This part of the project is aimed towards combining Atomic Force Microscopy (AFM) techniques with the fluorescent techniques already in use in this lab.
Atomic Force Microscopy technology allows us to position molecules with angstrom resolution, and measure the forces exerted on the molecule down to piconewtons. These capabilites compliment fluorescence techniques quite well, and the future promises an exciting and ambitious variety of experiments on proteins folding, vesicle fusion, fluoescent polymer properties, . . .
These are ambitious goals, an come with significant challenges. One must remember that the tip itself is a macromolecule, a large chunk of ceramic 10 - 40 nm wide, and we are trying to use that tip to manipulate slightly smaller biomolecules. Imagine trying to tie your shoes with a bowling ball glued to your hand (or even a baseball, if your tip is VERY sharp). With that in mind, here are some of our planned, more modest (?!) projects.
Here you can see photos from our AFM-setup.
Fluorescence as probe:
We know that mechanical rearrangements often accompany biological functions of molecules. The binding of some molecules to their substrates is associated with a change in the shape of the molecule. The question is, do the molecules preferentially bind to substrates in a particular state, or do they bind to the sustrate and drive the shape change.
FRET as ruler:
A typical and now oft performed experiment with the AFM is to stretch a molecule and measure the forces necessary to unfold it. Of course, the distance you've stretched the molecule is also measured, but that distance only refers to the endpoints of the molecule. Nobody knows exactly how different regions within the molecule responds to the applied force. Single FRET molecules tagged to different positions on the molecule, we hope to draw a local map of how individual domains of a molecule respond to applied forces and which ones unfold first.
Fluorescence as sample:
How do mechanical stresses change fluorescent properties? With the AFM, we can attach a flourescent molecule to the surface, stretch it, and watch how the flourescent and spectral properties change as a function of the applied load. This type of experiment has application in investigating and evaluating a growing number of fluoescent and/or conducting polymers being developed for commercial applications.