Calcium imaging

File:Calcium fluorescence imaging schematic.png

Chemical indicators are small molecules that can chelate calcium ions. All these molecules are based on an EGTA homologue called BAPTA, with high selectivity for calcium (Ca²⁺) ions versus magnesium (Mg²⁺) ions.

This group of indicators includes fura-2, indo-1, fluo-3, fluo-4, Calcium Green-1.

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. Once this form of the indicator is in the cell, cellular esterases will free the carboxyl groups and the indicator will be able to bind calcium. The free acid form of the dyes (i.e. without the acetoxymethyl ester modification) can also be directly injected into cells via a microelectrode or micropipette which removes uncertainties as to the cellular compartment holding the dye (the acetoxymethyl ester can also enter the endoplasmic reticulum and mitochondria). Binding of a Ca²⁺ ion to a fluorescent indicator molecule leads to either an increase in quantum yield of fluorescence or emission/excitation wavelength shift. Individual chemical Ca²⁺ fluorescent indicators are utilized for cytosolic calcium measurements in a wide variety of cellular preparations. The first real time (video rate) Ca²⁺ imaging was carried out in 1986 in cardiac cells using intensified video cameras.{{cite journal | vauthors = Cannell MB, Berlin JR, Lederer WJ | title = Intracellular calcium in cardiac myocytes: calcium transients measured using fluorescence imaging | journal = Society of General Physiologists Series | volume = 42 | pages = 201–214 | date = 1987-01-01 | pmid = 3505361 }} 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.{{cite journal | vauthors = Ivannikov MV, Macleod GT | title = Mitochondrial free Ca²⁺ levels and their effects on energy metabolism in Drosophila motor nerve terminals | journal = Biophysical Journal | volume = 104 | issue = 11 | pages = 2353–2361 | date = June 2013 | pmid = 23746507 | pmc = 3672877 | doi = 10.1016/j.bpj.2013.03.064 | bibcode = 2013BpJ...104.2353I }}

Calcium imaging, also referred to as calcium mapping, is also used to perform research on myocardial tissue.{{cite journal | vauthors = Jaimes R, Walton RD, Pasdois P, Bernus O, Efimov IR, Kay MW | title = A technical review of optical mapping of intracellular calcium within myocardial tissue | journal = American Journal of Physiology. Heart and Circulatory Physiology | volume = 310 | issue = 11 | pages = H1388–H1401 | date = June 2016 | pmid = 27016580 | pmc = 4935510 | doi = 10.1152/ajpheart.00665.2015 }} Calcium mapping is a ubiquitous technique used on whole, isolated hearts such as mouse, rat, and rabbit species.

