Emil Kartalov

Single-molecule measurements calibrate green fluorescent protein surface densities on transparent beads for use with 'knock-in' animals and other expression systems.

Quantitative aspects of synaptic transmission can be studied by inserting green fluorescent protein (GFP) moieties into the genes encoding membrane proteins. To provide calibrations for measurements on synapses expressing such proteins, we developed methods to quantify histidine-tagged GFP molecules (His6-GFP) bound to Ni-NTA moieties on transparent beads (80-120 microm diameter) over a density range comprising nearly four orders of magnitude (to 30000 GFP/microm2). The procedures employ commonly available Hg lamps, fluorescent microscopes, and CCD cameras. Two independent routes are employed: (1) single-molecule fluorescence measurements are made at the lowest GFP densities, providing an absolute calibration for macroscopic signals at higher GFP densities; (2) known numbers of His6-GFP molecules are coupled quantitatively to the beads. Each of the two independent routes provides linear data over the measured density range, and the two independent methods agree with root mean square (rms) deviation of 11-21% over this range. These satisfactory results are obtained on two separate microscope systems. The data can be corrected for bleaching rates, which are linear with light intensity and become appreciable at intensities > approximately 1 W/cm2. If a suitable GFP-tagged protein can be chosen and incorporated into a knock-in animal, the density of the protein can be measured with an absolute accuracy on the order of 20%.

Integrated microsystems for molecular pathology

We have integrated electronic, optical, magnetic, thermal and fluidic devices into systems to construct useful analysis tools. Over the past several years, we have developed soft lithography approaches to define microfluidic systems in which pico-Liter volumes can be manipulated. These fluidic delivery systems have more recently been integrated with optical and electronic devices. We have also developed thermal control systems with fast (>50oC/s) cooling and heating ramp speeds and excellent accuracy.

Optofluidic integration for medical diagnostics and spectroscopy

We show the capabilities of the applications of microlithography techniques optimized for the microelectronic industry for integrating optics with fluidics and electronics in integrated micro-chips. We also show the opportunities of silicon photonics to generate inexpensive optical systems for data communications and analysis.

Fluorescence apertureless near-field microscope: a step toward imaging information in DNA

Single molecule imaging with optical methods has become an important tool in biophysical studies. However, when imaging molecules at room temperature using far field optics, one can only resolve molecules that are separated by a distance greater than the diffraction limit of the microscope, about 220 nanometers. Near field techniques have allowed researchers to image with resolutions on the order of 30-50 nanometers. However, there are numerous reasons to try to push the resolution limit further. One that particular concerns our group is the notion to try to image information in DNA in order to measure sequence information. To that end, we have developed a new type of near field microscope, the fluorescence apertureless near field microscope.