Confocal imaging of [Ca2+] in cellular organelles by SEER, shifted excitation and emission ratioing of fluorescence

Abstract

Intracellular calcium signals regulate multiple cellular functions. They depend on release of Ca2+ from cellular stores into the cytosol, a process that appears to be tightly controlled by changes in [Ca2+] within the store. A method to image free [Ca2+] within cellular organelles was devised, which provided the first quantitative confocal images of [Ca2+] inside the sarcoplasmic reticulum (SR) of skeletal muscle. The method exploits, for greater sensitivity, the dual spectral shifts that some fluorescent dyes undergo upon binding Ca2+. It was implemented with mag-indo-1 trapped in the intracellular organelles of frog skeletal muscle and validated showing that it largely monitors [Ca2+] in a caffeine-sensitive compartment with the structure of the SR cisternae. A tentative calibration in situ demonstrated an increase in the dye's dissociation constant, not unlike that observed for other dyes in cellular environments. This increase, together with other characteristics of the ratioing method, placed the half-signal [Ca2+] near 1 mM, a value suitable for cellular stores. Demonstrated advantages of the technique include accuracy (that of a calibrated ratiometric method), dynamic range and sensitivity (from the combination of two spectral shifts), spatial and temporal resolution, and compatibility with a vast array of visible dyes to monitor diverse aspects of cellular function. SEER (shifted excitation and emission ratioing) also provides a [Ca2+]-independent measure of dye concentration in the cell. Store and mitochondrial [Ca2+] ([Ca2+]SR and [Ca2+]mito could be measured separately using the high spatial resolution of SEER. Evolution of [Ca2+]SR was followed upon changes in cytosolic [Ca2+] ([Ca2+]cyto). At [Ca2+]cyto = 100 nM, [Ca2+]mito remained near the lower limit of detection and [Ca2+]SR stabilized at values that were submillimolar according to our tentative calibration. Steady [Ca2+]SR was only slightly higher in 800 nM [Ca2+]cyto, and essentially did not decrease unless [Ca2+]cyto was reduced below 10 nM. While the increase of [Ca2+]SR was limited by loss through Ca2+ release channels, its decrease in low [Ca2+]cyto was largely dependent on leaks through the SR Ca2+ pump.

Figure 1. The basis of SEER

Fluorescence excitation and emission spectra of Ca²⁺-free (blue trace) or Ca²⁺-bound dye (red) determined by spectrofluorometry. Vertical violet and blue lines mark the position on excitation spectra of the argon laser lines (1, at 351 nm and 2, at 364 nm) used in SEER. Boxes 1 and 2 on the rainbow bar at top indicate the fluorescence emission ranges collected for images to be ratioed. The conventional practice would be to divide F₁₁ (obtained with excitation 1 in emission range 1) by F₁₂ (obtained with excitation 1 in emission range 2). By contrast, the SEER ratio is defined as F₁₁/F₂₂. Box 3 marks the collection range for fluorescence F₃₃ of rhod-2, excited at 543 nm, with emission spectrum in pink. Spectral curves reproduced with modifications from http://www.probes.com/servlets/spectra?fileid=1202ca

Figure 2. Spectra and calibration in solution

A, emission spectra measured with the experimental laser scanning microscope, aqueous solutions and chambers. Each point corresponds to emission in successive 10 nm-wide wavelength bands, recorded in solutions with 25 μm mag-indo-1, either free (open symbols), or saturated with Ca²⁺, excited at 351 nm (black) or 364 nm (red). B, data for 351 nm excitation on an expanded wavelength scale, showing an isosbestic point at 425 nm. The ranges represented by boxes 1 and 2 in the diagram at top represent the ranges used for measurements in cells, which did not agree in detail with the optimal ranges in solution. C, titration of the dye by Ca²⁺. Blue symbols: conventional emission ratios, namely ratios of fluorescence F₁₁ (excited at 351 nm in the 10 nm emission range centred at 408 nm) and fluorescence F₁₂ (excited at 351 nm in the 10 nm emission range centred at 492 nm). Green symbols: SEER ratioing, namely fluorescence F₁₁ divided by F₂₂, which is excited at 364 nm in the emission range centred at 492 nm. Dashed curves: best fits by eqn (8), with free parameters R_max/R_min (the asymptotic value) and K_D, the mid-signal concentration. Note that both parameters are more than 3 times greater with SEER.

