Standard fluorescent proteins as dual-modality probes for correlative experiments in an integrated light and electron microscope
Standard fluorescent proteins as dual-modality probes for correlative experiments in an integrated light and electron microscope
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DOI：
10.1007/s12154-015-0143-3
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发表时间：
2015-10-01
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影响因子：
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通讯作者：
Collinson, Lucy M.
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文献类型：
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作者：
Brama, Elisabeth;Peddie, Christopher J.;Collinson, Lucy M.
集成光学和电子显微镜（ILEMs）将实现新一代高精度相关成像实验。为了充分利用这些系统，样品必须包含双模态探针，以突出大分子在细胞超微结构中的位置。我们证明了荧光蛋白（FPs） GFP（绿色），YFP（黄色）和mCherry在使用树脂荧光（IRF）技术保存时可以作为ILEM的双模探针，该技术在轻度染色，树脂包埋的细胞和组织中提供稳定的活性荧光团。然而，我们发现ILEM中的真空压力会影响IRF截面中FPs的光物理特性。在这里，我们表明，降低真空压力会降低GFP和YFP的荧光强度，这是从样品中提取水的结果，并且在水蒸气（但不是氧气或氮气）的分压重建中是可逆的。我们还发现，虽然在200 Pa的分压下（使用水蒸气产生）荧光强度降低，但FP强度在真空中随时间的推移非常稳定，并且在成像过程中抗光漂白。因此，我们能够为标准FPs定义成像策略，作为单一“多色”集成显微镜系统中的双模探针。
Integrated light and electron microscopes (ILEMs) will enable a new generation of high-precision correlative imaging experiments. To fully exploit these systems, samples must contain dual-modality probes that highlight the position of macromolecules in the context of cell ultrastructure. We demonstrate that the fluorescent proteins (FPs) GFP (green), YFP (yellow) and mCherry can be used as dual-modality probes for ILEM when preserved using the inresin fluorescence (IRF) technique, which delivers stable active fluorophores in lightly stained, resin-embedded cells and tissues. However, we found that vacuum pressure in the ILEM affects the photophysics of FPs in IRF sections. Here, we show that reducing the vacuum pressure reduces fluorescence intensity of GFP and YFP, which is a consequence of water extraction from the sample and is reversible on recreation of partial pressure with water vapour (but not oxygen or nitrogen gas). We also find that, although fluorescence intensity is reduced at a partial pressure of 200 Pa (created using water vapour), the FP intensity is remarkably stable over time in vacuum and resistant to photobleaching during imaging. We are thus able to define imaging strategies for standard FPs acting as dual-modality probes in a single 'multi-colour' integrated microscope system.