研究目的
Investigating the use of DNA-based, exchangeable fluorophore labels for STED microscopy to bypass photobleaching and enable multi-color and 3D imaging of whole cells.
研究成果
The study demonstrates a simple and powerful experimental protocol for STED microscopy in cells that bypasses photobleaching using DNA-based, target-specific, exchangeable fluorophore labels. This concept allows for multi-color and 3D imaging with high contrast and is beneficial for live cell imaging with long acquisition times.
研究不足
The approach is currently limited to a small number of labels and not capable of targeting any protein in a cell. The resolution for dynamic labels is slightly worse than for covalent labels due to higher noise sensitivity.
1：Experimental Design and Method Selection:
The study employs confocal and STED microscopy with short, fluorophore-labeled oligonucleotides that transiently bind to complementary oligonucleotides attached to protein-specific antibodies.
2：Sample Selection and Data Sources:
U2OS cells were used for imaging the microtubule cytoskeleton and mitochondria.
3：List of Experimental Equipment and Materials:
Includes DNA-labeled antibodies, fluorophore-labeled oligonucleotides (imager strands), and commercial antibodies optimized for STED imaging.
4：Experimental Procedures and Operational Workflow:
Involves immunofluorescence labeling, time-lapse confocal microscopy, and STED microscopy with varying irradiation intensities.
5：Data Analysis Methods:
Includes image analysis routines for correcting sample movement and fluctuations in irradiation intensity, and Fourier Ring Correlation (FRC) for resolution measurement.
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