fluo {CalciOMatic}R Documentation

Convert Intracellular Calcium Concentration into Fluorescence Values

Description

The function fluo converts an intracellular calcium concentration to a photon count, depending on the values of the calibration parameters (R_min, R_max, K_eff and K_d) and the experiment-specific parameters (B_T, phi, S_B, T_stim, P and P_B)

Usage

fluo(Ca = 1, R_min = 0.136, R_max = 2.701, K_eff = 3.637, K_d = 0.583,
     B_T = 100, phi = 1.25, S_B = 10, T_stim = 0.015, P = 400, P_B = 400)

Arguments

Ca the intracellular calcium concentration (in muM)
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
K_d the dissociation constant of the dye in the cell (in muM). This parameter is obtained from calibration experiments
B_T the total dye concentration in the cell (in muM)
phi a dimensionless scaling experiment-specific parameter
S_B the background (+ dark current) fluorescence intensity (in count/pixel/sec)
T_stim the exposure time (in s)
P the number of pixels of the Region Of Interest (ROI)
P_B the number of pixels of the Background Region

Details

The calcium imaging technique makes use of the ability of a fluorescent dye (e.g. Fura) to bind with calcium ions presents inside a neural cell. Briefly, the photons emitted by a neural tissue are recorded by a CCD camera, following the illumination of the tissue at a relevant wavelength (corresponding to the excitation properties of the free and/or bound dye). The amount of photons emitted depends on the intracellular calcium concentration (with which the dye is bound), and, in the case of a ratiometric dye, a algebraic relationship links both variables. The latter is given by:

F_340 = (B_T*phi/(K_d+[Ca^{2+}]) * (R_min*K_eff+R_max*[Ca^{2+}]) + S_B,340) * T_stim,340 * P

F_380 = (B_T*phi/(K_d+[Ca^2+]) * (K_eff+[Ca^2+]) + S_B,380) * T_stim,380 * P

The function fluo determines photon counts according to one of these two equations, depending on the values of R_min, R_max and B_T

Value

An object of class "fluo_transient", which is a vector containing the fluorescence values calculated as described above. The object has several attributes, which are:

Ca a copy of argument Ca
R_min a copy of argument R_min
R_max a copy of argument R_max
K_eff a copy of argument K_eff
K_d a copy of argument K_d
B_T a copy of argument B_T
T_stim a copy of argument T_stim
P a copy of argument P
S_B a copy of argument S_B
phi a copy of argument phi

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

See Also

caBiExp, caMonoExp

Examples

## Parameters of the monoexponential calcium transient
tOn  <- 1
Time <- seq(0,12,length.out=160)
Ca0  <- 0.10
dCa  <- 0.25
tau  <- 1.5

## Calibrated parameters
R_min <- 0.136
R_max <- 2.701
K_eff <- 3.637
K_d <- 0.583

## Experiment-specific parameters
nb_B    <- 1
B_T     <- 100.0
T_340   <- 0.015
T_380   <- 0.006
P       <- 200
P_B     <- 200
phi     <- 2
S_B_340 <- 30
S_B_380 <- 80

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

## Define Background and Signal fluorescences at 340 and 380 nm
B_340 <- fluo(Ca=rep(0,nb_B), R_min=R_min, R_max=R_max, K_eff=K_eff, K_d=K_d,
              B_T=0, phi=phi, S_B=S_B_340, T_stim=T_340, P=P, P_B=P_B)

F_340 <- fluo(Ca=Ca, R_min=R_min, R_max=R_max, K_eff=K_eff, K_d=K_d,
              B_T=B_T, phi=phi, S_B=S_B_340, T_stim=T_340, P=P, P_B=P_B)
  
B_380 <- fluo(Ca=rep(0,nb_B), R_min=1, R_max=1, K_eff=K_eff, K_d=K_d,
              B_T=0, phi=phi, S_B=S_B_380, T_stim=T_380, P=P, P_B=P_B)

F_380 <- fluo(Ca=Ca, R_min=1, R_max=1, K_eff=K_eff, K_d=K_d,
              B_T=B_T, phi=phi, S_B=S_B_380, T_stim=T_380, P=P, P_B=P_B)

## Plot the fluorescence transients at 340 and 380 nm
layout(matrix(1:2,nrow=2))
plot(Time, F_340, type="l", bty="n")
plot(Time, F_380, type="l", bty="n")

[Package CalciOMatic version 1.1-3 Index]