Another area that we are interested in is using single-molecule fluorescence methods for studying protein folding reactions, protein-protein interactions and protein oligomer formation. Single-molecule methods can be used to probe protein oligomer formation and also, using fluorescence correlation spectroscopy, probe dynamics from the sub-microsecond to tens of seconds timescale. To enable these studies we have built a single-molecule microscope that can operate in FIFO (or time-tagged time-resolved mode, as some prefer to call it). There are a number of advantages to collecting data with this approach. With a recent upgrade to 4-card TCSPC detection system, one can monitor dynamics down to ps resolution. The system we are using was initially built by a WPI undergraduate in the lab, Danielle Jacobsen.
Our system is setup for fully automated operation. Samples can be loaded into 170 micron coverslip bottom microplates (either 96- or 384-well plate format). A syringe pump delivers water to the water objective automatically between wells. An algorithm automatically finds the coverslip position and positions the focal volume a specified distance above the coverslip. The data acquisition routine triggers the Becker&Hickl SPC card and waits for data collection to finish before proceeding to the next well. In this way, very reproducible data can be acquired on a number of different samples with the option to repeat as many times as the user needs.