DOI: 10.5061/dryad.zkh1893pv
title: Data from: Quantitative single-molecule FLIM and PIE-FRET imaging of biomolecular systems

description: The structural dynamics of proteins and nucleic acids are critical for
 their function in many biological processes, but investigating these
 dynamics is often challenging with traditional techniques. Time-correlated
 single photon counting (TCSPC) coupled with confocal microscopy is a
 versatile biophysical tool that enables real-time monitoring of
 biomolecular dynamics in a variety of systems, across many timescales.
 Quantitative single-molecule time-resolved fluorescence methods are
 uniquely positioned to investigate transient interactions and structural
 changes, yet application in complex biological systems remains limited by
 technical and analytical challenges. Combining fluorescence lifetime
 imaging microscopy (FLIM) with pulsed interleaved excitation Förster
 resonance energy transfer (PIE-FRET) offers a robust approach to overcome
 these barriers, enabling accurate distance measurements and dynamic
 studies across diverse sample types. In this study, we described practical
 workflows for implementing FLIM/PIE-FRET for quantitative measurements of
 nanoscale distances and dynamic processes in various biomolecular systems
 on a commercial microscope. Benchmark DNA constructs, RNA/DNA hybrids,
 liposome-encapsulated enzymes, and live Saccharomyces cerevisiae strains
 were prepared and imaged. Correction factors for FRET efficiency recovery
 were determined from diffusion-based experiments, and results were
 validated by direct comparison of intensity- and lifetime-based analyses.
 This data set contains raw TCSPC data (.ptu) or exported fluorescence
 intensity traces (.dat) for the different biomolecular samples.
description: # Data from: Quantitative single-molecule FLIM and PIE-FRET imaging of
 biomolecular systems Dataset DOI:
 [10.5061/dryad.zkh1893pv](https://doi.org/10.5061/dryad.zkh1893pv) ##
 Description of the data and file structure We describe practical workflows
 for implementing FLIM/PIE-FRET for quantitative measurements of nanoscale
 distances and dynamic processes in various biomolecular systems on a
 commercial microscope. ### Files and variables #### File:
 Hi_10pM_17_0712_acc.dat **Description:** High FRET benchmark DNA
 single-molecule acceptor trace  #### File: Hi_10pM_17_0712_donor.dat
 **Description:** High FRET benchmark DNA single-molecule donor trace  ####
 File: Lo_10pM_23_0821_DO.dat **Description:** Lo FRET benchmark DNA
 single-molecule donor only trace  #### File: Hi_10pM_27_0712_DO.dat
 **Description:** Hi FRET benchmark DNA single molecule donor only trace 
 #### File: Lo_10pM_27_0712_acc.dat **Description:** Lo FRET benchmark DNA
 single-molecule acceptor trace  #### File: Mid_10pM_28_0720_acc.dat
 **Description:** Mid FRET benchmark DNA single-molecule acceptor trace 
 #### File: Lo_10pM_27_0712_donor.dat **Description:** Lo FRET benchmark
 DNA single-molecule donor trace  #### File: Mid_10pM_28_0720_donor.dat
 **Description:** Mid FRET benchmark DNA single-molecule donor trace  ####
 File: Lo_10pM_28_0821_DO.dat **Description:** Lo FRET benchmark DNA
 single-molecule donor only trace  #### File: \__86_HighFRET.dat
 **Description:** encapsulated MutL single-molecule trace, High FRET ####
 File: None_99_100ms.dat **Description:** Mid FRET RNA/DNA hybrid substrate
 single-molecule trace #### File: Cluster_3_T100s_1.ptu
 **Description:** Nop7-GFP/mCherry-Rea1 cells #### File:
 Single_3_T61s_1.ptu **Description:** Nop7-GFP/mCherry-Rea1 cell #### File:
 800au_Hi_45min_3.ptu **Description:** Hi FRET benchmark DNA diffusing
 PIE-FRET, collected for 45 min, 2 nM #### File: 800au-45min_2nM_1.ptu
 **Description:** Mid FRET benchmark DNA diffusing PIE-FRET, collected for
 45 min, 2 nM #### File: Lo-800au-45min_2nM_2.ptu **Description:** Lo FRET
 benchmark DNA diffusing PIE-FRET, collected for 45 min, 2 nM #### File:
 Nop7_1_T0s_1.ptu **Description:** Nop7-GFP cell donor only #### File:
 Rea1-mCherry_1_T0s_1.ptu **Description:** Rea1-mCherry cell acceptor only
 #### File: JC_2colorPIE_20MHZ_PBS.mat **Description:** Profile needed to
 load .ptu files into PAM ## Code/software Open-source PIE Analysis with
 MATLAB (PAM) software
 ([https://pam.readthedocs.io/en/latest/GettingStarted.html](https://pam.readthedocs.io/en/latest/GettingStarted.html)) can open and process .ptu files. PAM software can be downloaded from GitLab ([https://gitlab.com/PAM-PIE/](https://gitlab.com/PAM-PIE/)). ## Access information Other publicly accessible locations of the data: * n/a Data was derived from the following sources: * SymPhoTime64 Software