Schapper Florian, Gonçalves José Tiago, Oheim Martin
Neurophysiology and New Microscopies, Ecole Supérieure de Physique et Chimie Industrielles (ESPCI), 10 rue Vauquelin, 75005 Paris, France.
Eur Biophys J. 2003 Nov;32(7):635-43. doi: 10.1007/s00249-003-0326-7. Epub 2003 Sep 3.
We demonstrate broad-field, non-scanning, two-photon excitation fluorescence (2PEF) close to a glass/cell interface by total internal reflection of a femtosecond-pulsed infrared laser beam. We exploit the quadratic intensity dependence of 2PEF to provide non-linear evanescent wave (EW) excitation in a well-defined sample volume and to eliminate scattered background excitation. A simple model is shown to describe the resulting 2PEF intensity and to predict the effective excitation volume in terms of easily measurable beam, objective and interface properties. We demonstrate non-linear evanescent wave excitation at 860 nm of acridine orange-labelled secretory granules in live chromaffin cells, and excitation at 900 nm of TRITC-phalloidin-actin/GPI-GFP double-labelled fibroblasts. The confined excitation volume and the possibility of simultaneous multi-colour excitation of several fluorophores make EW 2PEF particularly advantageous for quantitative microscopy, imaging biochemistry inside live cells, or biosensing and screening applications in miniature high-density multi-well plates.
我们通过飞秒脉冲红外激光束的全内反射，在靠近玻璃/细胞界面处展示了宽场、非扫描双光子激发荧光（2PEF）。我们利用2PEF对强度的二次依赖性，在明确的样品体积中提供非线性倏逝波（EW）激发，并消除散射背景激发。展示了一个简单模型来描述所得的2PEF强度，并根据易于测量的光束、物镜和界面特性预测有效激发体积。我们展示了在活嗜铬细胞中对吖啶橙标记的分泌颗粒进行860nm的非线性倏逝波激发，以及在TRITC-鬼笔环肽-肌动蛋白/GPI-GFP双标记成纤维细胞中进行900nm的激发。受限的激发体积以及同时对几种荧光团进行多色激发的可能性，使得EW 2PEF在定量显微镜、活细胞内成像生物化学或微型高密度多孔板中的生物传感和筛选应用中特别具有优势。