Figure 3. Validation of SEER

A and B, individual images F₁₁(x,y) and F₂₂(x,y) from a mag-indo-1-stained cell after 30 min in the ‘loading’ solution. C, ratio of A and B. D and E, corresponding images, and F, their ratio, after 2 min in release cocktail. The spatial aspects reverse upon depletion, with transversal structures of high fluorescence in F₁₁ (A) losing their fluorescence after depletion, but gaining high fluorescence in F₂₂ (E). Boxed areas in A–C are reproduced in Fig. 5 for further analysis. Identifiers: 120903b 445 and 451.

Figure 4. Spatial resolution of SEER imaging with mag-indo-1

A and B, averages of four images F₁₁ and F₂₂ at high spatial resolution in a cell with empty store. The sacs are transversal to the fibre axis. C, 3-D rendering of a stack of 15 F₂₂xy scans in same cell, deblurred to improve resolution (see Methods). The transversal structure of high staining is rendered as sacs in doublets. D, electron micrograph of thin section of guppy muscle, superimposed in C at the appropriate scale to show correspondence between the stained structure and SR terminal cisternae. Identifier: 072004a 98. Image D (from Franzini-Armstrong, 1999) was kindly provided by C. Franzini-Armstrong.

Figure 5. The distribution of dye in a stained cell

A–C, images F₁₁(x,y), F₂₂(x,y) and their ratio R, from a cell with loaded store (same as boxed portions of panels A–C in Fig. 3). D, dye concentration D_T (x,y), derived by linear combination of F₁₁ and F₂₂ (eqn (A12)). E, all-pixel histogram of D_T (x,y), which is distributed uni-modally around ∼20 μm. F, the histograms of R in regions with different D_T. Each curve corresponds to areas (x,y) where D_T is in each of the four quartiles of the distribution depicted in E. As D_T increases, R adopts slightly greater values. G, ratio, in same colour scale as in C, but restricted to the well-stained regions (upper quartile). H, [Ca²⁺] derived from G by eqn (1), showing the characteristic doublet structure of SR terminal cisternae. Horizontal bar in magnified area is 2 μm. Colour tables span the range 0–255 for A and B, 0–3.5 for C and E; 0–40 μm for D; 0–700 μm for F. Identifier: 120903b 445.

Figure 6. Calibrations in situ

A and B, F₁₁(x,y) and F₂₂(x,y) images, respectively, from a cell with permeabilized SR exposed to a nominally Ca²⁺-free solution. C, D_T(x,y), derived from A and B by eqn (A12). D, R=F₁₁/F₂₂, masked to areas in the two upper quartiles of the distribution of D_T (‘dye-restricted masking’). Identifier: 031605c_008. E and F, images F₁₁ and F₂₂, respectively, from a cell with intact SR, exposed to an internal solution with 30 μm[Ca²⁺] and 0.5 mm tetracaine. G, D_T(x,y), derived from E and F. H, ratio image, from E and F, masked to the two top quartiles of the distribution of D_T. Identifier: 031505c_014. Colour tables span the range 0–255 for A, B, E and F; 0–50 μm for C and G; 0–4.25 for D and H. I, histograms of the ratio images D (blue) and H (red), with gaussian fits (lines) of means 0.406 and 5.080. J, histogram of the ratio image from a fibre stained with indo-1 and treated as described for E–H. Gaussian fit (line) has mean of 4.98. Identifier: 31805a_024.

Figure 7. Calibrations in situ

A, evolution of mean ratio (R_SR) in a fibre with permeabilized SR exposed to different [Ca²⁺]_cyto as depicted in top diagram. Identifier: 031605b. B, summary results. Each symbol represents the average of R_SR in all images at the same [Ca²⁺]_cyto in the same experiment. Different types of symbols represent different fibres (n = 10). Continuous curve: best fit with eqn (1). The point at 0.7 mm[Ca²⁺] (diamond) is an average of values that decayed during the experiment and was not included in the fit. Best fit parameters: R_min= 0.410, γK_D= 802 μm. R_max was set to 5.08. Curves in dashed trace follow eqn (1) with the same R_min and R_max, and γK_D= 536 or 1068 μm.

