Super-resolution imaging of ciliary microdomains in isolated olfactory sensory neurons using a custom two-color stimulated emission depletion microscope.
Super-resolution imaging of ciliary microdomains in isolated olfactory sensory neurons using a custom two-color stimulated emission depletion microscope.
复制标题
使用定制的双色受激发射损耗显微镜对分离的嗅觉感觉神经元中的睫状微区进行超分辨率成像。
DOI：
10.1117/1.jbo.21.6.066017
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发表时间：
2016
影响因子：
3.5
通讯作者：
Gibson,EmilyA
中科院分区：
文献类型：
--
作者：
Meyer,StephanieA;Ozbay,BarisN;Potcoava,Mariana;Salcedo,Ernesto;Restrepo,Diego;Gibson,EmilyA
我们使用特制的显微镜对分离的嗅觉感觉神经元（OSNs）进行了刺激发射损耗（STED）成像。STED显微镜使用单一脉冲激光激发两个独立的荧光团，Atto 590和Atto 647N。门控定时电路结合不同颜色激发/STED激光脉冲的时间交错，过滤了两个通道的检测，极大地减少了串扰。我们量化了Atto 590和Atto 647N通道的仪器分辨率。测量到两个通道之间的空间间隔小于10 nm，远低于分辨率极限。定制的显微镜被整合到商业研究显微镜上，允许在同一视场上进行明场、微分干涉对比和荧光成像。我们用免疫标记的方法对小鼠的嗅转导纤毛膜蛋白进行图像定位。我们成像了不同纤毛中的可渗透环核苷酸门控（CNG）通道（Atto 594）和腺苷酸环化酶III型（ACIII） （Atto 647N）。STED成像解决了每个蛋白质分离良好的亚衍射限制簇。对于CNG和ACIII，我们分别量化了每个簇的大小，以获得and的平均值。STED成像显示在共聚焦图像中无法分辨的分离簇。
We performed stimulated emission depletion (STED) imaging of isolated olfactory sensory neurons (OSNs) using a custom-built microscope. The STED microscope uses a single pulsed laser to excite two separate fluorophores, Atto 590 and Atto 647N. A gated timing circuit combined with temporal interleaving of the different color excitation/STED laser pulses filters the two channel detection and greatly minimizes crosstalk. We quantified the instrument resolution to beand, for the Atto 590 and Atto 647N channels. The spatial separation between the two channels was measured to be under 10 nm, well below the resolution limit. The custom-STED microscope is incorporated onto a commercial research microscope allowing brightfield, differential interference contrast, and epifluorescence imaging on the same field of view. We performed immunolabeling of OSNs in mice to image localization of ciliary membrane proteins involved in olfactory transduction. We imaged-permeable cyclic nucleotide gated (CNG) channel (Atto 594) and adenylyl cyclase type III (ACIII) (Atto 647N) in distinct cilia. STED imaging resolved well-separated subdiffraction limited clusters for each protein. We quantified the size of each cluster to have a mean value ofand, for CNG and ACIII, respectively. STED imaging showed separated clusters that were not resolvable in confocal images.