Fluctuation methods to study protein aggregation in live cells: concanavalin A oligomers formation.

Prefibrillar oligomers of proteins are suspected to be the primary pathogenic agents in several neurodegenerative diseases. A key approach for elucidating the pathogenic mechanisms is to probe the existence of oligomers directly in living cells. In this work, we were able to monitor the process of aggregation of Concanavalin A in live cells. We used number and brightness analysis, two-color cross number and brightness analysis, and Raster image correlation spectroscopy to obtain the number of molecules, aggregation state, and diffusion coefficient as a function of time and cell location. We observed that binding of Concanavalin A to the membrane and the formation of small aggregates paralleled cell morphology changes, indicating progressive cell compaction and death. Upon protein aggregation, we observed increased membrane water penetration as reported by Laurdan generalized polarization imaging.

Complex wavelet filter improves FLIM phasors for photon starved imaging experiments.

Fluorescence lifetime imaging microscopy (FLIM) with phasor analysis provides easy visualization and analysis of fluorophores' lifetimes which is valuable for multiple applications including metabolic imaging, STED imaging, FRET imaging and functional imaging. However, FLIM imaging typically suffers from low photon budgets, leading to unfavorable signal to noise ratios which in many cases prevent extraction of information from the data. Traditionally, median filters are applied in phasor analysis to tackle this problem. This unfortunately degrades high spatial frequency FLIM information in the phasor analysis. These high spatial frequency components are typically edges of features and puncta, which applies to membranes, mitochondria, granules and small organelles in a biological sample. To tackle this problem, we propose a filtering strategy with complex wavelet filtering and Anscombe transform for FLIM phasor analysis. This filtering strategy preserves fine structures and reports accurate lifetimes in photon starved FLIM imaging. Moreover, this filter outperforms median filters and makes FLIM imaging with lower laser power and faster imaging possible.