Genetically encoded calcium indicators (GECIs) are powerful tools useful for in vivo imaging of cellular, developmental, and physiological processes.{{cite journal | vauthors = Hendel T, Mank M, Schnell B, Griesbeck O, Borst A, Reiff DF | title = Fluorescence changes of genetic calcium indicators and OGB-1 correlated with neural activity and calcium in vivo and in vitro | journal = The Journal of Neuroscience | volume = 28 | issue = 29 | pages = 7399–7411 | date = July 2008 | pmid = 18632944 | pmc = 6670390 | doi = 10.1523/JNEUROSCI.1038-08.2008 }}{{cite journal | vauthors = Gordon S, Dickinson MH | title = Role of calcium in the regulation of mechanical power in insect flight | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 103 | issue = 11 | pages = 4311–4315 | date = March 2006 | pmid = 16537527 | pmc = 1449689 | doi = 10.1073/pnas.0510109103 | bibcode = 2006PNAS..103.4311G | doi-access = free }}{{cite journal | vauthors = Kerr R, Lev-Ram V, Baird G, Vincent P, Tsien RY, Schafer WR | title = Optical imaging of calcium transients in neurons and pharyngeal muscle of C. elegans. | journal = Neuron | date = June 2000 | volume = 26 | issue = 3 | pages = 583–59 | doi = 10.1016/S0896-6273(00)81196-4 | pmid = 10896155 | s2cid = 311998 | doi-access = free }}{{cite journal | vauthors = Kim J, Lee S, Jung K, Oh WC, Kim N, Son S, Jo Y, Kwon HB, Heo WD | display-authors = 6 | title = Intensiometric biosensors visualize the activity of multiple small GTPases in vivo | journal = Nature Communications | volume = 10 | issue = 1 | pages = 211 | date = January 2019 | pmid = 30643148 | pmc = 6331645 | doi = 10.1038/s41467-018-08217-3 | bibcode = 2019NatCo..10..211K }} GECIs do not need to be acutely loaded into cells; instead the genes encoding for these proteins can be introduced into individual cells or cell lines by various transfection methods. It is also possible to create transgenic animals expressing the indicator in all cells or selectively in certain cellular subtypes. GECIs are used to study neurons,{{cite journal | vauthors = Afshar Saber W, Gasparoli FM, Dirks MG, Gunn-Moore FJ, Antkowiak M | title = All-Optical Assay to Study Biological Neural Networks | journal = Frontiers in Neuroscience | volume = 12 | pages = 451 | date = 2018 | pmid = 30026684 | pmc = 6041400 | doi = 10.3389/fnins.2018.00451 | doi-access = free }}{{cite journal | vauthors = Kovalchuk Y, Homma R, Liang Y, Maslyukov A, Hermes M, Thestrup T, Griesbeck O, Ninkovic J, Cohen LB, Garaschuk O | display-authors = 6 | title = In vivo odourant response properties of migrating adult-born neurons in the mouse olfactory bulb | journal = Nature Communications | volume = 6 | pages = 6349 | date = February 2015 | pmid = 25695931 | doi = 10.1038/ncomms7349 | doi-access = free | bibcode = 2015NatCo...6.6349K }} T-cells,{{cite journal | vauthors = Mues M, Bartholomäus I, Thestrup T, Griesbeck O, Wekerle H, Kawakami N, Krishnamoorthy G | title = Real-time in vivo analysis of T cell activation in the central nervous system using a genetically encoded calcium indicator | journal = Nature Medicine | volume = 19 | issue = 6 | pages = 778–783 | date = June 2013 | pmid = 23685843 | doi = 10.1038/nm.3180 | s2cid = 205391240 }} cardiomyocytes,{{cite journal | vauthors = Shinnawi R, Huber I, Maizels L, Shaheen N, Gepstein A, Arbel G, Tijsen AJ, Gepstein L | display-authors = 6 | title = Monitoring Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes with Genetically Encoded Calcium and Voltage Fluorescent Reporters | journal = Stem Cell Reports | volume = 5 | issue = 4 | pages = 582–596 | date = October 2015 | pmid = 26372632 | pmc = 4624957 | doi = 10.1016/j.stemcr.2015.08.009 }} and other cell types. Some GECIs report calcium by direct emission of photons (luminescence), but most rely on fluorescent proteins as reporters, including the green fluorescent protein GFP and its variants (eGFP, YFP, CFP).

