Aggregation distributions on cells determined by photobleaching image correlation spectroscopy

Area of Science:

• Cellular biology
• Biophysics
• Biochemistry

Background:

• Molecular organization into complexes is key in cellular biology.
• Analyzing macromolecular aggregation and dynamics is a biophysical challenge.
• Current methods require a brightness standard for absolute aggregation data.

Purpose of the Study:

• To develop a photobleaching-based image correlation spectroscopy (ICS) method.
• To quantify molecular aggregation and distribution on the submicron scale.
• To determine aggregation states in intact cells without a brightness standard.

Main Methods:

• Time-series analysis of confocal microscopy images using image correlation spectroscopy (ICS).
• Photobleaching ICS to analyze aggregation as a function of gradual photobleaching.
• Computer simulations and experimental application to Aβ peptide aggregation on nerve cells.

Main Results:

• Photobleaching ICS is sensitive to aggregation distribution on the submicron scale.
• The nonlinearity of apparent cluster density with bleaching relates to cluster size.
• Time-dependent increases in Aβ1-42 peptide aggregation were observed.
• Aggregation data fit monomer-dimer-tetramer-hexamer or monomer-dimer-trimer-pentamer models.

Conclusions:

• Photobleaching ICS effectively determines aggregation states on the supramolecular scale.
• This method eliminates the need for a brightness standard in cellular analysis.
• The study demonstrates a novel approach for studying molecular aggregation in vivo.