Total internal reflection fluorescence microscope optics.
Research
I am broadly interested in living matter and am currently focussing on its mechanical properties and motion at the molecular and cellular levels. Below you can find some more details about projects I am currently working on. Please direct inquiries to aebrown at physics dot upenn dot edu.
Hybrid TIRF/AFM Combines Molecular Specificity with High Resolution
New insights in biology often depend on new technologies that open windows on
nature on ever finer scales. To this end, I am working on a hybrid total internal reflection fluorescence and atomic force
microscopy (TIRF/AFM) that can both image and manipulate biological samples in their native aqueous environment with
nanometer precision.
A cartoon of the experimental geometry of our TIRF/AFM instrument is shown at the right. The flexible AFM cantilever is scanned over the surface to produce an image while a laser is used to excite sample fluorescence through the objective of an inverted optical microscope. In this way, a combined fluorescence (a) and topographic (b) image can be generated. Using specific fluorescent antibodies, TIRF imaging yields spatial and chemical information, in this case revealing the presence of microtubules in a chick axon. In contrast, AFM reveals finer structural details at the growth cone tip and also quantitative height information that can be used to determine the arrangement of microtubule bundles in the axon (white rectangle) that is not clear from the TIRF image alone.
Single Molecule Mechanics
AFM can also be used in force mode to investigate the mechanics of biological
molecules one at a time. In this experiment, a flexible cantilever with a sharp tip is repeatedly brought into contact with
a surface covered in a molecule of interest and retracted. When the tip is retracted, sometimes a non-specifically adsorbed
molecule is extended from the surface. By monitoring the deflection of the cantilever, we can generate a force-extension
curve that contains information about the mechanics of single molecules. Molecules of current interest include cytoskeletal
proteins like spectrin and filamin and fibrinogen, the protein that forms the scaffold of blood clots.
Microtubule Mechanics and Intracellular Transport
Microtubules are stiff biological polymers that form a critical
part of the cellular cytoskeleton. Microtubules are often thought of as relatively static highways used by molecular motors
like kinesin and dynein to transport cargoes from one end of the cell to another. In some cases, this can be a good
approximation, but we have found that microtubules writhe wildly inside of many cell types. These fluctuations are not
thermal, but are in fact driven by the action of molecular motors. The image on the left shows the standard deviation of
intensity at each pixel over the course of a fluorescent movie of microtubules in a drosophila S2 cell. Regions with
significant motion appear brighter giving the cell interior the appearance of an intense flame. Interestingly, these
fluctuations can be harnessed by cellular cargoes to speed their transport so in some cases a better analogy for
microtubules than a system of highways is a chaotic molecular stir-bar to enhance diffusion!