Table 4.

Summary of advantages and limitations of different microscopy systems suitable for Ca²⁺ imaging

Type of microscopy	Advantages	Limitations
Wide field microscopy	(i) Suitable for its use with FRET-based and single FP GECI; (ii) single cell resolution allowed with specific sample preparations (e.g. Guard cells in epidermal strips).	(i) Lack of optical sectioning.
Laser scanning confocal microscopy	(i) Optical sectioning allowed; (ii) spectral separation with multiple detectors; (iii) single cell analyses allowed in entire organs; (iv) most of the commercial configurations allow FRET analyses.	(i) Acquisition speed.
Spinning disk confocal microscopy	(i) Fast rate of acquisition; (ii) optical sectioning allowed; (iii) single cell analyses allowed in entire organs; (iv) lower phototoxicity.	(i) High price.
Light sheet fluorescence microscopy	(i) Fast rate of acquisition; (ii) optical sectioning allowed; (iii) single cell analyses allowed in entire organs; (iv) long-term analyses; (v) tailored design.	(i) Low versatility.