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Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy
Published on: December 9, 2013
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Molecule Counts in Localization Microscopy with Organic Fluorophores.
Christos Karathanasis1, Franziska Fricke1, Gerhard Hummer2,3
1Institute of Physical and Theoretical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 7, 60438, Frankfurt am Main, Germany.
Summary
Single-molecule localization microscopy (SMLM) enables counting of Alexa Fluor 647-labeled nucleic acids. This statistical method quantifies fluorophores per spot, facilitating quantitative super-resolution imaging.
Area of Science:
- Biophysics
- Molecular Biology
- Microscopy
Background:
- Single-molecule localization microscopy (SMLM) offers molecular counting capabilities beyond individual resolution.
- Organic fluorophores are crucial for biological imaging but can present challenges in quantitative analysis.
Purpose of the Study:
- To demonstrate SMLM-based molecule counting for Alexa Fluor 647-labeled nucleic acids.
- To develop a statistical model for extracting fluorophore counts from blinking events.
- To validate the counting method using single fluorophores and DNA origami structures.
Main Methods:
- Utilizing photoswitching conditions for Alexa Fluor 647 imaging in SMLM.
- Applying a statistical model to analyze fluorophore blinking event distributions.
- Validating counts against known single fluorophore and trimer configurations on DNA origami.
Main Results:
- Successfully demonstrated SMLM molecule counting for Alexa Fluor 647-labeled nucleic acids.
- Extracted accurate fluorophore counts per spot using the statistical model.
- Validated the method's precision with single fluorophores and Alexa Fluor 647 trimers.
Conclusions:
- A simple statistical strategy enables quantitative super-resolution imaging with organic fluorophores.
- SMLM molecule counting is effective for Alexa Fluor 647-labeled targets.
- This approach enhances the quantitative capabilities of super-resolution microscopy.
