One of the most powerful and widely used ways to observe structure and motion in unstained living cells and isolated organelles, whether normal or altered by disease, is video-enhanced differential interference contrast light microscopy (VE-DIC), which combines a DIC microscope, a video camera and a computer to improve image contrast. VE-DIC requires the microscopist to manually acquire and store a new background image every time conditions are changed. The goal of this project is to improve VE-DIC by switching the polarization of the light in alternate frames with a novel polarization modulator, then using a computer to subtract background by displaying the difference between alternate frames. The resultant polarization-modulated DIC (PM-DIC) difference image has twice the contrast and twice the signal-to-noise of VE-DIC. Background is automatically computed, which is a significant advantage. The principle underlying PM-DIC has already been verified by processing images off-line. The goal of the current proposal is to assemble and test a system that displays PM-DIC images in """"""""real time"""""""" (30 frames/s) while the specimen is in the microscope. The power of PM-DIC to observe more subtle dynamic features of cells and isolated cellular components will be tested with several biological systems, including the motor protein kinesin and the microtubule cable upon which it moves. In neurons, kinesin plays an especially critical role by transporting vesicles, synaptic membrane proteins, and mitochondria from the cell body to the tip of the axon. It has been shown in Drosophila that kinesin mutations cause neuron disease phenotypes which parallel the pathologies of some vertebrate motor neuron diseases, - including amyotrophic lateral sclerosis.
| Hill, D B; Macosko, J C; Holzwarth, G M (2008) Motion-enhanced, differential interference contrast (MEDIC) microscopy of moving vesicles in live cells: VE-DIC updated. J Microsc 231:433-9 |
| Holzwarth, G M; Hill, D B; McLaughlin, E B (2000) Polarization-modulated differential-interference contrast microscopy with a variable retarder. Appl Opt 39:6288-94 |
