Jason K. King

Maximum-likelihood position sensing and actively controlled electrokinetic transport for single-molecule trapping

A freely diffusing single fluorescent molecule may be scrutinized for an extended duration within a confocal microscope by actively trapping it within the femtoliter probe region. We present results from computational models and ongoing experiments that research the use of multi-focal pulse-interleaved excitation with time-gated single photon counting and maximum-likelihood estimation of the position for active control of the electrophoretic and/or electro-osmotic motion that re-centers the molecule and compensates for diffusion. The molecule is held within a region with approximately constant irradiance until it photobleaches and/or is replaced by the next molecule. The same photons used for determining the position within the trap are also available for performing spectroscopic measurements, for applications such as the study of conformational changes of single proteins. Generalization of the trap to multi-wavelength excitation and to spectrally-resolved emission is being developed. Also, the effectiveness of the maximum-likelihood position estimates and semi-empirical algorithms for trap control is discussed.

Three-dimensional anti-Brownian electrokinetic trapping of a single nanoparticle in solution

A microfluidic device with four electrodes in a tetrahedral arrangement is used to demonstrate three-dimensional (3D) trapping of an individual 40 nm fluorescent nanoparticle in solution. Astigmatic imaging is used to determine the particle position in 3D for real-time control of the electrode potentials, which regulate the magnitude and direction of the electric field and the resulting electrokinetic motion of the particle so as to counteract Brownian diffusion. Trapping within a radius of 5 μm for extended periods (>1 min) is exhibited for particles with diffusivity 5.2 μm2/s and could be improved by increasing the imaging rate of 30 Hz.

Microfluidic Device for the Electrokinetic Manipulation of Single Molecules

We discuss the construction and characterization of a microfluidic device for the electrokinetic manipulation of sub-micron particles. A tetrahedral arrangement of four electrodes with 100-micron separation is used to provide control in three dimensions.

Four-focus single-particle position determination in a confocal microscope

We discuss the capabilities for sub-diffraction, single-nanoparticle position determination in a confocal one- or twophoton microscope with four-focus pulse-interleaved excitation and time-gated single-photon counting. As the technique is scalable to multiple detectors for multi-color observations, it can be used to find the separations of differently colored molecules over a distance range that is complementary to that achievable by FRET. Also, there is a possibility for improved spatial localization by using the nonlinearity of saturation of the excitation or by using the technique together with imaging of the point spread function. Applications of two experimental set-ups for four-focus fluorescence excitation for studies of quantum dots and single-particle manipulation and trapping are also discussed.

Rapid, Single-Molecule Assays in Nano/Micro-Fluidic Chips with Arrays of Closely Spaced Parallel Channels Fabricated by Femtosecond Laser Machining

Cost-effective pharmaceutical drug discovery depends on increasing assay throughput while reducing reagent needs. To this end, we are developing an ultrasensitive, fluorescence-based platform that incorporates a nano/micro-fluidic chip with an array of closely spaced channels for parallelized optical readout of single-molecule assays. Here we describe the use of direct femtosecond laser machining to fabricate several hundred closely spaced channels on the surfaces of fused silica substrates. The channels are sealed by bonding to a microscope cover slip spin-coated with a thin film of poly(dimethylsiloxane). Single-molecule detection experiments are conducted using a custom-built, wide-field microscope. The array of channels is epi-illuminated by a line-generating red diode laser, resulting in a line focus just a few microns thick across a 500 micron field of view. A dilute aqueous solution of fluorescently labeled biomolecules is loaded into the device and fluorescence is detected with an electron-multiplying CCD camera, allowing acquisition rates up to 7 kHz for each microchannel. Matched digital filtering based on experimental parameters is used to perform an initial, rapid assessment of detected fluorescence. More detailed analysis is obtained through fluorescence correlation spectroscopy. Simulated fluorescence data is shown to agree well with experimental values.

Ultrasensitive fluorescence correlation spectroscopy of highly parallelized microfluidic devices

Reducing reagent needs and costs while increasing throughput constitute important needs for assays in pharmaceutical drug discovery. We are developing an ultrasensitive, fluorescence-based detection system in highly parallel microfluidic channels with kHz readout rates in each channel. Prototype microfluidic devices with an array of >150 microchannels have been fabricated by direct femtosecond laser machining of fused silica substrates. A device is placed in a custombuilt, wide-field microscope where a line-generating red diode laser provides uniform epi-illumination just a few microns high across a 500 micron field of view. Single-molecule levels in the probe volumes can be attained by flowing suitably dilute aqueous solutions (~pM) of fluorescently labeled biomolecules through the microchannels. Fluorescence is detected with an electron-multiplying CCD camera allowing readout rates up to 7 kHz for each microchannel. Rapid initial assessment of detected fluorescence signals is performed through digital filtering derived from simulations based on experimental parameters. Fluorescence correlation spectroscopy can then provide more detailed analysis of the sample within each microchannel. Optimized microfluidic devices could be mass-produced in low-cost polymers using imprint lithography.