caFromRatio {CalciOMatic} | R Documentation |
The function caFromRatio
applies the ratiometric transformation
to vectors of fluorescence (including background fluorescence) and
returns the corresponding intracellular calcium concentration.
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)
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 |
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)
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).
Sebastien Joucla sebastien.joucla@parisdescartes.fr
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
## (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")