Ludwig-Maximilians-Universitat Munchen, Walter-Brendel-Zentrum fur Experimentelle Medizin, Marchioninistrasse 27, Munchen, D-81377 Germany.
J Biomed Opt. 2010 Mar-Apr;15(2):026017. doi: 10.1117/1.3374337.
In conventional fluorescence or confocal microscopy, emitted light is generated not only in the focal plane but also above and below. The situation is different in multiphoton-induced fluorescence and multiphoton-induced higher harmonic generation. Here, restriction of signal generation to a single focal point permits that all emitted photons can contribute to image formation if collected, regardless of their path through the specimen. Often, the intensity of the emitted light is rather low in biological specimens. We present a method to significantly increase the fraction of photons collected by an epi (backward) detector by placing a simple mirror, an aluminum-coated coverslip, directly under the sample. Samples investigated include fluorescent test slides, collagen gels, and thin-layered, intact mouse skeletal muscles. Quantitative analysis revealed an intensity increase of second- and third-harmonic generated signal in skeletal muscle of nine- and sevenfold respectively, and of fluorescent signal in test slides of up to twofold. Our approach thus allows significant signal improvement also for situations were a forward detection is impossible, e.g., due to the anatomy of animals in intravital microscopy.
在传统的荧光或共聚焦显微镜中，发射光不仅在焦平面上产生，而且在焦平面上方和下方也产生。在多光子诱导荧光和多光子诱导高次谐波产生中，情况则不同。在这里，信号产生的限制仅限于单个焦点，这使得所有发射的光子如果被收集，都可以有助于形成图像，而不管它们通过样本的路径如何。通常，生物样本中发射光的强度相当低。我们提出了一种通过在样品下方直接放置一个简单的镜子（铝涂层盖玻片）来显著增加反向（背向）探测器收集的光子分数的方法。所研究的样品包括荧光测试载玻片、胶原蛋白凝胶和薄层层状的完整小鼠骨骼肌。定量分析显示，第二和第三谐波产生的信号在骨骼肌中的强度分别增加了九倍和七倍，而在测试载玻片中的荧光信号则增加了两倍。因此，我们的方法也允许在无法进行正向检测的情况下（例如，由于活体显微镜中动物的解剖结构）显著改善信号。