Figure 8. SEER resolves mitochondria

A, ratio image of a cell loaded with mag-indo-1 and rhod-2, then membrane-permeabilized and immersed in a 100 nm [Ca²⁺] solution. (Image is masked to the three upper quartiles of D_T.) B, simultaneously acquired image of rhod-2 fluorescence. Note detailed correspondence between low [Ca²⁺] regions in A and rhod-2 stained mitochondria in B, but greater heterogeneity in mitochodrial staining by rhod-2. Identifier: 022504a_11. C, ratio image of a cell loaded with mag-indo-1, membrane-permeabilized and exposed to a cytosolic solution with 100 nm[Ca²⁺] and Mitrotracker green. D, simultaneously acquired image of Mitotracker fluorescence. Again, there is detailed correspondence between mitochondrial staining by Mitotracker and regions of low [Ca²⁺] in C. Identifier:092403b_9. Colour table spans the range 0–3.5 for A and C; 0–255 for B and D.

Figure 9. SR loading at elevated [Ca²⁺]_cyto

A, ratio image after 30 s loading in 100 nm[Ca²⁺]_cyto. Image masked to two upper quartiles of D_T. Note mitochondria in superficial region. B, image in different region of same cell, after 60 s in 400 nm[Ca²⁺]_cyto. C, image obtained immediately after B, at lower magnification, to show ‘cuff’ of mitochondria. D and E, histograms of ratio in images A and B. Lines, best fits by sum of two gaussian terms of means R_m and R_SR. Mean values were: for A, R_m= 1.20, R_SR= 2.02; for B, R_m= 1.12, R_SR= 2.64. Identifiers: 051904a_14, 26 and 27.

Figure 10. Evolution of [Ca²⁺]_SR upon changes in [Ca²⁺]_cyto

Time course of mean ratio R_SR in successive images, while [Ca²⁺]_cyto was changed as depicted at top. Intervals labelled ‘0’ were spent in relaxing solution. [Ca²⁺]_SR on right axis calculated from ratio values according to eqn (1) with parameters listed in legend of Fig. 7. Note very slow decay of [Ca²⁺]_SR upon change to 100 nm[Ca²⁺]_cyto, and rapid decay in relaxing solution. Identifier: 071504a.

Figure 11. Summary of the evolution of [Ca²⁺]_SR

A, mean ratio R_SR measured at set times after the change from relaxing solution (less than 10 nm[Ca²]_cyto) to 100 nm (black) or 800 nm (red), averaged over all comparable experiments (numbers in parentheses). Bars are s.e.m.[Ca²⁺]_SR scaled calculated as described for Fig. 10. Dashed lines: exponential fits, with parameters given in the text. B, evolution of R_SR after reducing [Ca²⁺]_cyto as shown at top. Values from different experiments were averaged as described for A. Note that the changes from high [Ca²⁺]_cyto (green and red) were to a 100 nm [Ca²⁺] solution, and led to a very slow reduction in [Ca²⁺]_SR. The descent from 100 nm [Ca²⁺] (black) was to nominally Ca²⁺-free solution and resulted in faster decay of [Ca²⁺]_SR. C, green symbols, evolution of [Ca²⁺]_SR upon increase to 800 nm[Ca²⁺]_cyto in the presence of 0.5 mm tetracaine. Ratio images obtained at intervals of 5 s. The asymptotic value of the exponential fit (dashed line), 3.76, corresponds to a 3-fold greater [Ca²⁺]_SR than in the absence of tetracaine in 100 nm Ca²⁺ (black) or 800 nm Ca²⁺ (Panel B). Identifier: 031505a_s12.

Figure 12. SR leak pathways include the pump

A, evolution of R_SR (blue), [Ca²⁺]_SR (scale at right) and D_T (grey) upon changing the cytosolic solution from 100 nm to one nominally Ca²⁺-free. Upon the first change, [Ca²⁺]_SR fell rapidly. A second change, after exposing the cell to TBQ, failed to induce decay of [Ca²⁺]_SR. Sets of images obtained every 1.6 s. B–E, images D_T(x,y) at times b–e (calculated by eqn (A12)) showing the bleaching effects of repeated imaging at high frequency. Identifier: 031105b, series 14 and 19.

Figure 13. Pump–leak model of evolution of [Ca²⁺]_SR

A depicts an SR model compartment with a pump and a leak pathway. B, evolution of [Ca²⁺]_SR upon changes in [Ca²⁺]_cyto represented at top of panel. Properties of pump and leak are given in Methods. Both calculations started in the nominally [Ca²⁺]-free and ATP-free relaxing solution, modelled as having [Ca²⁺]= 10 nm and [ATP]= 1 μm. To simulate experimental changes, after 100 nm[Ca²⁺] the system was returned to values representing the relaxing solution. After 800 nm[Ca²⁺] the system was changed to 100 nm[Ca²⁺].