DOI: 10.5061/dryad.zkh1893nd
title: Quantitative comparison of fluorescent proteins using protein nanocages in live cells

description: To standardize comparison of fluorescent protein performance on a
 molecule-by-molecule basis in a physiological intracellular environment,
 we constructed fluorescent protein tagged I3-01 peptides that
 self-assemble into stable sixty subunit dodecahedrons inside live
 mammalian cells. We were especially interested in determining which of the
 recently published monomeric StayGold variants is best for live microscopy
 in mammalian cells. Combining nanocage brightness and photobleaching
 measurements into a single metric, mStayGold stood out as far superior to
 all other green and red fluorescent proteins we tested with a functional
 lifetime that is at least 8-10-fold longer compared with EGFP or mEmerald.
 Analysis of intracellular nanocage diffusion further confirmed the
 monomeric nature of mStayGold and we demonstrate that mStayGold-tagged
 nanocages can serve as highly photostable nanoparticles to analyze
 intracellular biophysical properties. Analysis of frequently used red
 fluorescent proteins was less encouraging and recent mScarlet or mRuby
 variants did not perform substantially better than mCherry on a typical
 spinning disk confocal microscope system, highlighting the importance of a
 standardized method to benchmark fluorescent proteins to make optimal
 choices for specific experimental setups.
description: # Quantitative comparison of fluorescent proteins using protein nanocages
 in live cells #### Description This dataset contains raw data and analysis
 used to produce figures and conclusions associated with the Journal of
 Cell Science paper
 ([https://doi.org/10.1242/jcs.263858](https://doi.org/10.1242/jcs.263858)). A detailed description of the data and analysis methodology can be found in the associated manuscript. Raw 16-bit microscopy data are provided as Nikon ND2 files that are native to Nikon Elements microscopy software. ND2 files can also be opened with ImageJ/Fiji with the Bioformats plugin (Note: microscopy metadata may not be imported correctly into ImageJ), or with a freely available Nikon Elements Viewer. Data analysis on which manuscript figures are based is provided as Microsoft Excel spreadsheets. Data are organized by figure panel in the manuscript with .ZIP files containing both raw data and analysis for each figure. See manuscript for full experimental detail on each figure. Outlined below is where to find which kind of data with additional explanation on Excel spreadsheets containing all data used to generate the corresponding figure panels. #### Figure_1 **Fig1_F_G_H.zip**: Organized into folders named with the green fluorescent protein name containing subfolders relating to independent experiments. Data are short z-stacks of D-Mannitol treated RPE cells expressing the indicated FP-tagged I3-01 nanocages used to quantify nanocage fluorescence intensity by 2D Gaussian fitting as described in the manuscript. In this folder, **Fig1_G.xlsx**: Individual nanocage fluorescence intensity measurements of the five green fluorescent proteins as indicated organized into groups of 10 indicating measurements in different cells and groups of three indicating different experiments (identified by different colors: black, red, green). In this folder, **Fig1_H.xlsx**: Total fluorescence intensity measurements of the same cells and average nanocage intensity measurements per cell used to estimate the number of I3-01 nanocages per cell. **Fig1_I.zip**: Subfolders contain short z-stacks of D-Mannitol treated RPE cells expressing the indicated single- and tandem-tagged mEmerald and mStayGold I3-01 nanocages used to quantify nanocage fluorescence intensity by 2D Gaussian fitting as described in the manuscript. in this folder, **Fig1_I.xlsx**: Individual nanocage fluorescence intensity measurements of the indicated single- and tandem-tagged mEmerald and mStayGold I3-01 nanocages organized into groups of 10 indicating measurements in different cells. #### Figure_2 **Fig2_B_C.zip**: In this folder, **Fig2_B_C.xlsx**: Each tab is named for the green fluorescent protein expressed and contains whole cell intensity measurements for the cells indicated in columns B-U with elapsed time in column A. Columns W-AB contain single exponential decay fitting results: rate constant k, starting intensity x0, baseline xs (converging to the camera dark offset), the goodness of fit rsquare, the half-life th = ln(2) / k, and the number of outliers. **Fig2_D_E_F.zip**: Organized into folders containing data for each of the single and tandem mEmerald and mStayGold nanocages measured. Each folder contains subfolders with the TIRF microscopy time-lapse series to measure nanocage diffusion and the corresponding nanocage tracking data output from u-track ([https://github.com/DanuserLab/u-track](https://github.com/DanuserLab/u-track)). The Matlab 'Channel_1_tracking_result.mat' files contain the list of tracks with the nanocage coordinates saved in the tracksCoordAmpCG tab. Within this tab in each group of eight, the first two numbers represent the x and y coordinates for each timepoint in a given track used to calculate nanocage mean square displacement curves. Please see manuscript for additional details on nanocage tracking. In this folder, **Fig2_E.xlsx**: Tabs contain the single and tandem mStayGold-tagged nanocage average mean square displacement curves for each cell generated using the @msdanalyzer package ([https://tinevez.github.io/msdanalyzer/](https://tinevez.github.io/msdanalyzer/)). Each column is one cell with elapsed time in column A. Please see manuscript for details on MSD analysis. In this folder, **Fig2_F.xlsx**: Each tab contains diffusion coefficients (D), number of tracks analyzed and the 95% confidence interval of the measured diffusion coefficient for the indicated single and tandem mEmerald and mStayGold nanocages determined using the @msdanalyzer package ([https://tinevez.github.io/msdanalyzer/](https://tinevez.github.io/msdanalyzer/)). Please see manuscript for details on MSD analysis. #### Figure 3 **Fig3_B_C.zip**: Organized into folders named with the red fluorescent protein name containing subfolders relating to independent experiments. Data are short z-stacks of D-Mannitol treated RPE cells expressing the indicated FP-tagged I3-01 nanocages used to quantify nanocage fluorescence intensity by 2D Gaussian fitting as described in the manuscript. Note that there are two sets of mCherry data corresponding to the comparison to the mScarlet variants and separately to mRuby3 on different days. In this folder, **Fig_3C.xlsx**: Individual nanocage fluorescence intensity measurements of the five red fluorescent proteins as indicated organized into groups of 10 indicating measurements in different cells and groups of three indicating different experiments (identified by different colors: black, red, green). mScarlet and mRuby data are from different experiments and are compared to the corresponding mCherry measurements from the same day. **Fig3_D.zip**: In this folder, **Fig_3D.xlsx**: Each tab is named for the red fluorescent protein expressed and contains whole cell intensity measurements for the cells indicated in columns B-K with elapsed time in column A. Columns M-R contain single exponential decay fitting results: rate constant k, starting intensity x0, baseline xs (converging to the camera dark offset), the goodness of fit rsquare, the half-life th = ln(2) / k, and the number of outliers.