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Correcting chromatic offset in multicolor superresolution localization microscopy
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DOI：10.1364/OE.21.010978.png)
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Localization based super-resolution microscopy techniques require precise drift correction methods because the achieved spatial resolution is close to both the mechanical and optical performance limits of modern light microscopes. Multi-color imaging methods require corrections in addition to those dealing with drift due to the static, but spatially-dependent, chromatic offset between images. We present computer simulations to quantify this effect, which is primarily caused by the high-NA objectives used in super-resolution microscopy. Although the chromatic offset in well corrected systems is only a fraction of an optical wavelength in magnitude (<50 nm) and thus negligible in traditional diffraction limited imaging, we show that object colocalization by multi-color super-resolution methods is impossible without appropriate image correction. The simulated data are in excellent agreement with experiments using fluorescent beads excited and localized at multiple wavelengths. Finally we present a rigorous and practical calibration protocol to correct for chromatic optical offset, and demonstrate its efficacy for the imaging of transferrin receptor protein colocalization in HeLa cells using two-color direct stochastic optical reconstruction microscopy (dSTORM). (C) 2013 Optical Society of America
Keyword:
FLUORESCENCE NANOSCOPY
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期刊
IF:
3.3
论文数:
6.1W
被引数:
14.3W
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引用论文
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