ABSTRACT
We present methods for making and testing the membrane biophysics of model lipid droplets (LDs). Methods are described for imaging LDs ranging in size from 0.1 to 40 µm in diameter with high-resolution microscopy and spectroscopy. With known LD compositions, membrane binding, sorting, diffusion, and tension were measured via fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging microscopy (FLIM), atomic force microscopy (AFM), and imaging flow cytometry. Additionally, a custom, small-volume pendant droplet tensiometer is described and used to measure the association of phospholipids to the LD surface. These complementary, cross-validating methods of measuring LD membrane behavior reveal the interplay of biophysical processes on lipid droplet monolayers.
Subject(s)
Lipid Droplets , Lipid Droplets/metabolism , Lipid Droplets/chemistry , Microscopy, Atomic Force/methods , Microscopy, Fluorescence/methods , Fluorescence Recovery After Photobleaching/methods , Humans , Flow Cytometry/methods , Spectrometry, Fluorescence/methodsABSTRACT
The mechanisms by which the lipid droplet (LD) membrane is remodeled in concert with the activation of lipolysis incorporate a complex interplay of proteins, phospholipids, and neutral lipids. Model LDs (mLDs) provide an isolated, purified system for testing the mechanisms by which the droplet composition, size, shape, and tension affects triglyceride metabolism. Described here are methods of making and testing mLDs ranging from 0.1 to 40 µm diameter with known composition. Methods are described for imaging mLDs with high-resolution microscopy during buffer exchanges for the measurement of membrane binding, diffusion, and tension via fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging microscopy (FLIM), atomic force microscopy (AFM), pendant droplet tensiometry, and imaging flow cytometry. These complementary, cross-validating methods of measuring LD membrane behavior reveal the interplay of biophysical processes in triglyceride metabolism.
ABSTRACT
Although Kramers' theory for diffusive barrier crossing on a 1D free energy profile plays a central role in landscape theory for complex biomolecular processes, it has not yet been rigorously tested by experiment. Here we test this 1D diffusion scenario with single molecule fluorescence measurements of DNA hairpin folding. We find an upper bound of 2.5 µs for the average transition path time, consistent with the predictions by theory with parameters determined from optical tweezer measurements.
Subject(s)
DNA, Single-Stranded/chemistry , Macromolecular Substances/chemistry , Models, Chemical , Spectrometry, Fluorescence/methods , Diffusion , Nucleic Acid Conformation , Optical Tweezers , ThermodynamicsABSTRACT
We demonstrate optical control of the geometric phase acquired by one of the spin states of an electron confined in a charge-tunable InAs quantum dot via cyclic 2pi excitations of an optical transition in the dot. In the presence of a constant in-plane magnetic field, these optically induced geometric phases result in the effective rotation of the spin about the magnetic field axis and manifest as phase shifts in the spin quantum beat signal generated by two time-delayed circularly polarized optical pulses. The geometric phases generated in this manner more generally perform the role of a spin phase gate, proving potentially useful for quantum information applications.
