SPDM:: light microscopy with single-molecule resolution at the nanoscale
SPDM:: light microscopy with single-molecule resolution at the nanoscale
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DOI：
10.1007/s00340-008-3152-x
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发表时间：
2008-10-01
影响因子：
2.1
通讯作者：
Cremer, C.
中科院分区：
文献类型：
--
作者：
Lemmer, P.;Gunkel, M.;Cremer, C.
基于精确确定物体位置的远场荧光技术有可能绕过衍射理论给出的直接成像的光学分辨率限制。为了利用局域化来获得远低于衍射极限的结构信息，必须独立地检测结构中的点状成分，即使它们的距离低于传统的光学分辨率极限。这一目标可以通过利用荧光标记的各种光物理性质(光谱特征)来实现。在最初的实验中，光谱精密距离显微镜/光谱位置确定显微镜(SPDM)被限制在相对较少的要在观察体积内分辨的成分。最近，光可转化分子的引入极大地增加了可独立定域的组分的数量。在这里，我们提出了SPDM概念的扩展，利用了荧光蛋白质的可逆光漂白提供的新的光谱特征。结合空间调制照明(SMI)显微镜，在现阶段，我们已经获得了估计的有效光学分辨率，横向约为20 nm，轴向约为50 nm，或约为激发波长的1/25-1/10。
Far-field fluorescence techniques based on the precise determination of object positions have the potential to circumvent the optical resolution limit of direct imaging given by diffraction theory. In order to use localization to obtain structural information far below the diffraction limit, the 'point-like' components of the structure have to be detected independently, even if their distance is lower than the conventional optical resolution limit. This goal can be achieved by exploiting various photo-physical properties of the fluorescence labeling ('spectral signatures'). In first experiments, spectral precision distance microscopy/spectral position determination microscopy (SPDM) was limited to a relatively small number of components to be resolved within the observation volume. Recently, the introduction of photoconvertable molecules has dramatically increased the number of components which can be independently localized. Here, we present an extension of the SPDM concept, exploiting the novel spectral signature offered by reversible photobleaching of fluorescent proteins. In combination with spatially modulated illumination (SMI) microscopy, at the present stage, we have achieved an estimated effective optical resolution of approximately 20 nm in the lateral and 50 nm in the axial direction, or about 1/25th-1/10th of the exciting wavelength.