Calcium imaging
A bright single-wavelength calcium dye (Kd ≈ 345 nM) excited at 488 nm and loaded as an AM ester, with a fluorescence increase of up to about 100-fold on binding calcium. Standard for cultured cells and high-throughput assays.
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Fluo-4
A bright single-wavelength calcium dye (Kd ≈ 345 nM) excited at 488 nm and loaded as an AM ester, with a fluorescence increase of up to about 100-fold on binding calcium. Standard for cultured cells and high-throughput assays.
How it differs from the standard method
- Load 1–5 µM AM ester with Pluronic F-127, 20–45 min, then de-esterify 20–30 min.
- Not ratiometric: calibrate with ionomycin (Fmax) or report ΔF/F₀.
- Leaks and compartmentalises over time; image within an hour of loading.
1Turning calcium into light
Free calcium in the cytosol sits near 100 nM at rest and rises ten-fold or more within milliseconds when channels open or stores release it. Almost every kind of cell signalling and every action potential in a neuron passes through such a rise, so watching calcium is a way of watching activity.
Kd sets the range
An indicator responds best between about a tenth and ten times its Kd. A high-affinity indicator detects small rises but saturates during large ones; a low-affinity one stays linear but gives a smaller signal per event.
The indicator is a buffer
Every indicator molecule binds calcium that would otherwise be free. Too much indicator shrinks and slows the very transient it reports.
Rise and decay
The trace is the calcium signal filtered by the indicator's binding kinetics. Frames must come several times per decay time or transients are missed.
From ΔF/F to nM
Single-wavelength dyes give [Ca²⁺] = Kd (F − Fmin)/(Fmax − F); Fmax from ionomycin at the end of the run, Fmin from the dye's dynamic range or a Ca²⁺-free solution.
2Loading dyes well
AM esters cross the membrane and are trapped when cellular esterases cleave them. Until they are cleaved the dye is fluorescent but calcium-blind, so cells need 20–30 min after washing before imaging.
3Choosing a genetically encoded indicator
GCaMP variants trade sensitivity against speed and linearity. GCaMP6s responds strongly to a single action potential and decays slowly, which makes it good at detecting sparse spikes but saturating in bursts; GCaMP6f is faster and less saturating at the cost of a smaller response per spike; newer variants (jGCaMP7 and 8) push both.
Key terms
Practise these as flashcards →Change in fluorescence divided by the baseline fluorescence; the standard readout of single-wavelength indicators.
Calcium concentration at which half the indicator is bound; sets the range of concentrations it reports.
Acetoxymethyl ester form of a dye that crosses membranes and is trapped after esterase cleavage.
Removal of the AM groups inside the cell, required before the dye binds calcium.
Bound calcium added per free calcium added; an indicator adds its own κ to the cell's, blunting transients.
A calcium ionophore that floods cells with calcium, used to obtain Fmax and as a positive control.
A genetically encoded indicator built from circularly permuted GFP, calmodulin and the M13 peptide.
The tangle of processes around cell bodies; its fluorescence contaminates somatic ROIs in tissue.
When to use it
- Following signalling responses (receptor activation, store release, entry) in many cultured cells at once: a synthetic dye such as Fluo-4.
- Recording activity of identified neurons or other cells over days, in culture, slices or animals: a genetically encoded indicator such as GCaMP.
- Estimating absolute calcium concentrations: a ratiometric dye (Fura-2) or a calibrated single-wavelength dye.
Compared with alternatives
Records membrane potential and currents directly with sub-millisecond resolution, one cell at a time.
Detects spikes from many neurons electrically, faster than any indicator, but without cell identity.
Measures calcium responses as a population average in multi-well plates for screening.
FRET-based calcium sensors (cameleons) give ratiometric readouts less sensitive to expression level.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
A bright single-wavelength calcium dye (Kd ≈ 345 nM) excited at 488 nm and loaded as an AM ester, with a fluorescence increase of up to about 100-fold on binding calcium. Standard for cultured cells and high-throughput assays.
- Load 1–5 µM AM ester with Pluronic F-127, 20–45 min, then de-esterify 20–30 min.
- Not ratiometric: calibrate with ionomycin (Fmax) or report ΔF/F₀.
- Leaks and compartmentalises over time; image within an hour of loading.