Nanoscope for Individual Molecules in Biophysics and Biotechnology

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Many essential biological signals, ranging from DNA transcription to immunological responses to infection, can be activated and controlled by a single or a few molecules or in the cell; therefore, the capability for measurement of dynamical functions of individual biomolecules in vivo and in vitro is necessary for effective biophysical research. The objective of this award is to create effective instrumentation for observing and understanding these sensitive life processes, and as a byproduct to enhance the molecular sensitivity of analytical microchip biotechnology. Non-invasive optical detection of individual biomolecules and location with nanometer precision and measurement of the dynamics of individual molecular functions in vivo or in vitro is needed. Optical microscopic imaging of fluorescence is a promising approach, but access to single molecule processes is tenuous, although individual molecules have been detectable in special cases for many years. The aim of this award is to develop a user-friendly, benign optical nanoscope for single molecule detection and localization at nanometer scales on living cells for biophysical research. Recent research on fluorescence of individual molecules has revealed photophysical pathology that confounds many biophysical applications at the single molecule level, but fortunately promising research approaches have been discovered that may yield satisfactory optical probes. The optical nanoscope development relies on multiphoton laser microscopy (MPM) and fluorescence correlation spectroscopy (FCS). Combining these methods enhanced by precision nanostructure fabrications, new optimized optics and new software developments provides the basis to design and construct a purpose-designed optical Nanoscope complex for optimized single molecule dynamical measurements using FCS with MPM imaging for nanometer molecular localization in cellular and tissue structures. The equipment to be purchased consists of the components required to develop, construct and optimize the optical nanoscope on a timeline of about three years. The development of single molecule optical probes utilizes photophysical instrumentation to measure optical dynamics from hundreds of picoseconds to hundreds of seconds with time correlated single photon counting, fluorescence correlation spectroscopy, photon burst analysis, and spectral recording, all at the single molecule level. Human resource development comprises an inherent byproduct of this program through interdisciplinary training of participating predoctoral and postdoctoral students and visiting scientists in several disciplines. They can be expected on the basis of our past experience to become effective biophysical researchers as academic faculty, technology managers, and research and development leaders.