Single-molecule quantification of lipotoxic expression of activating transcription factor 3.

Activating transcription factor 3 (ATF3) is a member of the mammalian activation transcription factor/cAMP, physiologically important in the regulation of pro- and anti-inflammatory target genes. We compared the induction of ATF3 protein as measured by Western blot analysis with single-molecule localization microscopy dSTORM to quantify the dynamics of accumulation of intranuclear ATF3 of triglyceride-rich (TGRL) lipolysis product-treated HAEC (Human Aortic Endothelial Cells). The ATF3 expression rate within the first three hours after treatment with TGRL lipolysis products is about 3500 h(-1).

Quantifying molecular colocalization in live cell fluorescence microscopy.

One of the most challenging tasks in microscopy is the quantitative identification and characterization of molecular interactions. In living cells this task is typically performed by fluorescent labeling of the interaction partners with spectrally distinct fluorophores and imaging in different color channels. Current methods for determining colocalization of molecules result in outcomes that can vary greatly depending on signal-to-noise ratios, threshold and background levels, or differences in intensity between channels.

Direct comparison of fatty acid ratios in single cellular lipid droplets as determined by comparative Raman spectroscopy and gas chromatography.

Cellular lipid droplets are the least studied and least understood cellular organelles in eukaryotic and prokaryotic cells. Despite a significant body of research studying the physiology of lipid droplets it has not yet been possible to fully determine the composition of individual cellular lipid droplets. In this paper we use Raman spectroscopy on single cellular lipid droplets and least-squares fitting of pure fatty acid spectra to determine the composition of individual lipid droplets in cells after treatment with different ratios of oleic and palmitic acid.

Correlation-matrix analysis of two-color coincidence events in single-molecule fluorescence experiments.

We introduce a robust and relatively easy-to-use method to evaluate the quality of two-color (or more) fluorescence coincidence measurements based on close investigation of the coincidence correlation-matrix. This matrix contains temporal correlations between the number of detected bursts in individual channels and their coincidences. We show that the Euclidian norm of a vector Γ derived from elements of the correlation matrix takes a value between 0 and 2 depending on the relative coincidence frequency.