Of the fluorescent reporters, calcium indicator systems can be classified into single fluorescent protein (FP) systems, and paired fluorescent protein systems. Camgaroos were one of the first developed variants involving a single protein system. Camgaroos take advantage of calmodulin (CaM), a calcium binding protein. In these structures, CaM is inserted in the middle of yellow fluorescent protein (YFP) at Y145. Previous mutagenesis studies revealed that mutations at this position conferred pH stability while maintaining fluorescent properties, making Y145 an insertion point of interest. Additionally, the N and C termini of YFP are linked by a peptide linker (GGTGGS). When CaM binds to Ca2+, the effective pKa is lowered, allowing for chromophore deprotonation.{{cite journal | vauthors = Baird GS, Zacharias DA, Tsien RY | title = Circular permutation and receptor insertion within green fluorescent proteins | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 96 | issue = 20 | pages = 11241–11246 | date = September 1999 | pmid = 10500161 | pmc = 18018 | doi = 10.1073/pnas.96.20.11241 | bibcode = 1999PNAS...9611241B | doi-access = free }} This results in increased fluorescence upon calcium binding in an intensiometric fashion. Such detection is in contrast with ratiometric systems, in which there is a change in the absorbance/emission spectra as a result of Ca2+ binding.{{cite book | vauthors = Park JG, Palmer AE | chapter = Quantitative Measurement of Ca2+ and Zn2+ in Mammalian Cells Using Genetically Encoded Fluorescent Biosensors | title = Fluorescent Protein-Based Biosensors | series = Methods in Molecular Biology | volume = 1071 | pages = 29–47 | date = 2014 | pmid = 24052378 | pmc = 4051312 | doi = 10.1007/978-1-62703-622-1_3 | isbn = 978-1-62703-621-4 }} A later developed single-FP system, dubbed G-CaMP, also invokes circularly permuted GFP. One of the termini is fused with CaM, and the other termini is fused with M13 (the calmodulin binding domain of myosin light kinase){{cite journal | vauthors = Rodriguez EA, Campbell RE, Lin JY, Lin MZ, Miyawaki A, Palmer AE, Shu X, Zhang J, Tsien RY | display-authors = 6 | title = The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins | journal = Trends in Biochemical Sciences | volume = 42 | issue = 2 | pages = 111–129 | date = February 2017 | pmid = 27814948 | pmc = 5272834 | doi = 10.1016/j.tibs.2016.09.010 }} The protein is designed such that the termini are close in space, allowing for Ca2+ binding to cause conformational changes and chromophore modulation, allowing for increased fluorescence. G-CaMP and its refined variants have nanomolar binding affinities.{{cite journal | vauthors = Nakai J, Ohkura M, Imoto K | title = A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein | journal = Nature Biotechnology | volume = 19 | issue = 2 | pages = 137–141 | date = February 2001 | pmid = 11175727 | doi = 10.1038/84397 | s2cid = 30254550 }} A final single protein variant is the CatchER, which is generally considered to be a lower affinity indicator. Its calcium binding pocket is quite negative; binding of the cation helps to shield the large concentration of negative charge and allows for recovered fluorescence.{{cite journal | vauthors = Pérez Koldenkova V, Nagai T | title = Genetically encoded Ca(2+) indicators: properties and evaluation | journal = Biochimica et Biophysica Acta (BBA) - Molecular Cell Research | volume = 1833 | issue = 7 | pages = 1787–1797 | date = July 2013 | pmid = 23352808 | doi = 10.1016/j.bbamcr.2013.01.011 }}

In contrast to these systems are paired fluorescent protein systems, which include the prototypical Cameleons. Cameleons consist of two different fluorescent proteins, CaM, M13, and a glycylglycine linker. In the absence of Ca2+, only the donor blue-shifted fluorescent protein will be fluorescent. However, a conformational change caused by calcium binding repositions the red-shifted fluorescent protein, allowing for FRET (Förster resonance energy transfer) to take place. Cameleon indicators produce a ratiometric signal (i.e. the measured FRET efficiency depends on the calcium concentration). Original variants of cameleons were originally more sensitive to Ca2+ and were acid quenched.{{cite journal | vauthors = Miyawaki A, Griesbeck O, Heim R, Tsien RY | title = Dynamic and quantitative Ca2+ measurements using improved cameleons | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 96 | issue = 5 | pages = 2135–2140 | date = March 1999 | pmid = 10051607 | pmc = 26749 | doi = 10.1073/pnas.96.5.2135 | bibcode = 1999PNAS...96.2135M | doi-access = free }} Such shortcomings were abrogated by Q69K and V68L mutations. Both of these residues were close to the buried anionic chromophore and these mutations probably hinder protonation, conferring greater pH resistance.

