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.



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.



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.



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.




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.



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.




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.