Jonas Korlach

Fluorescence correlation spectroscopy with single molecule sensitivity on cell and model membranes.

We report on the successful application of fluorescence correlation spectroscopy (FCS) to the analysis of single fluorescently labeled lipid analogue molecules diffusing laterally in lipid bilayers, as exemplified by time traces of fluorescence bursts of individual molecules entering and leaving the excitation area. FCS measurements performed on lipid probes in rat basophilic leukemia cell membranes showed deviations from two-dimensional Brownian motion with a single uniform diffusion constant. Giant unilamellar vesicles were employed as model systems to characterize diffusion of fluorescent lipid analogues in both homogeneous and mixed lipid phases with diffusion heterogeneity. Comparing the results of cell membrane diffusion with the findings on the model systems suggests possible explanations for the observations: (a) anomalous subdiffusion in which evanescent attractive interactions with disparate mobile molecules modifies the diffusion statistics; (b) alternatively, probe molecules are localized in microdomains of submicroscopic size, possibly in heterogeneous membrane phases.

Conformational changes of calmodulin upon Ca2+ binding studied with a microfluidic mixer

A microfluidic mixer is applied to study the kinetics of calmodulin conformational changes upon Ca2+ binding. The device facilitates rapid, uniform mixing by decoupling hydrodynamic focusing from diffusive mixing and accesses time scales of tens of microseconds. The mixer is used in conjunction with multiphoton microscopy to examine the fast Ca2+-induced transitions of acrylodan-labeled calmodulin. We find that the kinetic rates of the conformational changes in two homologous globular domains differ by more than an order of magnitude. The characteristic time constants are ≈490 μs for the transitions in the C-terminal domain and ≈20 ms for those in the N-terminal domain of the protein. We discuss possible mechanisms for the two distinct events and the biological role of the stable intermediate, half-saturated calmodulin.

Nanofabricated Device for Fluorescence Correlation Spectroscopy in Sub-Femtoliter Volumes

We present fabrication and testing of a microfluidics/integrated optics device. We present a fabrication method for microfluidic devices and in the development of new tools for fluorescence based experiments. The fabrication technique, relying on the use of sacrificial layers to form microfluidic circuitry is fully compatible with current processing techniques of the semiconductor industry. This bonding-free method opens the door to the integration of conventional CMOS electronics with microfluidics systems.

Zero-mode waveguides for single molecule analysis and fast DNA sequencing

Summary form only given. Zero-mode waveguides enable the observation of which fluorescent nucleotide analog is being added to the double stranded product in the active site of the enzyme while excluding signal from freely diffusing fluorescent species. The effectiveness of zero-mode waveguides is demonstrated by real-time observation of single molecules of DNA polymerase immobilized inside zero-mode waveguides, using micromolar concentrations of fluorescently labeled nucleotide analogs.

Confinement of Fluorescence Excitation for Single Molecule Detection at High Concentrations

Nanofabrication techniques are employed to make metal-clad zero-mode optical waveguides to confine the excitation volume in single-molecule fluorescence detection experiments. Fluorescence correlation spectroscopy is used to characterize the effective volume of the system, yielding a smallest observed volume of 20 zeptoliters (20×10−21 liters).

DNA Fragment Sizing and Fluorescence Correlation Spectroscopy in Nanofabricated Channels.

A device is fabricated by our sacrificial layer technique [1]. It is used to show how this method provides a way to measure fluorescence from single molecules with a good signal to noise ratio [2]. The size of single DNA molecules is measured by using fluorescence bursts. The channel with submicron dimension allows to probe all the solution that flows through the channel and to have all the molecules in focus on the detector.