使用单平面照明显微镜的三维荧光寿命成像可提供更高的信噪比。

Three-dimensional Fluorescence Lifetime Imaging with a Single Plane Illumination Microscope provides an improved signal to noise ratio.

作者信息

Greger Klaus, Neetz Manuel J, Reynaud Emmanuel G, Stelzer Ernst H K

机构信息

Cell Biology and Biophysics Unit (CBBU), EMBL, Meyerhofstraße 1, D-69117 Heidelberg, Germany.

出版信息

Opt Express. 2011 Oct 10;19(21):20743-50. doi: 10.1364/OE.19.020743.

DOI:10.1364/OE.19.020743

PMID:21997084

Abstract

We designed a widefield frequency domain Fluorescence Lifetime Imaging Microscopy (FLIM)setup, which is based on a Single Plane Illumination Microscope (SPIM). A SPIM provides an inherent optical sectioning capability and reduces photobleaching compared to conventional widefield and confocal fluorescence microscopes. The lifetime precision of the FLIM was characterized with Rhodamine 6G solutions of different quencher concentrations [KI]. We demonstrate the high spatial resolution of the SPIM-FLIM combination in the intensity domain as well as in the lifetime domain with latex bead samples and multiple recordings of three-dimensional live Madine-Darby Canine Kidney (MDCK) cysts. We estimate that the bleaching rate after 600 images have been recorded is below 5%.

摘要

我们设计了一种基于单平面照明显微镜（SPIM）的宽场频域荧光寿命成像显微镜（FLIM）装置。与传统的宽场和共聚焦荧光显微镜相比，SPIM具有固有的光学切片能力，并减少了光漂白现象。我们使用不同猝灭剂浓度[KI]的罗丹明6G溶液对FLIM的寿命精度进行了表征。我们通过乳胶珠样品以及对三维活体马-达二氏犬肾（MDCK）囊肿的多次记录，展示了SPIM-FLIM组合在强度域和寿命域的高空间分辨率。我们估计，在记录600张图像后，漂白率低于5%。