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Ultrahigh-resolution multicolor colocalization of single fluorescent probes
T D Lacoste1, X Michalet, F Pinaud
1Material Sciences and Physical Biosciences Divisions, Lawrence Berkeley National Laboratory, CA 94720, USA.
Summary
This study introduces an optical ruler using unique fluorescent probes and a specialized microscope for ultrahigh-resolution imaging. It achieves precise distance measurements below 10 nm, bridging nanoscale and microscale imaging gaps.
Area of Science:
- Nanotechnology
- Optical Physics
- Biophysics
Background:
- Accurate nanoscale distance measurements are crucial for understanding molecular interactions and material properties.
- Existing techniques like fluorescence resonance energy transfer (FRET) have limitations in range and resolution.
- Conventional far-field microscopy often lacks the resolution required for precise nanometer-scale measurements.
Purpose of the Study:
- To develop and demonstrate an optical ruler with ultrahigh resolution for precise distance measurements.
- To overcome chromatic aberrations in multicolor imaging of fluorescent probes.
- To bridge the gap between near-field and far-field imaging techniques.
Main Methods:
- Utilized two distinct fluorophore families: energy-transfer fluorescent beads (TransFluoSpheres) and semiconductor nanocrystal quantum dots.
- Constructed a multicolor sample-scanning confocal microscope with piezo-scanner for nanometer-scale steps.
- Employed precise localization of fluorescent spots by fitting to the microscope's excitation point-spread function.
Main Results:
- Achieved two-dimensional colocalization of TransFluoSpheres (40 nm) and nanocrystals (3-10 nm).
- Demonstrated distance measurement accuracy better than 10 nm using conventional far-field optics.
- Successfully imaged fluorescent emitters free of chromatic aberrations.
Conclusions:
- The developed optical ruler provides unprecedented accuracy for nanoscale distance measurements.
- This technique integrates principles of FRET and advanced microscopy for versatile applications.
- The ruler spans a measurement range from a few nanometers to tens of micrometers, offering a significant advancement in optical metrology.