We demonstrate three-dimensional (3D) super-resolution microscopy in whole fixed cells using photoactivated localization microscopy (PALM). The use of the bright, genetically expressed fluorescent marker photoactivatable monomeric (m)Cherry (PA-mCherry1) in combination with near diffraction-limited confinement of photoactivation using two-photon illumination and 3D localization methods allowed us to investigate a variety of cellular structures at <50 nm lateral and <100 nm axial resolution. Compared to existing methods, we have substantially reduced excitation and bleaching of unlocalized markers, which allows us to use 3D PALM imaging with high localization density in thick structures. Our 3D localization algorithms, which are based on cross-correlation, do not rely on idealized noise models or specific optical configurations. This allows instrument design to be flexible. By generating appropriate fusion constructs and expressing them in Cos7 cells, we could image invaginations of the nuclear membrane, vimentin fibrils, the mitochondrial network and the endoplasmic reticulum at depths of greater than 8 μm.
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We thank N. Morgan and A. Gillespie for training and use of their spin coater; G. Patterson (National Institute of Biomedical Imaging and Bioengineering) for the gift of purified PA-mCherry1 and mEos2 and for the use of his cell culture facilities; A. Jin for measuring the thickness of our quantum dot films; E. Ramko for help with preparing the PA-mCherry1 fusion vectors; K. Kilborn (Intelligent Imaging Innovations) for loaning us the Vector Point Scanning 2P system; and S. Parekh and H. Eden for feedback and suggestions on the manuscript. This work was supported by the Intramural Research Programs of the National Institute of Biomedical Imaging and Bioengineering.
The authors declare no competing financial interests.
Supplementary Figures 1–16, Supplementary Table 1 and Supplementary Notes 1–3 (PDF 2999 kb)
z-stack of PA-mCherry1-mito fusions, to accompany Figure 3. Histogram bin size is 60 nm, individual frames are separated by 60 nm z steps. Smoothing of σ = 0.4 pixels in each dimension was applied before plotting data. (MOV 1173 kb)
z-stack of PA-mCherry1-ER fusions, to accompany Figure 4. Histogram bin size is 60 nm, individual frames are separated by 60 nm z steps. Smoothing of σ = 0.6 pixels in each dimension was applied before plotting data. (MOV 1519 kb)
z-stack of PA-mCherry1-vimentin fusions, to accompany Figure 5. Histogram bin size is 60 nm, individual frames are separated by 60 nm z steps. Smoothing of σ = 0.6 pixels in each dimension was applied before plotting data. (MOV 4243 kb)
z-stack of PA-mCherry1-lamin fusions, to accompany Figure 6. Histogram bin size is 50 nm, individual frames are separated by 50 nm z steps. Smoothing of σ = 0.75 pixels in each dimension was applied before plotting data. (MOV 7782 kb)
z-stack of PA-mCherry1-lamin fusions, extending over > 8.5 μm imaging depth. Histogram bin size is 60 nm, individual frames are separated by 60 nm z steps. Smoothing of σ = 0.75 pixels in each dimension was applied before plotting data. (MOV 3267 kb)
Supplementary Software (ZIP 51 kb)
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York, A., Ghitani, A., Vaziri, A. et al. Confined activation and subdiffractive localization enables whole-cell PALM with genetically expressed probes. Nat Methods 8, 327–333 (2011). https://doi.org/10.1038/nmeth.1571
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