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. NIR GECIs rely on biliverdin-binding fluorescent proteins, which are largely derived from bacterial phytochromes. NIR systems are similar to inverse pericams in that both experience a decrease in fluorescence upon Ca2+ binding. RCaMPs and RGECOs are functional at 700+ nm, but are quite dim.{{cite journal | vauthors = Qian Y, Piatkevich KD, Mc Larney B, Abdelfattah AS, Mehta S, Murdock MH, Gottschalk S, Molina RS, Zhang W, Chen Y, Wu J, Drobizhev M, Hughes TE, Zhang J, Schreiter ER, Shoham S, Razansky D, Boyden ES, Campbell RE | display-authors = 6 | title = A genetically encoded near-infrared fluorescent calcium ion indicator | journal = Nature Methods | volume = 16 | issue = 2 | pages = 171–174 | date = February 2019 | pmid = 30664778 | pmc = 6393164 | doi = 10.1038/s41592-018-0294-6 }} A Cameleon analog involving NIR FRET has been successfully constructed as well.{{cite journal | vauthors = Shemetov AA, Monakhov MV, Zhang Q, Canton-Josh JE, Kumar M, Chen M, Matlashov ME, Li X, Yang W, Nie L, Shcherbakova DM, Kozorovitskiy Y, Yao J, Ji N, Verkhusha VV | display-authors = 6 | title = A near-infrared genetically encoded calcium indicator for in vivo imaging | journal = Nature Biotechnology | volume = 39 | issue = 3 | pages = 368–377 | date = March 2021 | pmid = 33106681 | pmc = 7956128 | doi = 10.1038/s41587-020-0710-1 }}

A special class of GECIs are designed to form a permanent fluorescent tag in active neurons. They are based on the photoswitchable protein Eos which turns from green to red through photocatalyzed (with violet light) backbone cleavage.{{cite journal | vauthors = Nienhaus K, Nienhaus GU, Wiedenmann J, Nar H | title = Structural basis for photo-induced protein cleavage and green-to-red conversion of fluorescent protein EosFP | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 102 | issue = 26 | pages = 9156–9159 | date = June 2005 | pmid = 15964985 | pmc = 1166600 | doi = 10.1073/pnas.0501874102 | bibcode = 2005PNAS..102.9156N | doi-access = free }} Combined with the CaM, violet light photoconverts only neurons that have elevated calcium levels. SynTagMA is a synapse-targeted version of CaMPARI2.{{cite journal | vauthors = Fosque BF, Sun Y, Dana H, Yang CT, Ohyama T, Tadross MR, Patel R, Zlatic M, Kim DS, Ahrens MB, Jayaraman V, Looger LL, Schreiter ER | display-authors = 6 | title = Neural circuits. Labeling of active neural circuits in vivo with designed calcium integrators | journal = Science | volume = 347 | issue = 6223 | pages = 755–760 | date = February 2015 | pmid = 25678659 | doi = 10.1126/science.1260922 | s2cid = 206562601 }}

