Widefield/Total Internal Reflection Microscopy (WF/TIRF)

Our home-built WF/TIRF Setup is equipped with 6 different laser diodes (445 nm, 488 nm, 514 nm, 561 nm, 594 nm, 642 nm) and 3 Andor DU-897 EMCCD cameras including one motorized filterwheel to allow multicolor measurements with up to 4 different dyes. We use a self-written Labview program to control our hardware via an FPGA unit by applying electronic triggers and by using the integrated 400 MHz clock for synchronization. This makes our measurement completely independent of the computer and any additional software-related delays. Two shutters in the widefield and TIRF lightpath can be triggered electronically to alter between widefield and TIRF excitation on a frame by frame basis (up to 400 Hz). Image data from each camera is directly streamed onto individual solid-state drives (SSDs). Thus we can acquire dual-color TIRF image series with very high framerates of ~33 ms, which are only limited by the camera readout times or by low signal-to-noise ratios of the experiment. If switching between WF and TIRF or filterwheels are used the framerates are limited by the respective hardware switching times and are therefore higher than 33 ms. The Setup is as well equipped with a PECON incubation chamber to allow long-term live-cell imaging at 37°C and 5 % CO2 and a motorized translation stage to store cell positions for automated serial measurements. During prolonged imaging, the perfect focus system integrated into the Nikon Eclipse Ti body continuously corrects for potential z-focal drift. The setup is ideally suited for single virus tracing