Supplementary document for Alignment and Characterisation of Remote-Refocusing Systems - 6600964.pdf

Supplementary Materia

Media 2: Fibre-coupled multiphoton microscope with adaptive motion compensation

Originally published in Biomedical Optics Express on 01 May 2015 (boe-6-5-1876

Media 1: Fibre-coupled multiphoton microscope with adaptive motion compensation

Originally published in Biomedical Optics Express on 01 May 2015 (boe-6-5-1876

Fluorescence decay parameters obtained from the fitting of ΔC11CFP and mTFP1.

<p><τ> is the average lifetime calculated as described by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049200#pone.0049200.e007" target="_blank">equation (7)</a>. χ² is the fit quality criterion calculated by the fitting software (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049200#pone.0049200.e006" target="_blank">equation (6)</a>). Errors are the 67% confidence intervals returned by the TRFA analysis software. FP: fluorescent protein.</p

Fluorescence Lifetime Readouts of Troponin-C-Based Calcium FRET Sensors: A Quantitative Comparison of CFP and mTFP1 as Donor Fluorophores

<div><p>We have compared the performance of two Troponin-C-based calcium FRET sensors using fluorescence lifetime read-outs. The first sensor, TN-L15, consists of a Troponin-C fragment inserted between CFP and Citrine while the second sensor, called mTFP-TnC-Cit, was realized by replacing CFP in TN-L15 with monomeric Teal Fluorescent Protein (mTFP1). Using cytosol preparations of transiently transfected mammalian cells, we have measured the fluorescence decay profiles of these sensors at controlled concentrations of calcium using time-correlated single photon counting. These data were fitted to discrete exponential decay models using global analysis to determine the FRET efficiency, fraction of donor molecules undergoing FRET and calcium affinity of these sensors. We have also studied the decay profiles of the donor fluorescent proteins alone and determined the sensitivity of the donor lifetime to temperature and emission wavelength. Live-cell fluorescence lifetime imaging (FLIM) of HEK293T cells expressing each of these sensors was also undertaken. We confirmed that donor fluorescence of mTFP-TnC-Cit fits well to a two-component decay model, while the TN-L15 lifetime data was best fitted to a constrained four-component model, which was supported by phasor analysis of the measured lifetime data. If the constrained global fitting is employed, the TN-L15 sensor can provide a larger dynamic range of lifetime readout than the mTFP-TnC-Cit sensor but the CFP donor is significantly more sensitive to changes in temperature and emission wavelength compared to mTFP and, while the mTFP-TnC-Cit solution phase data broadly agreed with measurements in live cells, this was not the case for the TN-L15 sensor. Our titration experiment also indicates that a similar precision in determination of calcium concentration can be achieved with both FRET biosensors when fitting a single exponential donor fluorescence decay model to the fluorescence decay profiles. We therefore suggest that mTFP-based probes are more suitable for FLIM experiments than CFP-based probes.</p> </div

Titration model parameters.

<p>These parameters are obtained from fitting the theoretical titration model to the dataset. The error on the concentration measured at [<i>Ca²⁺</i>]<i> = 1 µM</i> is shown in the last column on the right.</p

Temperature dependence of the average fluorescence lifetime of ΔC11CFP and mTFP1.

<p>A triple exponential model was used to describe ΔC11CFP fluorescence decays and a double exponential model for mTFP1. The average lifetimes were calculated using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049200#pone.0049200.e007" target="_blank">equation (7)</a>. Errors are the 67% confidence intervals returned by the TRFA analysis software. The straight lines represent the linear regression of the corresponding dataset with a slope of −0.048 ns/°C for ΔC11CFP and −0.019 ns/°C for mTFP1.</p

Fluorescence decays of eGFP at 21°C.

<p>(A) eGFP from cytosol preparation of HEK293T cells expressing eGFP. (B) purified eGFP. IRF: Instrument Response Function. Res. : residuals.</p

Fluorescence decays of TN-L15.

<p><b>(A) and mTFP-TnC-Cit (D) in 0 µM and 40 µM of free calcium.</b> The resulting fit quality criteria (χ²) from the global fits are 1.128 and 1.316 for the TN-L15 and mTFP-TnC-Cit datasets respectively. (B) and (C) show the corresponding fit residuals at 0 µM and 40 µM for TN-L15. (E) and (F) show the fit residuals for mTFP-TnC-Cit at 0 µM and 40 µM. Res. : residuals.</p

TN-L15 and mTFP-TnC-Cit fluorescence lifetimes obtained from a single exponential fit.

<p>The fitting criteria are χ² = 22.867 for TN-L15 and χ² = 4.569 for mTFP-TnC-Cit. The error bars show the standard deviation calculated from three replicate decay acquisitions.</p