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.{{cite journal | vauthors = Baubet V, Le Mouellic H, Campbell AK, Lucas-Meunier E, Fossier P, Brúlet P | title = Chimeric green fluorescent protein-aequorin as bioluminescent Ca2+ reporters at the single-cell level | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 97 | issue = 13 | pages = 7260–7265 | date = June 2000 | pmid = 10860991 | pmc = 16533 | doi = 10.1073/pnas.97.13.7260 | bibcode = 2000PNAS...97.7260B | doi-access = free }} Such systems may rely on aequorin and the luciferin coelenterazine. Ca2+ binding causes a conformational change that facilitates coelenterazine oxidation. The resultant photoproduct emits blue light as it returns to the ground state. Colocalization of aequorin with GFP facilitates BRET/CRET (Bioluminescence or Chemiluminescence Resonance Energy Transfer), resulting in a 19 - 65 brightness increase. Such structures can be used to probe millimolar to nanomolar calcium concentrations. A similar system invokes obelin and its luciferin coelenteramide, which may possess faster calcium response time and Mg2+ insensitivity than its aqueorin counterpart.{{cite book | vauthors = Illarionov BA, Frank LA, Illarionova VA, Bondar VS, Vysotski ES, Blinks JR | chapter = Recombinant obelin: Cloning and expression of cDNA, purification, and characterization as a calcium indicator | title = Bioluminescence and Chemiluminescence Part C | series = Methods in Enzymology | volume = 305 | pages = 223–249 | date = 2000 | pmid = 10812604 | doi = 10.1016/s0076-6879(00)05491-4 | isbn = 9780121822064 }} Such systems can also leverage the self-assembly of luciferase components. In a system dubbed “nano-lantern,” the luciferase RLuc8 is split and placed on different ends of CaM. Calcium binding brings the RLuc8 components in close proximity, reforming luciferase, and allowing it to transfer to an acceptor fluorescent protein.

To minimize damage to the visualized cells, two-photon microscopy is often invoked to detect the fluorescence from the reporters.Oh, J., Lee, C., & Kaang, B. K. (2019). Imaging and analysis of genetically encoded calcium indicators linking neural circuits and behaviors. The Korean journal of physiology & pharmacology : official journal of the Korean Physiological Society and the Korean Society of Pharmacology, 23(4), 237–249. https://doi.org/10.4196/kjpp.2019.23.4.237 The use of near-IR wavelengths and minimization of axial spread of the point function{{cite journal | vauthors = Kaminer I, Nemirovsky J, Segev M | title = Optimizing 3D multiphoton fluorescence microscopy | journal = Optics Letters | volume = 38 | issue = 19 | pages = 3945–3948 | date = October 2013 | pmid = 24081095 | doi = 10.1364/OL.38.003945 | bibcode = 2013OptL...38.3945K }} allows for nanometer resolution and deep penetration into the tissue. The dynamic range is often determined from such measurements. For non-ratiometric indicators (typically single protein indicators), it is the ratio of the fluorescence intensities obtained under Ca2+ saturated and depleted conditions, respectively. However, for ratiometric indicators, the dynamic range is the ratio of the maximum FRET efficiency ratio (calcium saturated) to the minimum FRET efficiency ratio (calcium depleted). Yet another common quantity used to measure signals produced by calcium concentration fluxes is the signal-to-baseline ratio (SBR), which is simply the ratio of the change in fluorescence (F - F0) over the baseline fluorescence. 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.

Regardless of the type of indicator used, the imaging procedure is generally very similar. Cells loaded with an indicator, or expressing it in the case of a GECI,{{Citation |last1=Akerboom |first1=Jasper |title=Engineering and Application of Genetically Encoded Calcium Indicators |date=2012 |work=Genetically Encoded Functional Indicators |pages=125–147 |editor-last=Martin |editor-first=Jean-René |url=https://doi.org/10.1007/978-1-62703-014-4_8 |access-date=2024-03-15 |place=Totowa, NJ |publisher=Humana Press |language=en |doi=10.1007/978-1-62703-014-4_8 |isbn=978-1-62703-014-4 |last2=Tian |first2=Lin |last3=Marvin |first3=Jonathan S. |last4=Looger |first4=Loren L.|url-access=subscription }} can be viewed using a fluorescence microscope and captured by a Scientific CMOS (sCMOS){{cite journal | vauthors = Nguyen JP, Shipley FB, Linder AN, Plummer GS, Liu M, Setru SU, Shaevitz JW, Leifer AM | display-authors = 6 | title = Whole-brain calcium imaging with cellular resolution in freely behaving Caenorhabditis elegans | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 113 | issue = 8 | pages = E1074–E1081 | date = February 2016 | pmid = 26712014 | pmc = 4776509 | doi = 10.1073/pnas.1507110112 | arxiv = 1501.03463 | doi-access = free | bibcode = 2016PNAS..113E1074N }} camera or CCD camera. 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. Images are analyzed by measuring fluorescence intensity changes for a single wavelength or two wavelengths expressed as a ratio (ratiometric indicators). If necessary, the derived fluorescence intensities and ratios may be plotted against calibrated values for known Ca²⁺ levels to measure absolute Ca²⁺ concentrations. Light field microscopy methods{{cite journal | vauthors = Pégard NC, Liu HY, Antipa N, Gerlock M, Adesnik H, Waller L | title = Compressive light-field microscopy for 3D neural activity recording. | journal = Optica | date = May 2016 | volume = 3 | issue = 5 | pages = 517–24 | doi = 10.1364/optica.3.000517 | bibcode = 2016Optic...3..517P | doi-access = free }} extend functional readout of neural activity capabilities in 3D volumes.

