Dataset DOI: 10.5061/dryad.9ghx3ffrh

Single-molecule fluorescence images of individual Cy5-labeled enzyme molecules binding and unbinding from cellulose fibrils were imaged using 488 nm and 637 nm excitation laser reflected by a multiline dichroic mirror (FF500/646-Di01, Semrock) and focused at the back aperture of a 1.49 NA 60X oil-immersion objective (Olympus) to provide total-internal-reflection (TIR) excitation at the cover glass / water interface across a ~50 µm diameter area in the object plane. Sample emission was collected and imaged by the same objective onto the 512x512-pixel sensor of an electron multiplying CCD (EMCCD) camera (Photonmax, Princeton Instruments). A 37 nm wide bandpass filter centered at 676 nm was used to isolate Cy5 fluorescence excited at 637 nm. For the binding time measurements, the overall magnification of the imaging system (73X) mapped each EMCCD pixel to a 220x220 nm area in the object plane.  Relatively low power (~0.2 mW @ 637 nm) excitation was used to reduce the effect of Cy5 photobleaching on the binding time measurements. Under similar imaging conditions, fluorescence lifetimes of most (90%) of Cy5 fluorophores exhibited an extended characteristic decay time of 1101 s in the GODCAT oxygen scavenging buffer system (Supplemental Information 1). Image stacks were collected at EMCCD camera integration times of 1 second.

The table below indicate enzyme and reaction conditions associated with each datafile.

Description of the data and file structure

Descriptions of the data and file structure are provided in the attached Datafile_structure.csv

Code/software

Both .spe and .tif files contain image stacks (multiple images collected in sequence over a period of time) that can be directly opened in ImageJ (File->Open and browse to select file, or drag and drop). The image stacks can be played to watch movies of fluorescently-labeled cellulose binding and unbinding on the surface with cellulose fibrils. It is helpful to match brightness and contrast of the images (Image->Adjust->Brightness/Contrast) when comparing multiple data sets.