The technique development proposed here will result in new optical microscopy methods that have sub-20 nm spatial resolution, single molecule detection limits, Angstrom level sectioning capabilities, simultaneous fluorescence and force sensitivity, and the ability to carry out all of these measurements on living cellular systems under physiological conditions. The goal is to develop new techniques and methods that combine attributes from near-field scanning optical microscopy (NSOM), atomic force microscopy (AFM), and fluorescence resonance energy transfer (FRET) to enhance imaging capabilities for single molecule studies viable biological tissue. The NSOM/AFM and NSOM/FRET probes to be developed in specific aims 1 and 2 will be compatible with many commercial AFM instruments, adding important new contrast mechanisms for those currently conducting AFM measurements. Once tested and validated on control samples, the nanotechnology developed under specific aims 1 and 2 will be applied to studies on the nuclear membrane in specific aim 3. The high resolution and sensitivity of these techniques will be utilized to unlock key outstanding issues concerning the role and identity of the """"""""plug"""""""" observed in nuclear pore complexes, the plug response to lumenal calcium depletion, the distribution and correlation of nuclear pores with calcium channels in the membrane, and the dynamics of plug motion in the nuclear pore complex. These experiments will be done on viable nuclear membranes under physiological conditions, something heretofore not possible with the near-field technique
