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Counting Proteins in Single Cells with Addressable Droplet Microarrays
Published on: July 6, 2018
Chapter 9: Counting proteins in living cells by quantitative fluorescence microscopy with internal standards
Jian-Qiu Wu1, Chad D McCormick, Thomas D Pollard
1Department of Molecular Genetics , The Ohio State University, Columbus, Ohio 43210, USA.
Methods in Cell Biology
|January 3, 2009
Summary
Confocal microscopy quantifies fluorescent molecules in live cells. This method offers a wide dynamic range for precise molecular counting in single cells, aiding researchers in various life science fields.
Area of Science:
- Cell biology
- Biophysics
- Biochemistry
Background:
- Accurate quantification of macromolecules in live cells is crucial for understanding molecular pathways.
- Traditional methods may lack the dynamic range or precision for in vivo measurements.
Purpose of the Study:
- To present a method for measuring the absolute number of fluorescent molecules in live cells using confocal microscopy.
- To provide a practical guide for implementing this technique in biological research.
Main Methods:
- Treating a confocal microscope as a spectrophotometer.
- Measuring fluorescence intensity to determine molecular counts.
- Detailed step-by-step protocol for in vivo concentration measurements.
Main Results:
- Demonstrated a dynamic range of over three orders of magnitude for molecular quantification.
- Enabled accurate counting of fluorescent molecules within single live cells.
- Provided practical considerations for successful implementation.
Conclusions:
- Confocal microscopy can serve as a spectrophotometer for absolute molecular quantification in live cells.
- This technique offers a valuable tool for cell biologists, biochemists, and biophysicists.
- Facilitates the study of macromolecular dynamics in living systems.
