Quantitative Photo Activated Localization Microscopy: Unraveling the Effects of Photoblinking

In this work we discuss how to use photophysical information for improved quantitative measurements using Photo Activated Localization Microscopy (PALM) imaging. We introduce a method that reliably estimates the number of photoblinking molecules present in a biological sample and gives a robust way to quantify proteins at the single-cell level from PALM images. We apply this method to determine the amount of β2 adrenergic receptor, a prototypical G Protein Coupled Receptor, expressed on the plasma membrane of HeLa cells

Universal emission intermittency in quantum dots, nanorods, and nanowires

Virtually all known fluorophores, including semiconductor nanoparticles, nanorods and nanowires exhibit unexplainable episodes of intermittent emission blinking. A most remarkable feature of the fluorescence intermittency is a universal power law distribution of on- and off-times. For nanoparticles the resulting power law extends over an extraordinarily wide dynamic range: nine orders of magnitude in probability density and five to six orders of magnitude in time. The exponents hover about the ubiquitous value of -3/2. Dark states routinely last for tens of seconds, which are practically forever on quantum mechanical time scales. Despite such infinite states of darkness, the dots miraculously recover and start emitting again. Although the underlying mechanism responsible for this phenomenon remains an enduring mystery and many questions remain, we argue that substantial theoretical progress has been made.Comment: 9 pages, 2 figures, Accepted versio

FRET as a biomolecular research tool — understanding its potential while avoiding pitfalls

International audienceThe applications of Förster resonance energy transfer (FRET) grow with each year. However, different FRET techniques are not applied consistently, nor are results uniformly presented, which makes implementing and reproducing FRET experiments challenging. We discuss important considerations for designing and evaluating ensemble FRET experiments. Alongside a primer on FRET basics, we provide guidelines for making experimental design choices such as the donor-acceptor pair, instrumentation and labeling chemistries; selecting control experiments to unambiguously demonstrate FRET and validate that the experiments provide meaningful data about the biomolecular process in question; analyzing raw data and assessing the results; and reporting data and experimental details in a manner that easily allows for reproducibility. Some considerations are also given for FRET assays and FRET imaging, especially with fluorescent proteins. Our goal is to motivate and empower all biologists to consider FRET for the powerful research tool it can be