fluo {CalciOMatic} | R Documentation |
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
)
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)
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 |
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
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 |
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
## 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")