Calcium imaging
These dyes are often used with the chelator carboxyl groups masked as acetoxymethyl esters, in order to render the molecule lipophilic and to allow easy entrance into the cell.
[3] Later development of the technique using laser scanning confocal microscopes revealed sub-cellular Ca2+ signals in the form of Ca2+ sparks and Ca2+ blips.
Relative responses from a combination of chemical Ca2+ fluorescent indicators were also used to quantify calcium transients in intracellular organelles such as mitochondria.
Genetically encoded calcium indicators (GECIs) are powerful tools useful for in vivo imaging of cellular, developmental, and physiological processes.
Previous mutagenesis studies revealed that mutations at this position conferred pH stability while maintaining fluorescent properties, making Y145 an insertion point of interest.
However, a conformational change caused by calcium binding repositions the red-shifted fluorescent protein, allowing for FRET (Förster resonance energy transfer) to take place.
Of growing importance in calcium detection are near-IR (NIR) GECIs, which may open up avenues for multiplexing different indicator systems and allowing deeper tissue penetration.
[23] While fluorescent systems are widely used, bioluminescent Ca2+ reporters may also hold potential because of their ability to abrogate autofluorescence, photobleaching [no excitation wavelength is needed], biological degradation and toxicity, in addition to higher signal-to-noise ratios.
Colocalization of aequorin with GFP facilitates BRET/CRET (Bioluminescence or Chemiluminescence Resonance Energy Transfer),[18] resulting in a 19 - 65 brightness increase.
A similar system invokes obelin and its luciferin coelenteramide, which may possess faster calcium response time and Mg2+ insensitivity than its aqueorin counterpart.
Calcium binding brings the RLuc8 components in close proximity, reforming luciferase, and allowing it to transfer to an acceptor fluorescent protein.
[26] The use of near-IR wavelengths and minimization of axial spread of the point function[27] allows for nanometer resolution and deep penetration into the tissue.
This can be related to the SNR (signal to noise ratio) by multiplying the SBR by the square root of the number of counted photons.
[18] A special class of genetically encoded calcium indicators are designed to form a permanent fluorescent tag in active neurons.
Confocal and two-photon microscopes provide optical sectioning ability so that calcium signals can be resolved in microdomains such as dendritic spines or synaptic boutons, even in thick samples such as mammalian brains.
Methods such as fiber photometry,[45][46] miniscopes[47] and two-photon microscopy[48][49] offer calcium imaging in freely behaving and head-fixed animal models.
