步完成了任意二維圖形之微加工而加工時間只僅僅需要幾秒鐘,與傳統多光子單點掃描加工方式相互比較,其加工速度提升了百倍以上。
2. Three-dimensional Photolithography (三維光微影製程技術)
。Specific 3D microstructures (特殊的三維微結構製作)
Three-dimensional (3D) multilayer protein microstructures have been precisely fabricated via multiphoton excited photochemistry by using a developed femtosecond laser direct writing system with repetition positioning and vector scanning techniques. Extracellular matrix (ECM) proteins such as fibronectin (FN) are difficult to stack 3D structure due to their flat shape. Herein, to fabricate complex 3D microstructure with FN, bovine serum albumin (BSA) is first formed to a designed complex 3D scaffold, and then FN is fabricated at specific locations on the BSA scaffold; hence, the fabricated ECM microstructure can be utilized to help and guide cells in 3D environment. This 3D multilayer protein fabrication technique holds the potential for cell studies in designed complex 3D ECM scaffolds.
結合多光子激發影像定位以及雷射直寫向量掃描技術的系統開發,可應用在任意形狀之三維生物支架的高精細度結構製造,且優化製造所時間以及提升結構之完整度,藉由聲光調變器來控制脈衝數,可以脈衝寬度調變的方式來控制製作時雷射能量的參雜度來控制蛋白質在結構中鍵結的濃度,以達到提升其實用性與結構複雜度,並建構出人造之三維利基環境給細胞生長與貼附。
To develop the multiphoton fabrication capability, including 3D polymerized polyacrylamide or crosslinked bovine serum albumin (BSA) microstructures containing gold nanorods (AuNRs). The following figure shows an AuNRs-doped 3D woodpile BSA microstructure imaged with (a) 3D TPL, (b) 2D bright field, and (c) SEM.
發展多光子激發微製作技術應用於具有金奈米柱 (gold nanorods,AuNRs) 之3D微米polyacrylamide和牛血清蛋白 (bovine serum albumin,BSA) 結構,也證實其具有表面電漿子 (surface plasmons) 之特性。下圖中展示摻雜金奈米柱 (AuNRs) 於3D木堆之牛血清蛋白(BSA)的微觀結構影像(a)三維雙光子致光;(b)明視野;以及(c)掃描式電子顯微鏡(SEM)。
Graphene-based materials have been interesting for its unique properties. In our lab, we used multiphoton lithography to develop 3D graphene oxide freeform microstructures, and then apply them in 3D microelectronic device, photonics crystal, and tissue scaffold.
石墨烯因突破性的特殊性質使其在近幾年快速發展。本實驗室藉多光子技術開發出三维氧化石墨烯微觀結構,並將應用它們在三維微電子器件、光子晶體和組織支架等。
。High-throughput microfabrication (高通量的微製作)
One of the limits of conventional scanning multiphoton excitation (MPE) microfabrication is its low throughput due to point-by-point processing. To surpass this limit, a MPE microfabrication system based on temporal focusing and patterned excitation has been developed to quickly provide 3D freeform microstructures. Compared to conventional scanning MPE, this approach offers freeform microstructures and a greater than three-order increase in fabrication speed. Furthermore, the system integrated with a digital micromirror device for locally controlling the laser pulse numbers which can provide high-throughput fabrication of 3D gray-level bio-microstructures.
傳統的多光子激發(MPE)微製作技術是採用點掃描的方式來進行,因此、製作速度慢所造成的低產量是其最大缺點。為了改善這個限制,我們發展了一套具時域聚焦與圖形化照射的MPE微製作系統。與傳統的掃描式MPE相比,此方法不僅可製作出任意形狀的微結構,在製作的速度上更可提升約三個數量級。此外,這套系統整合了一個數位微面鏡裝置可用來針對特定的區域,局部地控制雷射脈衝的數量而同時製作出多個不同灰階的三維蛋白質結構。
High-throughput multiphoton-induced reduction and ablation of graphene oxide (GO) films have also been studied using this system. The degree of reduction and ablation can be precisely implemented via controlling the laser wavelength, power, and pulse number. Compared to point-by-point scanning laser direct writing, this method offers a high-throughput and multiple-function approach to accomplish large-area and micro-scale patterns on GO films, which matches the requirement of mass production for GO-based applications in microelectronic devices.
我們利用此系統對氧化石墨烯(GO)薄膜進行高通量多光子誘發還原與剝離的研究。其還原的程度以及剝離的過程可以很精準地被操控藉由調控雷射的波長、功率以及脈衝數量。相對於逐點雷射掃描的方式,此方法具備了高產能與多功能的優點,進而可以在GO的薄膜上製作出具有微米特徵尺度的大面積圖案,將有助於GO相關材料在微電子裝置上的應用。