Seeming Impossible Biological Problems

Several of these innovations: Multiphoton Microscopy (MPM), Fluorescence Correlation Spectroscopy (FCS), nanoscopic molecular tracking and most recently, nanostructured molecular dynamic probes are being applied to some of these seeming impossible biological problems. Over the years, about 35 of our publications have focused on the challenges of neuroscience, including: molecular mechanisms and physics of auditory transduction, the first successful single channel recording of reconstituted natural ion channels and on their structural fluctuations and mechano-sensitivity, signal delays along neural processes in neural networks, detection and imaging of serotonin and its secretion, imaging the development of the lesions of Alzheimer's Disease in transgenic mice, and most recently (now in press) successful optical imaging of action potentials with time resolutions corresponding to patch clamp recordings which promises to supplement the usual application of MPM to calcium signals as a method of choice for neural response measurements in live neural networks.

Membrane Heterogeneity

Our early emphasis on optical measurement of molecular mobility in cell membranes led to the engineering of Fluorescence Photobleaching Recovery, also called FRAP and later to the first nanoscopic tracking of the individual cell surface receptor molecules in the complete population on living cells, which led eventually to evidence for the membrane heterogeneity now known as "membrane rafts" in the form of our discovery of anomalous subdiffusion and diversity of characteristics of tracking trajectories on the living cell surfaces. We have recently resumed research on the fundamentals of membrane heterogeneity, motivated by the chronic violations of the elementary paradigms of chemical physics in its current biological discussions. We have recently analyzed the behavior of large multiphase bilayer vesicles to measure the interphase energies (line tension) for the first time, detect the effects of the Gaussian curvature energy of membranes and discover the facilitation of vesicle budding by interphase tensions. This research also demonstrated the onset of critical fluctuations in these two-dimensional fluids as the temperature approached the line of critical points where the two phases merge and the energy cost of fluctuations and the interphase tension vanish. It is ironical that the three-dimensional analog of precisely this problem was first observed and studied in our laboratory nearly 40 years ago.

DNA-Protein Crosslinking

We have recently developed and initiated a major collaborative program to apply ultrafast ultraviolet multiphoton excitation of molecular photochemistry to DNA-protein crosslinking. Our objective is to develop efficient methods to utilize this scheme, which has been shown to be 1,000 times faster than existing methods, in order to discover and map the complex population of protein transcription factors that regulate the copying of template DNA, thus initiating protein synthesis during cell growth and duplication for the second set of chromosomes required for successful mitosis. It is thought that at least 2,000 transcription factors are involved in the human genome, but only about 10 have so far been significantly understood. Our initial projects involve measurements of the photochemical specificity of the crosslinking process between the 20 amino acids and 4 nucleotides, as well as their sequence sensitivity in order to determine the generality of the method. Photophysical analysis of the crosslinking mechanisms, starting with our demonstrated femtosecond pulse effectiveness which we attribute to short lifetimes of the first excited state of the nucleotides is a second fundamental objective. Simultaneously, biological research applications of our crosslinking procedures are being applied with our collaborating molecular biologists.

Enzyme Kinetics

We have also recently developed methods for detection and measurements of enzyme kinetics with single molecule sensitivity to measure enzyme kinetics fluctuations, individual particle detection sensitivity and molecular size scaling even to attomolar concentrations, and convenient small volume chemical kinetics with fast enough mixing for one microsecond time resolution (presently we reach about 30 microseconds).

Clinical Medicine

As our biophysical research has evolved, we have come closer to realizing direct applications of our techniques in clinical medicine. Thus, our current multiphoton imaging research focuses on in vivo imaging, particularly on disease states generated in transgenic animal models of human diseases and on potential medical tools such as multiphoton endoscopy. This strategy now impinges on the realm of biomedical engineering.