Methods such as fiber photometry,{{cite journal | vauthors = Wang Y, DeMarco EM, Witzel LS, Keighron JD | title = A selected review of recent advances in the study of neuronal circuits using fiber photometry | journal = Pharmacology, Biochemistry, and Behavior | volume = 201 | pages = 173113 | date = February 2021 | pmid = 33444597 | doi = 10.1016/j.pbb.2021.173113 | s2cid = 231579597 }}{{cite journal | vauthors = Sych Y, Chernysheva M, Sumanovski LT, Helmchen F | title = High-density multi-fiber photometry for studying large-scale brain circuit dynamics | journal = Nature Methods | volume = 16 | issue = 6 | pages = 553–560 | date = June 2019 | pmid = 31086339 | doi = 10.1038/s41592-019-0400-4 | s2cid = 152283372 | url = https://www.zora.uzh.ch/id/eprint/184956/1/s41592-019-0400-4.pdf }} miniscopes{{cite journal | vauthors = Resendez SL, Jennings JH, Ung RL, Namboodiri VM, Zhou ZC, Otis JM, Nomura H, McHenry JA, Kosyk O, Stuber GD | display-authors = 6 | title = Visualization of cortical, subcortical and deep brain neural circuit dynamics during naturalistic mammalian behavior with head-mounted microscopes and chronically implanted lenses | journal = Nature Protocols | volume = 11 | issue = 3 | pages = 566–597 | date = March 2016 | pmid = 26914316 | doi = 10.1038/nprot.2016.021 | pmc = 5239057 }} and two-photon microscopy{{cite journal | vauthors = Jung JC, Mehta AD, Aksay E, Stepnoski R, Schnitzer MJ | title = In vivo mammalian brain imaging using one- and two-photon fluorescence microendoscopy | journal = Journal of Neurophysiology | volume = 92 | issue = 5 | pages = 3121–3133 | date = November 2004 | pmid = 15128753 | pmc = 2826362 | doi = 10.1152/jn.00234.2004 }}{{cite journal|author1-link=Karel Svoboda (scientist) | vauthors = Svoboda K, Yasuda R | title = Principles of two-photon excitation microscopy and its applications to neuroscience | journal = Neuron | volume = 50 | issue = 6 | pages = 823–839 | date = June 2006 | pmid = 16772166 | doi = 10.1016/j.neuron.2006.05.019 | s2cid = 7278880 | doi-access = free }} offer calcium imaging in freely behaving and head-fixed animal models.

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• {{cite news | veditors = Nuccitelli R |title=A Practical guide to the study of calcium in living cells |series=Methods in Cell Biology |publisher=Academic Press |location=Boston |year=1994 |isbn=978-0-12-564141-8 |url=http://www.sciencedirect.com/science/bookseries/0091679X/40 }}

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Category:Cell imaging