Dynamic FRET example videos related to "Mars, a molecule archive suite for reproducible analysis and reporting of single-molecule properties from bioimages"

Videos of dynamic switching between iso-I and iso-II conformations of a holiday junction at 50 mM Magnesium resulting in high and low FRET from Cy3 and Alexa647 labels positioned on the arms. Holiday junctions are surface immobilized through a biotin attachment and imaged using TIRF microscopy. The camera sensor is split using a dual view so that the acceptor emission is on the top and the donor emission is on the bottom. Videos from each position are provided as compressed zip files containing a sequence of tif files and associated metadata text file. Image sequences were collected using Micro-Manager 2.0 using ALEX or alternating laser excitation with alternating 637 and 532 pulses separated as two different channels. Beam profile images are provided for 637 and 532 excitation allowing for correction of the non-uniform beam profiles. The following 2D affine transformation matrix can be used to transform from the top acceptor emission region to the bottom donor emission region during processing.

Affine 2D transformation from top to bottom: (m00, m01, m02, m10, m11, m12), (1.00276, 0.000208, 1.01236, 0.000267, 1.00312, 507.21025)

A detailed image processing workflow for this dataset using Mars can be found under the example section at https://duderstadt-lab.github.io/mars-docs/ or directly at https://duderstadt-lab.github.io/mars-docs/examples/FRET_dynamic/