Sub-diffraction imaging on standard microscopes through photobleaching microscopy with non-linear processing

被引:25
作者
Munck, Sebastian [1 ,2 ,3 ]
Miskiewicz, Katarzyna [1 ,2 ]
Sannerud, Ragna [1 ,2 ]
Menchon, Silvia A. [1 ,2 ]
Jose, Liya [1 ,2 ]
Heintzmann, Rainer [4 ,5 ,6 ]
Verstreken, Patrik [1 ,2 ]
Annaert, Wim [1 ,2 ]
机构
[1] VIB Ctr Biol Dis, B-3000 Louvain, Belgium
[2] Katholieke Univ Leuven, Ctr Human Genet, B-3000 Louvain, Belgium
[3] VIB, LiMoNe, B-3000 Louvain, Belgium
[4] Univ Jena, Inst Phys Chem, D-07743 Jena, Germany
[5] Inst Photon Technol, D-07745 Jena, Germany
[6] Kings Coll London, NHH, Randall Div Cell & Mol Biophys, London SE1 1UL, England
基金
欧洲研究理事会;
关键词
Bleaching; Super-resolution microscopy; Method; PiMP; SUBDIFFRACTION-RESOLUTION; ILLUMINATION MICROSCOPY; FLUORESCENCE MICROSCOPY; LIMIT; RECONSTRUCTION; DEPLETION; NANOSCOPY; BREAKING;
D O I
10.1242/jcs.098939
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
Visualization of organelles and molecules at nanometer resolution is revolutionizing the biological sciences. However, such technology is still limited for many cell biologists. We present here a novel approach using photobleaching microscopy with non-linear processing (PiMP) for sub-diffraction imaging. Bleaching of fluorophores both within the single-molecule regime and beyond allows visualization of stochastic representations of sub-populations of fluorophores by imaging the same region over time. Our method is based on enhancing the probable positions of the fluorophores underlying the images. The random nature of the bleached fluorophores is assessed by calculating the deviation of the local actual bleached fluorescence intensity to the average bleach expectation as given by the overall decay of intensity. Subtracting measured from estimated decay images yields differential images. Non-linear enhancement of maxima in these diffraction-limited differential images approximates the positions of the underlying structure. Summing many such processed differential images yields a super-resolution PiMP image. PiMP allows multi-color, three-dimensional sub-diffraction imaging of cells and tissues using common fluorophores and can be implemented on standard wide-field or confocal systems.
引用
收藏
页码:2257 / 2266
页数:10
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