Lifetime and spectral screen of candidate sensors to create G-Ca-FLITS.

Sensor variants are indicated by their mutations with respect to Tq-Ca-FLITS. The calcium-bound state (sat) is measured in presence of 0.1 mM CaCl2 and the calcium-free state (apo) after addition of 9.5 mM EDTA.

*Emission and excitation maxima are only indicated if a red shift of the spectrum with respect to Tq-Ca-FLITS was observed. See also Figure S3.

**Phase lifetimes were measured at 37 °C by FD-FLIM. The lifetime change is calculated as the lifetime in the calcium-bound state minus the calcium-free state.

Lifetime contrast of improved green versions of Tq-Ca-FLITS.

*’AD’ stands for the mutations V27A and N271D, ‘VS’ for V27 and N271S, ‘AS’ for V27A and N271S.

**The fluorescence lifetime of bacterial lysates was measured at RT with by FD-FLIM after addition of 0.1 mM CaCl2 and after addition of 9.5 mM EDTA. Mean phase lifetimes of the full field of view of the microscope are indicated.

***The fluorescence lifetime of HeLa cells expressing the different sensor variants was measured without stimulation and after addition of 5 μg/mL ionomycin and 5 mM calcium to the medium, measured at 37 °C. The mean and standard deviation (sd) of the fluorescence lifetime of all cells (n) are indicated.

Photophysical properties of G-Ca-FLITS and intermediate variant.

Apo – calcium-free state (10 mM EGTA), sat – calcium-bound state (39 μM free Ca2+), ε - Extinction coefficient, QY – quantum yield with 95% confidence interval between curly brackets.

Excitation and emission spectra of G-Ca-FLITS and Tq-Ca-FLITS.

The lighter lines indicate the calcium-free state (10 mM EGTA) and the darker lines the calcium-bound state (39 μM free Ca2+). The Tq-Ca-FLITS is shown as a reference and was reported before (van der Linden et al., 2021). Spectra are normalized to the maximum of the calcium-free state for G-Ca-FLITS.

Influence of pH on phase lifetime of the proteins.

Fluorescence lifetime of proteins diluted in pH buffer was measured (n=3). In case of G-Ca-FLITS, this was done in presence (gray, 0.1 mM CaCl2) or absence of calcium (black, 5 mM EGTA). A smooth curve is fitted through the data using the loess method, using α = 0.4. The gray band indicates the 95% confidence interval of the smooth fit.

Calcium calibration of G-Ca-FLITS in vitro at 37°C.

(A) The measured fluorescence lifetime of protein isolate of G-Ca-FLITS in a range of calcium concentrations is plotted in a polar space (n=3), with the color indicating the concentration. The measurements fall on a straight line (in gray) on the polar plot between the lowest and highest concentration (indicated by an X). (B) For each measurement, the fraction of sensors in the calcium-bound state (the low lifetime) is determined, taking the intensity contribution of the two extreme states into account. The fraction is plotted against the concentration of free calcium to obtain a calibration curve.

Calcium calibration parameters of G-Ca-FLITS.

The calibration was performed with purified protein at two temperatures. The phase and modulation lifetime of the sensor are indicated for the two states of the sensor. apo – in calcium-free buffer (10 mM EGTA), sat – in buffer with 39 mM free CaCl2, τφ and τM – phase lifetime and modulation lifetime, KD – calcium affinity with 95% confidence interval indicated between curly brackets, n – Hill coefficient with 95% confidence interval indicated between curly brackets.

Brightness analysis of green fluorescent proteins and biosensors in HeLa cells using a co-expressed mScarlet-I.

The ratio GFP/RFP was determined 24 h after transfection both in resting, unstimulated cells (pre) and cell stimulated with 5 μg/mL ionomycin and 5 mM calcium to reach saturation of the sensors (post). The ratios were normalized to cells expressing EGFP. Dots show individual cell data (n ≥ 99) and the large dot the average of each biological replicate (N = 2, except for EGFP; N=3).

Comparison of the signal-to-noise ratio of the phase lifetime of G-Ca-FLITS and Tq-Ca-FLITS in HeLa cells.

HeLa cells were measured in a resting state and after addition of 5 μg/mL ionomycin and 5 mM calcium. Measurements of individual cells are indicated by colored dots (n=18 for G-Ca-FLITS, n=9 for Tq-Ca-FLITS). Lines connect the measurements of the same cell. The average of all cells is shown in black. For each cell, 400 pixels were analyzed for the mean lifetime and intensity, and for the sd of the lifetime. SNR = mean lifetime / sd lifetime. Comparable phase lifetimes are found for all cells expressing a construct but the SNR is lower for lower intensity cells and varies less between the two states for G-Ca-FLITS.

Measuring calcium concentrations in HeLa cells and BOECs using G-Ca-FLITS targeted to various organelles.

(A) Localization of G-Ca-FLITS in HeLa cells and (B) in BOECs. Images are taken with a 63x magnification. (C) Measured calcium concentration in various organelles in HeLa cells and (D) BOECs. Measurements of single cells are indicated by circles, gray for unstimulated cells and black for cells after addition of 5 μg/mL ionomycin and 5 mM calcium. The mean of all cells is indicated by a black line. (E) Changing free calcium concentration after stimulation with 1 μM histamine or addition of 5 μg/mL ionomycin and 5 mM calcium in the cytosol and (F) in the mitochondria. Each line represents a single cell. Arrows indicate the moments of addition.

Quantitative imaging of spontaneous calcium dynamics in mitochondria.

(A, B) FLIM images were acquired with a Leica Stellaris8 every 60 seconds and at the end of the sequence 10 µM digitonin was added to obtain a maximal response. The lifetime images were converted to calcium concentration using the KD determined in vitro and the extremes from the lifetime image. False colors reflect the calcium concentration, according to the scale bar on the left. Calcium image at t=1 (A) and t=11 minutes (B), showing the three regions where calcium concentrations were quantified and displayed over time (C).

Two-photon TCSPC signals from G-Ca-FLITS and Tq-Ca-FLITS, and comparison to jGCAMP7f, in an intact Drosophila brain.

(A) Schematic of a head-fixed live Drosophila melanogaster female, imaged with standard saline for two minutes before perfusing a high [K+] saline variant over the brain.

(B) Single example traces of flies expressing jGCaMP7f (pink, left), Tq-Ca-FLITS (turquoise, center) or G-Ca-FLITS (green, right) in the EPG neurons under the control of R60D05-Gal4. Top row: Tq-Ca-FLITS and G-Ca-FLITS show strong FLIM changes in response to elevated [K+] while jGCaMP7f shows very little. Bottom row: jGCaMP7f and Tq-Ca-FLITS exhibit large changes in fluorescence while G-Ca-FLITS becomes moderately dimmer. Gray region indicates time when high K+ saline was perfused.

(C) Intensity traces from all flies. Vertical scalebars: 0.200 nanoseconds for G-Ca-FLITS and Tq-Ca-FLITS, 5 ΔF/F for jGCaMP7f. Horizontal scalebars: 1 minute.

(D) Fluorescence (left) and FLIM (right) images of example flies from panel B before [K+] elevation (baseline, 2 minute average, top row) and for the 30 second surrounding the peak change in calcium (second row). Lifetime images are masked to show only automatically-determined “foreground” pixels. Third row: photon arrival time histograms for the data of the above rows.

(E-G) Summary statistics for all flies (from panel C). (E) shows changes in fluorescence intensity, (F) shows absolute lifetime measurements, and (G) reflects the change in lifetime from baseline to plateau for each of the three indicators.