Bridging length scales to measure polymer assembly.

Area of Science:

• Biophysics
• Cell Biology
• Biochemistry

Background:

• Quantitative measurement of polymer concentration is crucial for understanding protein polymerization pathways.
• Existing techniques are limited, especially for in vivo applications.
• There is a need for methods applicable within complex cellular environments.

Purpose of the Study:

• To develop and validate a novel methodology for time-resolvable quantitative polymer concentration measurements in cells and cell extracts.
• To overcome the limitations of traditional microscopy and spectroscopy methods for in vivo studies.
• To enable precise quantification of dynamic polymer assembly processes within complex biological systems.

Main Methods:

• Development of a hybrid microscopy-spectroscopy technique.
• Quantification of the relationship between microscopy and spectroscopy signals across multiple locations.
• Application using two-photon microscopy with Fluorescence Lifetime Imaging Microscopy (FLIM) of Förster Resonance Energy Transfer (FRET).
• Validation in tissue culture cells and Xenopus egg extracts.

Main Results:

• The developed methodology quantitatively measures polymer concentration, specifically microtubule assembly.
• The relationship between FRET and two-photon intensity measurements aligns with theoretical predictions.
• Measurements accurately reflect changes in acquisition time, labeling ratios, and polymer concentration.
• Successful application in complex biological samples like cells and egg extracts.

Conclusions:

• The combined microscopy-spectroscopy approach provides accurate, time-resolvable quantitative measurements of polymer concentration in vivo.
• This methodology is effective for studying microtubule assembly dynamics in complex cellular environments.
• The technique has broad applicability for investigating nucleation and assembly pathways of various polymers.