Fluorescence Techniques to Study Lipid Dynamics

Abstract

Biological research has always tremendously benefited from the development of key methodology. In fact, it was the advent of microscopy that shaped our understanding of cells as the fundamental units of life. Microscopic techniques are still central to the elucidation of biological units and processes, but equally important are methods that allow access to the dimension of time, to investigate the dynamics of molecular functions and interactions. Here, fluorescence spectroscopy with its sensitivity to access the single-molecule level, and its large temporal resolution, has been opening up fully new perspectives for cell biology. Here we summarize the key fluorescent techniques used to study cellular dynamics, with the focus on lipid and membrane systems.T o elucidate cellular processes in their native dynamic environment has been one of the main issues in cell biology over the past decades. The lack of appropriate techniques has long been the main limiting step for the research on dynamic systems, because it was impossible to acquire real time information with the well-known biochemical techniques. The key challenge in dynamically observing biological systems is to combine the ability to resolve moderate to very low concentrations of molecules-because they are simply limited in living cells-on relevant timescales. Relevant timescales in cell biology can be minutes and hours, on a systemic level of cell metabolism, down to the microsecond and even nanosecond regime in which molecular and intramolecular rearrangements take place. With respect to lipidic systems, relevant dynamics range from the local movements of lipids by diffusion to the mechanical transformations of whole membranes, spanning several orders of magnitude in time to be covered. Like for other cellular processes, the investigation of lipids and membranes also in general benefited greatly from the introduction of fluorescence microscopy and spectroscopy to biology. After the 1960s, great technological inventions based on the phenomenon of fluorescence were made, such as confocal microscopy, fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), Förster resonance energy transfer (FRET), total internal reflection fluorescence (TIRF), and two-photon microscopy, that not only revolutionized imaging but also yielded access to dynamics on previously inaccessible timescales. Another very big step was certainly taken after the introduction of fluorescent proteins, which again accelerated the use of these techniques in living cells and organisms. Nowadays, the technical advancements of fluorescence-based methods allow us to explore systems as small as single molecules with temporal resolution down to the nanoseconds regime. Lately, even the resolution limit of optical microscopy, for a long time being one of the fundamental barriers in elucidating cellular processes, has been overcome by smart applications of the phenomenon of fluorescence.This article aims at giving a short overview on mainly fluorescence-based metho...

Membrane Organization and Lipid Rafts

“…The concept of sphingolipid-sterol-protein rafts that were smaller than the resolution of the optical microscope stimulated the search for novel methodologies. Recent studies using different imaging and spectroscopic methods have revealed interesting glimpses of how the lipids and proteins behave in the crowded bilayer (Sezgin and Schwille 2011). These studies have confirmed the existence of cholesteroldependent nanoscale assemblies of sphingolipids and GPI-anchored proteins in the plasma membrane of living cells.…”

The mystery of membrane organization: composition, regulation and roles of lipid rafts

“…Although confocal microscopy studies have reported co-localization of certain molecules with putative lipid raft markers (such as cholera toxin) as evidence of their raft association 44 , in general the resolution of such systems is insufficient to directly assay raft domain structure and composition. To overcome this limitation, several optical tools have recently been developed 45,46 and applied to investigate nano-scale structures and dynamics in cells. For instance, super-resolution optical microscopy approaches such as photoactivated localization microscopy (PALM), stimulated emission depletion (STED) microscopy (Supplementary Box S2), and near-field scanning optical microscopy (NSOM) have been used to visualize lipid-mediated protein clustering 47–50 .…”

A comparative study on fluorescent cholesterol analogs as versatile cellular reporters

“…Figure 2B compares values of the partitioning coeffi cient %Lo for the different Chol analogs. Due to the compositional complexity, the molecular Here, d ( P STED ) and D ( P STED ) are the diameter of the observation spot and the average transit time at the STED power, P STED , and d ( P STED = 0) and D ( P STED = 0) the respective parameters at confocal recordings, where the diameter of the confocal spot [ d ( P STED = 0) = 200 nm for 488 nm and 240 nm for 635 nm excitation] has been determined from imaging fl uorescent beads or from theory ( 36 ). Shortening of the transit time D due to photobleaching from the STED laser is unlikely, as has been shown by several control measurements before, i.e., the decrease in d with STED power P STED is not a result of photobleaching by the STED laser [e.g., ( 37,38 )].…”