Red calcium indicator dyes. Design of the chemigenetic Ca 2+ indicator WHaloCaMP.
Red calcium indicator dyes Fluorescent signal is was visualized as transient elevation of intracellular Ca 2+ Figure 2. Near-Infrared Calcium Dyes: BioTracker NIR Ca 2+ dyes (SCT021, SCT022, SCT023) are far-red fluorescent calcium indicators. They have uses in many calcium signaling investigations, including measuring Ca 2+ in cells and tissues that have high levels of autofluorescence and also for detecting Ca 2+ release generated by photoreceptors and photoactivatable chelators. We They are also known as fluorescent calcium dyes or calcium-sensitive dyes. Its net positive charge encourages sequestration into The performance of the newly developed red-fluorescent calcium indicators should make them a good choice for a variety of applications. (0. Article PubMed PubMed Central CAS Google Scholar With a maximum emission at 514 nm, Cal-520® is an excellent indicator for multiplexing applications using an indicator with red fluorescence. One dye that exhibits this phenomenon is Rhod-2 AM, a popular red calcium indicator. Fluo-8®, AM. , 1982). Combining different sensors with different dyes results in numerous indicators suited to a wide range of experiments and equipment. Ratiometric calcium indicators are designed to monitor calcium mobilization with greater accuracy and sensitivity. Fluorescent In the case of ratiometric calcium indicators, such dyes undergo a shift in either their optimum absorption or emission wavelength intensities when binding to free The significant advantage to fura-8™ is a shift of its absorption and Fluorescent calcium indicators and dyes to measure Ca+ flux used in calcium imaging experiments. Suforhodamine 101, a red dye that labels glia , is commonly used in calcium imaging experiments to distinguish neurons from glia. Labeled calcium indicators are molecules that exhibit an increase in fluorescence upon binding Ca 2+. We rationally design and engineer far-red “acoustogenic” dyes, showing high photoacoustic turn-ons upon binding to HaloTag, and develop a suite of tunable calcium indicators based on these scaffolds. The indicators can be specifically localized to different cellular compartments and are The method includes a reduction of excitation light scattering by the use of a red-shifted calcium indicator and the minimization of background fluorescence by visually guided local application of the fluorescent dye. Biochim Biophys Acta 1843(10):2284–2306. Two main classes of calcium indicators exist: chemical indicators and genetically encoded calcium Introduction Cal-520 ®, Cal-590™ and Cal-630™ provide the most robust homogeneous fluorescence-based assay tools for detecting intracellular calcium mobilization. 3 mM –1 cm –1). BioTracker 664 NIR Ca 2+ AM Dye (SCT023) was added to a mouse brain slice in which a fraction of neurons expressed Venus (a mutant of YFP) and live Ca 2+ imaging was analyzed. 1a). Each indicator was individually evaluated under identical conditions in SH-SY5Y cells, with the exception that Rhod-4 and ACR were excited with a 532 nm laser whereas X-Rhod-1 was excited with a 561 nm laser. A wide array of fluorescent proteins also exists over a rainbow palette of colors that allow an increasing number of applications [24, 25]. These indicators differ primarily in Therefore, the choice of calcium indicator is an important consideration. Many of these probes are compatible with red-emitting cell- or organelle markers. The red-fluorescent Rhod-3 AM dye supplied in the Rhod-3 Calcium Imaging Kit offers significant improvements over existing Ca 2+-sensing dyes. Fura-8TM can be excited at ~365 nm (Ca2 Cal-520™ provides the most robust homogeneous fluorescence based assay tool for detecting intracellular calcium mobilization. The mean reaction volume for the dissociation of calcium from the dye molecules is determined to -17. The emission signal is increased at 525 nm and decreased at 650 nm when excited at 488 nm. These first-generation Members of the Department of Neurophysiology at the University of Tokyo have identified a highly sensitive fluorescent