5 Davey Lab
Penn State UniversityContacts:
Paul S. Weiss: stm@psu.edu
Anne Counterman: aec@stm1.chem.psu.edu
Julia Heetderks: jjh272@psu.edu
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The second of two Nikon inverted optical microscopes in the lab, this instrument is also dedicated to study morphology and properties of lipid bilayer vesicles. It is used for both fluorescence and phase contrast (DIC) methods of imaging. Projects on this microscope involve studying the three-dimensional morphology of giant unilamellar vesicles (GUVs), and learning to manipulate vesicle shape using microtubule growth and decay. We have used these methods to reproducibly form shapes not predicted by theory, and are further investigating the physical basis for their existence.
This microscope is used for all tubulin experiments. Unpolymerized tubulin is introduced into vesicles, where it then forms microtubules. This stretches the vesicle membrane to form long, narrow protrusions. The degradation of the microtubules allows a subsequent relaxation of the membrane into a spherical shape, on a measureable time scale. The membrane may temporarily form a semi-stable “pearled” shape during relaxation. Different vesicle components and environmental factors affect this process, giving us some degree of control.
We have also observed free floating GUVs with one or more flattened edges, in contrast to the normal spherical shape. These we call D-shaped vesicles, because in cross section they appear as the letter D. The flat edge is usually composed of a different, higher melting point composition of lipids. This phenomenon is also under investigation for biological significance.
This instrument features a joystick controlled three-axis micromanipulation stage. It is a widefield microscope, but can be used to attain three-dimensional images (similar to confocal microscopy) by capturing multiple images at different focal depths. These are stacked and rendered into a complete vesicle image, viewable from any angle.
References:
D'Onofrio TG, Binns CW, Muth EH, Keating CD, Weiss PS.
2002. Controlling and measuring local composition and properties in lipid
bilayer membranes. Journal of Biological Physics 28(4):605-617.
D'Onofrio TG, Hatzor A, Counterman AE, Heetderks JJ, Sandel
MJ, Weiss PS. 2003.
Controlling and Measuring the Interdependence
of Local Properties in Biomembranes.
Langmuir,
in press.