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Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells
Published on: December 11, 2021
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Imaging fluorescence (cross-) correlation spectroscopy in live cells and organisms
Jan W Krieger1, Anand P Singh2,3,4, Nirmalya Bag2,3,5
1German Cancer Research Center (DKFZ), Heidelberg, Germany.
Nature Protocols
|November 6, 2015
Summary
This study introduces imaging fluorescence (cross-) correlation spectroscopy (FCS/FCCS) using SPIM or TIRF microscopy. This technique offers spatially resolved mobility and interaction maps with reduced photodamage for bioimaging applications.
Area of Science:
- Biophysics
- Microscopy
- Quantitative Bioimaging
Background:
- Fluorescence (cross-) correlation spectroscopy (FCS/FCCS) is a powerful technique for studying molecular dynamics.
- Conventional confocal microscopy-based FCS/FCCS can be limited by photodamage and spatial resolution.
- There is a need for advanced imaging techniques to provide spatially resolved molecular information.
Purpose of the Study:
- To provide guidelines for implementing imaging FCS/FCCS using SPIM or TIRF microscopy.
- To enable quantitative bioimaging with high spatial resolution.
- To reduce photodamage compared to conventional methods.
Main Methods:
- Combining single-plane illumination (SPIM) or total internal reflection fluorescence (TIRF) microscopy with fast, single-molecule-sensitive cameras.
- Developing protocols for sample preparation, setup calibration, data acquisition, and evaluation.
- Applying the technique to both in vitro and in vivo samples.
Main Results:
- Generation of spatially resolved mobility and interaction maps with hundreds to thousands of pixels.
- Demonstration of reduced photodamage compared to confocal microscopy-based FCS/FCCS.
- Successful application across diverse sample types.
Conclusions:
- Imaging FCS/FCCS using SPIM/TIRF is a powerful, quantitative bioimaging tool.
- The protocol allows for efficient data acquisition and analysis within approximately one day.
- This method advances the study of molecular dynamics in biological systems.
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