High-resolution video-enhanced light microscopy

Abstract

Video-enhanced differential interference contrast (VE-DIC) methods can produce high contrast images of isolated macromolecular complexes and cellular fine structures invisible by eye or photographic recording methods through the light microscope. Objects 10 nm - 20 nm wide, much smaller than the wavelength of green light, can be seen at high contrast, although lateral resolution is limited by the Abbe diffraction limit (about 200 nm for 550 green light through objective NA = 1.4). In addition, VE-DIC microscopy can produce thin optical sections through transparent thick specimens with z-axis resolution less than 200 nm. We have been developing this technology to provide video assays (30 frames per sec) of the dynamics of individual molecular complexes. Applications include measurement of the polymerization kinetics of individual 25 nm diameter microtubules in living cells and in reconstituted preparations, the assembly of individual polymers of sickle cell hemoglobin in vitro, the translation of mechanochemical enzymes like dynein and kinesin along individual microtubules in vitro and the movements of individual sister kinetochores during mitosis. Lateral movements of diffraction limited images of spherical particles can be tracked with 1 nm precision. We have used a semi-automatic version of the Gelles tracking algorithm to measure the movements of individual kinetochores and centrosomes in living cells at 1 sec intervals with 100-200 nm precision.