20.109(S07):sample journal article summary

We know what properties we'd like sensors to have. In no particular order: You've read that genetically encoded calcium sensors have great benefits but how well do they compare to synthetic (aka chemical) sensors like Fura-2, Fluo4-FF or X-Rhod-5F?
 * it should be bright
 * it should respond linearly and quickly to large range of inputs, [Ca2+] for today
 * it should be sensitive to even subtle, single cellular stimuli
 * it should be inexpensive
 * it should not be disruptive to other cellular activities
 * it should be easy to use

Consider the comparison made in the article by Pologruto, Yasuda, and Svoboda J Neurosci (2004)24:9572. These authors
 * 1) try to correlate fluorescence with cellular activity by comparing fluorescence and chemical indicator (finding: fluorescence is nonlinear indicator at low activity levels)
 * 2) try to correlate fluorescence with [Ca2+] (finding: complex relationship)
 * 3) compare readout in cells with in vitro values since other CaM exist in cells and may influence sensitivity (finding: diffusion not influenced by CaM-binding proteins).

terms

 * F
 * fluorescence from indicator (for GECI and for chemical indicators)
 * factors influencing F
 * Ca2+ fluctuation…so waited until reached equilibrium, defined F0 as baseline, average F for 200 msec after equilibration and before stimulation
 * photobleaching of indicator…measured as ~40% after 50 minutes
 * noise in PMT…measured “dark” noise for 50 msec with shutter closed then subtracted mean
 * phi
 * degree to which fluorescence is saturated
 * Rf
 * dynamic range of the indicator
 * = Fmax/Fmin
 * previously experimentally determined
 * Kd
 * dissociation constant of Ca2+ from indicator
 * previously experimentally determined as concentration of Ca2+ for 1/2 phi
 * n
 * Hill coeff, measure of coopertivity
 * also need to define “alpha” as scaling term and “beta” as non-specific term to solve for phi in terms of Kd, [Ca2+] and n

results

 * 1) single stimuli (pg 9574)
 * 2) *“In response to a single action potential, the synthetic indicator produced robust, rapid onset fluorescence changes….In contrast, [two GECI] produced only very small fluorescence responses; these were detected above the noise only when averaging over many (8-16) trials.”
 * 3) *Fig 2A
 * 4) variable patterns of stimuli (pg 9575)
 * 5) *“both [chemical indicators] respond to Ca2+ elevations sufficiently quickly to follow the stimulus patterns reliably. In contrast, GECI power spectra did not reveal a clear peak above the noise at the stimulus frequency, even under the most favorable conditions. Thus, unlike synthetic indicators, GECIs respond too slowly to follow individual action potentials within a burst.”
 * 6) *Fig 3
 * 7) as quantitative measure of Ca2+ (pg 9575)
 * 8) *“GECIs have idiosyncratic and complex fluorescence saturation curves, making their use for quantitative [Ca2+] imaging problematic.”
 * 9) *Fig 5
 * 10) interaction of GECI with CaM-binding proteins in cell (pg 9576)
 * 11) *“Because CaM (and hence GECI) properties are changed by interactions with CaM-binding proteins, assessing GECI mobility is important for the interpretation of GECI signals”
 * 12) *“In all cases, after bleaching, fluorescence recovered to >95% of the baseline fluorescence.”
 * 13) *“We conclude that GECIs are mostly freely diffusible”