David DeRosier, Ph.D.
Abraham S. and Gertrude Burg
Chair of Life Sciences
Cryo-PALMing the Synapse
Science is firmly rooted in observation, and it is observation that ultimately serves as the foundation of the scientific method. For centuries light microscopy has allowed biologists to observe the structure or form present at very fine scales enabling them to hypothesize how these observed structures might be linked to important life functions. So it is appropriate that the last speaker at this year's retreat was Dr. David DeRosier, who has been developing ways to enhance light microscopy that may allow us to observe biological processes on an even smaller scale than previously thought possible.
Dr. DeRosier's work centers on determining the organization for the hundreds of different proteins at the synapse, a question that is of great interest in the field of neuroscience. A fundamental problem with examining the location of these proteins lies within the physical limitations of various imaging techniques. For example, the electron microscope is limited in its ability to produce images of "large" structures like the synapse. Furthermore, electron microscopy severely restricts a researcher's ability to examine proteins of interest. However, electron microscopy can provide images of the morphological features at molecular resolution. Conversely, super-resolution, fluorescence light microscopy, not being limited by Rayleigh resolution, can visualize at least two fluorescently labeled proteins at once, as well as survey many synapses in a single field of view and can localize the labeled proteins within a few nanometers. However, it cannot visualize the protein components but, rather, only determine their locations. Thus a combination of the two methods provides what each method alone cannot. Dr. DeRosier is developing a cold stage that will enable researchers to carry out super-resolution fluorescence microscopy while preserving the specimen for subsequent electron cryo-microscopy.
Super-resolution fluorescence light microscopy has one main underlying idea: the centroid of a photon can be mapped based on the distribution of light coming from a single fluorophore. This method works best at extremely cold temperatures. Lowering the temperature increases the number of photons each fluorophore emits prior to bleaching. The large increase in the number of photons means an improvement in the precision of fluorophore localization. To accomplish this goal, Dr. DeRosier and colleagues have designed a cold stage, which cools the specimen during imaging but allows the user to operate the microscope at room temperature. The cryo-stage, when complete, will be compatible with a conventional upright fluorescence microscope. The plan is to set up a steady-state situation in which cold gas flows under the specimen to keep it cold, while room-temperature immersion water flows between the objective and the cover slip so as to prevent the water from freezing. The stage consists of a copper finger cooled by cold-flowing nitrogen gas. The frozen-hydrated sample sits atop the copper finger. A collar is fitted over the objective lens, and the immersion water flows between the objective lens and the cover slip, which forms the bottom of the collar. The objective and cover slip move as a unit relative to the specimen. A cryogenic liquid optically couples the bottom of the cover slip to the frozen-hydrated sample so that the full numerical aperture of the objective lens is available. Dr. DeRosier has built a prototype version of this stage and is currently in the process of testing it.