| 摘要: | 多光子顯微術由於激發過程是非線性，所以擁有良好的縱向及橫向解析度，且激發光波長較單光子顯微術長，因此樣本穿透深度也較深，其中倍頻訊號(如二倍頻及三倍頻)的激發過程中並無實際能階躍遷，滿足動量守恆，因此較不易產生光漂白及破壞樣本。
不同於螢光訊號，因為倍頻訊號屬於無實際能階躍遷，無法如螢光訊號般進行強度的調制，所以不適用螢光的超解析顯微術。1994年，S. W. Hell提出了基態耗損顯微術(Ground state depletion, GSD)這項技術，藉由降低基態電子數目，影響樣本的吸收，進而調制螢光的訊號強度。本論文利用倍頻訊號的一項特性：若樣本存在多光子吸收能階，將會增強倍頻訊號的強度，因此嘗試利用GSD來降低樣本的吸收，使倍頻的增益減弱，來達到調制倍頻訊號。
由於倍頻的激發需要極高的光強度，本論文將以二維點掃描顯微系統為架構來取得三倍頻影像，並導入耗損光(depletion light)，使之與激發光重合產生激態耗損，因三倍頻會滿足光子動量的方向，將以順向方式架設收光系統，使用濾波片將激發光及耗損光濾除，再使用光電倍增管接收三倍頻訊號。本研究以血紅素、黑色素為樣本進行基態耗損的測試，系統以1064 nm的飛秒雷射作為激發光，根據樣本的吸收光譜，以綠光連續波(Continuous wave, CW)雷射作為耗損光。為能進一步產生具有週期結構的耗損光，耗損光經過空間光調制器(Spatial Light Modulator, SLM)產生繞射，使兩道+1與1的繞射光在樣本的焦平面干涉，產生餘弦波的結構照明，如此將能以不同的耗損光強度產生週期性的三倍頻強度調制。;Due to nonlinear excitation, multi-photon microscopy could provide well performance at axial and lateral resolution. In addition, the wavelength of excitation light is longer than the wavelength of single-photon microscopy relatively. Hence, it has better penetration depth. Take harmonic generation for an instance, during the excitation process, there is no transition of real energy state. Complying with the conservation of momentum. Accordingly, cause less optical bleaching or damage to the interacted tissues.
Harmonic generation is different from fluorescence signal. There is no transition of real energy state. As a result, the super resolution microscopy of fluorescence is not
suitable for harmonic generation. Ground state depletion microscopy was published by S. W. Hell at 1994. By decreasing the number of electron of ground, affect the absorption of the sample. As a consequence, modulate the intensity of fluorescence. This essay apply a characteristic of harmonic generation: if there is real energy state of multi-photon absorption, it will enhance the intensity of harmonic generation. Therefore, we can decade the enhancement of harmonic generation by applying GSD. Finally, we can modulate the enhance the signal of harmonic generation.
Owing to the high intensity requirement of multi-photon. This essay will apply 2-D point scanning system to obtain THG image. Furthermore, involving the depletion light make the sample GSD。THG comply with the momentum of the insert photon. The system will set up as a forward system. THG will be detected by a photomultiplier tube, which is mounted with a UV filter.
This research consider melanin and hemoglobin as sample for GSD testing. Our system take 1064 nm femto-second laser as excitation laser. According to the absorption spectrum of sample, use 532 nm continuous wave laser as depletion light. In order to generate a structured illumination depletion light. Use Spatial Light Modulator (SLM) to diffract the light. Make use of the +1 and 1 order diffraction light to interfere at the focal plane of the sample. Consequently, generate the sinusoidal structured illumination. In this way, we can modulate the THG at different intensity of depletion light. |
