Development of a Cryogenic Temperature Confocal Laser Scanning Microscope for Investigating Molecular Electric Fields

Abstract

Molecular electric fields and potentials govern the chemical and physical behavior of molecules. In many biological systems, processes involving charge separation and transport such as enzymatic activity [1, 2], photosynthesis [3] and the electrostatic steering of ligands towards active sites, have been attributed to these fields. Gaining a detailed, quantitative description of the effects molecular electric fields play, will provide for a more precise mechanistic understanding of such chemical phenomena. Until now, obtaining information concerning these fields and potentials from experiments has been a problematic task. It is the principle objective of this investigation to quantitatively determine and describe the molecular electric fields at the oxygen binding site in the heme proteins myoglobin and hemoglobin. By utilizing single molecule and hole-burning spectroscopies, Stark effect measurements will be employed; to study the impact internal electric field distributions play. To this end, we have developed and constructed a cryogenic temperature version of a confocal laser scanning microscope; to be used for Stark spectroscopy investigations on these heme proteins using high resolution techniques such as single molecule or hole burning spectroscopy. At this stage, room and low temperature imaging and calibration of the microscope have been performed using 1μm fluorescent beads and AFM grids. The next step entails imaging single molecules of Protoporphyrin IX and Stark investigations of various metal substituted porphyrins in glasses. The primary focus of work thus far has been on assembly and design of the microscope and on sample preparation for use in molecular electric field examinations.1) E.D. Getzoff, et.al, Nature 358, (1992) 347.2) J. P. Hosler, et.al,Biochemistry 35, (1996) 10776.3) A. P. de Silva, et.al.,Chem. Commun., (1999) 163.