Eissing Nathalie, Heger Lukas, Baranska Anna, Cesnjevar Robert, Büttner-Herold Maike, Söder Stephan, Hartmann Arndt, Heidkamp Gordon F, Dudziak Diana
Friedrich-Alexander Universität Erlangen-Nürnberg, Universitätsklinikum Erlangen, Department of Dermatology, Laboratory of Dendritic Cell Biology, Erlangen, Germany.
Friedrich-Alexander Universität Erlangen-Nürnberg, Universitätsklinikum Erlangen, Department of Paediatric Cardiac Surgery, Erlangen, Germany.
Immunol Lett. 2014 Sep;161(1):1-5. doi: 10.1016/j.imlet.2014.04.003. Epub 2014 Apr 12.
Confocal laser scanning microscopy is an advanced technique for imaging tissue samples in vitro and in vivo at high optical resolution. The development of new fluorochrome variants do not only make it possible to perform multicolor flow cytometry of single cells, but in combination with high resolution laser scanning systems also to investigate the distribution of cells in lymphoid tissues by confocal immunofluorescence analyses, thus allowing the distinction of various cell populations directly in the tissue. Here, we provide a protocol for the visualization of at least six differently fluorochrome-labeled antibodies at the same time using a conventional confocal laser scanning microscope with four laser lines (405 nm, 488 nm, 555 nm, and 639 nm laser wavelength) in both murine and human tissue samples. We further demonstrate that compensation correction algorithms are not necessary to reduce spillover of fluorochromes into other channels when the used fluorochromes are combined according to their specific emission bands and the varying Stokes shift for co-excited fluorochromes with the same laser line.
共聚焦激光扫描显微镜是一种先进技术，可在体外和体内以高光学分辨率对组织样本进行成像。新型荧光染料变体的开发不仅使单细胞多色流式细胞术成为可能，而且与高分辨率激光扫描系统相结合，还能通过共聚焦免疫荧光分析研究淋巴组织中细胞的分布，从而能够直接在组织中区分各种细胞群。在此，我们提供了一种方案，可使用具有四条激光线（405 nm、488 nm、555 nm和639 nm激光波长）的传统共聚焦激光扫描显微镜，同时在小鼠和人类组织样本中可视化至少六种不同荧光染料标记的抗体。我们进一步证明，当根据所用荧光染料的特定发射带以及同一激光线激发的荧光染料的不同斯托克斯位移来组合使用荧光染料时，无需补偿校正算法来减少荧光染料向其他通道的溢出。
