caFromRatio {CalciOMatic}R Documentation

Get Calcium Concentration From Fluorescence Signals, Using the Ratiometric Transformation

Description

The function caFromRatio applies the ratiometric transformation to vectors of fluorescence (including background fluorescence) and returns the corresponding intracellular calcium concentration.

Usage

caFromRatio(adu_B_340, adu_340,
            adu_B_380, adu_380,
            T_340 = 0.015, T_380 = 0.006,
            P, P_B,
            R_min = 0.136, R_max = 2.701, K_eff = 3.637,
            Plot = FALSE)

Arguments

adu_B_340 a vector of background fluorescence values (photon counts) recorded at 340 nm
adu_340 a vector of fluorescence values recorded at 340 nm
adu_B_380 a vector of background fluorescence values recorded at 380 nm
adu_380 a vector of fluorescence values recorded at 380 nm
T_340 the exposure time at 340 nm (in s)
T_380 the exposure time at 380 nm (in s)
P the number of pixels of the Region Of Interest (ROI)
P_B the number of pixels of the Background Region
R_min the minimum fluorescence ratio between the measurements at 340 and 380 nm. This parameter is obtained from calibration experiments
R_max the maximum fluorescence ratio between the measurements at 340 and 380 nm. This parameter is obtained from calibration experiments
K_eff the effective dissociation constant of the dye in the cell (in muM). This parameter is obtained from calibration experiments
Plot a logical value: Set to TRUE to plot the calcium transient estimated by the ratiometric transformation

Details

The calcium imaging technique makes use of the ability of a fluorescent dye (e.g. Fura) to bind with calcium ions present inside the cell. Briefly, the photons emitted by the calcium-free and calcium-bound forms of the dye are recorded by a CCD camera, following the illumination of the tissue by a light source at relevant wavelengths (corresponding to maxima of excitation of the free and bound forms of the dye). In the case of a ratiometric dye, an algebraic relationship links the intracellular calcium concentration to the photon counts at both wavelengths (340 and 380 nm, in the case of Fura-2). It is thus possible to retrieve the intracellular calcium concentration from the ratio of the photon counts recorded at these two wavelengths (after subtraction of the background fluorescence): This is the ratiometric transformation. The ratio R is defined as:

R = (1/P*adu_340-1/P_B*adu_B_340)/(1/P*adu_380-1/P_B*adu_B_380)*T_380/T_340 = (R_min*K_eff+R_max*[Ca^2+])/(K_eff+[Ca^2+]).

Then, the intracellular calcium concentration is given by:

Ca^2+ = K_eff * (R-R_min) / (R_max-R)

Value

A vector of intracellular calcium concentration calculated with the ratiometric transformation described above. This vector comes with the estimated covariance matrix as attribute (see Joucla et al. (2009) for more details).

Author(s)

Sebastien Joucla sebastien.joucla@parisdescartes.fr

References

Joucla S, Pippow A, Kloppenburg P and Pouzat C (2009) Quantitative estimation of calcium dynamics from ratiometric measurements: a direct, non-ratioing, method, Journal of Neurophysiology, in revision

Examples

## (0) "Experimental" parameters

## Parameters of the monoexponential calcium transient
tOn <- 1
Time <- seq(0,10,0.1)
Ca0 <- 0.10
dCa <- 0.25
tau <- 1.5

## Calibration parameters
R_min <- list(value=0.136, mean=0.136, se=0.00363, USE_se=TRUE)
R_max <- list(value=2.701, mean=2.701, se=0.151,   USE_se=TRUE)
K_eff <- list(value=3.637, mean=3.637, se=0.729,   USE_se=TRUE)
K_d   <- list(value=0.583, mean=0.583, se=0.123,   USE_se=TRUE)

## Experiment-specific parameters
nb_B    <- 5
B_T     <- 100.0
T_340   <- 0.015
T_380   <- 0.006
P       <- 1000
P_B     <- 1000
phi     <- 1.25
S_B_340 <- 100/P/T_340
S_B_380 <- 100/P/T_380

## (1) Create a monoexponential calcium decay
Ca_Mono <- caMonoExp(t = Time, tOn = tOn,
                     Ca0 = Ca0, dCa = dCa, tau = tau)

## (2) Create the background and transient fluorescence signals
adu_B_340 <- rep(fluo(Ca=rep(0,nb_B),
                      R_min=R_min$value, R_max=R_max$value,
                      K_eff=K_eff$value, K_d=K_d$value,
                      B_T=0, phi=phi, S_B=S_B_340, T_stim=T_340, P=P, P_B=P_B))
  
adu_340 <- rep(fluo(Ca=Ca_Mono,
                    R_min=R_min$value, R_max=R_max$value,
                    K_eff=K_eff$value, K_d=K_d$value,
                    B_T=B_T, phi=phi, S_B=S_B_340, T_stim=T_340, P=P, P_B=P_B))

adu_B_380 <- rep(fluo(Ca=rep(0,nb_B),
                      R_min=1, R_max=1, K_eff=K_eff$value, K_d=K_d$value,
                      B_T=0, phi=phi, S_B=S_B_380, T_stim=T_380, P=P, P_B=P_B))
  
adu_380 <- rep(fluo(Ca=Ca_Mono,
                    R_min=1, R_max=1, K_eff=K_eff$value, K_d=K_d$value,
                    B_T=B_T, phi=phi, S_B=S_B_380, T_stim=T_380, P=P, P_B=P_B))

## (3) Get the noisy calcium transient from the ratiometric transformation
Ca <- caFromRatio(adu_B_340, adu_340,
                  adu_B_380, adu_380,
                  T_340 = 0.015, T_380 = 0.006,
                  P, P_B,
                  R_min = R_min, R_max = R_max, K_eff = K_eff,
                  Plot = TRUE)

## (4) Superimpose the original calcium transient
lines(Ca_Mono, lty=2, col="red")

[Package CalciOMatic version 1.1-3 Index]