In addition to the localization coordinates, other parameters such as the number of photons emitted per molecule, the width of the PSF and the fluorescent background in the immediate vicinity of the detected molecule are recorded from which fluorophore characteristics such as on-off duty cycles and bleaching rates can be extracted.
ArcLight fluorescence from single molecules was recorded by an Evolve 128 EMCCD camera (Photometrics, Tucson, Arizona) attached to a home-built TIRF setup based on an Olympus IX71 inverted microscope (Center Valley, Pennsylvania) with a 60X/1.45 NA microscope objective (Olympus).
Vibration rotational absorption lines of CH4, H2O, CO2 and CO molecules were recorded in the near infrared spectral region.
In each subvolume the number of molecules is recorded at each time step and molecules can jump between neighboring subvolumes to model diffusion.
New time resolved vibrational spectrum of Br2 molecules are recorded using a single 532 nm, pulses of 7 ns duration at 10 Hz repetition rate obtained from Q-switched Nd YAG laser.
Areas covered with individual molecules were recorded at a 50,000 times magnification.
While the fluorescence images of the red mKikGR molecules were recorded with the 568 nm excitation light (100 msec integration, 1 Hz), the 405 nm light was introduced into the microscope between the 568 nm pulses to photoswitch mKikGR molecules from the green to red form.
The green mKikGR molecules were photoswitched into the red mKikGR with a single pulse of 405 nm light, and fluorescence images of individual red mKikGR molecules were recorded every second with 568 nm excitation (Figure 6A). Figure 6B shows a time course of integrated fluorescence intensities.
Concurrently, also aggregates between proteins and salt molecules are recorded.
