Botchway Stanley W, Scherer Kathrin M, Hook Steve, Stubbs Christopher D, Weston Eleanor, Bisby Roger H, Parker Anthony W
Central Laser Facility, STFC, Rutherford Appleton Laboratory, Research Complex at Harwell, Harwell Oxford, Didcot, UK.
J Microsc. 2015 Apr;258(1):68-78. doi: 10.1111/jmi.12218. Epub 2015 Feb 9.
Multiphoton microscopy is widely employed in the life sciences using extrinsic fluorescence of low- and high-molecular weight labels with excitation and emission spectra in the visible and near infrared regions. For imaging of intrinsic and extrinsic fluorophores with excitation spectra in the ultraviolet region, multiphoton excitation with one- or two-colour lasers avoids the need for ultraviolet-transmitting excitation optics and has advantages in terms of optical penetration in the sample and reduced phototoxicity. Excitation and detection of ultraviolet emission around 300 nm and below in a typical inverted confocal microscope is more difficult and requires the use of expensive quartz optics including the objective. In this technical note we describe the adaptation of a commercial confocal microscope (Nikon, Japan E-C1 or E-C2) for versatile use with Ti-sapphire and OPO laser sources and the addition of a second detection channel that enables detection of ultraviolet fluorescence and increases detection sensitivity in a typical fluorescence lifetime imaging microscopy experiment. Results from some experiments with this setup illustrate the resulting capabilities.
多光子显微镜在生命科学中被广泛应用，它利用低分子量和高分子量标记物的外在荧光，其激发和发射光谱位于可见光和近红外区域。对于激发光谱在紫外区域的内在和外在荧光团成像，使用单激光或双激光的多光子激发避免了对可透过紫外线的激发光学器件的需求，并且在样品的光学穿透和降低光毒性方面具有优势。在典型的倒置共聚焦显微镜中，激发和检测300nm及以下的紫外发射更加困难，并且需要使用包括物镜在内的昂贵石英光学器件。在本技术说明中，我们描述了如何对商用共聚焦显微镜（尼康，日本E-C1或E-C2）进行改装，使其能够与钛宝石和光学参量振荡器（OPO）激光源通用，以及增加第二个检测通道，该通道能够检测紫外荧光并提高典型荧光寿命成像显微镜实验中的检测灵敏度。使用该设置进行的一些实验结果展示了由此产生的能力。