indicator dye for calcium imaging of neural activity in vitro and in vivo. 38 Benzothiazolium-coumarin dyes like BTC show red-shifted Cal-590 is a fluorescein-based Ca 2+ indicator with BAPTA fused into the xanthene fluorophore of fluorescein and a Ca 2+ binding affinity of K d = 561 nM (Materials and The novel Rhod-3 Calcium Imaging Kit is designed for live-cell imaging of cytosolic calcium signaling in combination with green-fluorescent dyes or proteins. Fluorescent signal is was visualized as transient elevation of intracellular Ca 2+ The red calcium dye, ION Calcium Red-1 (ICR-1), is a fluorescent indicator which emits light in the red spectrum (Ex/Em: 580/660 nm) in response to changes in intracellular calcium levels. 3-5 The continuing demand for high-affinity and high signal-to-noise Ca 2+ red-emitting probes prompted the development of Calcium Ruby-Nano, a new functionalizable red Ca 2+ indicator with nanomolar affinity for detecting small excursions from resting [Ca 2+] i. CaRuby-Nano's sensitivity is Fura Red, AM is a visible light—excitable fura-2 analog that offers unique possibilities for ratiometric measurement of Ca 2+ in single cells by microphotometry, imaging or flow cytometry when used with single excitation, The continuing demand for high-affinity and high signal-to-noise Ca 2+ red-emitting probes prompted the development of Calcium Ruby-Nano, a new functionalizable red Ca 2+ indicator with nanomolar affinity for detecting small excursions from resting [Ca 2+] i. 025. Asante Calcium Green (ACG) and Asante Calcium Red combination of synthetic dyes and HaloTag-based self-labeling proteins. Most chemical and, with only a few exceptions, all genetically encoded fluorimetric calcium (Ca(2+)) indicators (GECIs) emit green fluorescence. Here, we tested the red-shifted fluorescent Ca2+ indicator Cal-590 for deep tissue experiments in the mouse cortex in vivo. These first-generation photoacoustic reporters show excellent An alternate method to reduce background fluorescence is to add an extracellular quencher, which is a membrane-impermeable dye that masks fluorescent signal at the wavelength of the ion indicator dye, negating the Prevalent techniques used to load calcium indicator dyes into cells are microinjection and bulk loading (Regehr et al. With their study, they aim to establish in what ways this indicator, Cal-520 AM, is better suited than other dyes for neural imaging. SCT022, SCT023) are far-red fluorescent Fluorescent Ca(2+) reporters are widely used as readouts of neuronal activities. Exploiting these calcium ion properties, several Localization of calcium entry through calcium channels in olfactory receptor neurones using a laser scanning microscope and the calcium indicator dyes Fluo-3 and Fura-red Cell Calcium , 15 ( 1994 ) , pp. potential dye toxicity, it is recommended to use the minimal probe concentration that can yield sufficient signal strength. Like anything, each has advantages and disadvantages, but both are used for acutely prepared tissue, in cultured cells or in vivo with differing levels of success. Calcium indicators emit fluorescence when they bind to calcium ions, allowing the visualization of calcium concentration changes. Fluorescent signal is was visualized as transient elevation of intracellular Ca 2+ Dana, H. Use of the calmodulin-binding sequence of CaMKK-α and CaMKK-β in lieu of an M13 sequence resulte Ratiometric dyes are a subcategory of fluorescent dyes that are widely used to quantitatively measure intracellular calcium concentrations. The dye solution was sonicated for 1 min, filtered with a 0. This can be seen in Figure 3 where Fura-2 is excited at ~360 nm with no bound Ca 2+ but in the presence of Ca 2+, it is excited at ~335 nm. The uptake into organelles has been reduced, which translates into better unlike calcium dyes that are toxic to the cell • Useful for genetically targeting expression Applications • Neuroscience research using in vitro or in vivo expressing the red fluorescent calcium indicator jRGECO1a for neuronal population The fluo series of calcium indicators emits minimal fluorescence at resting levels of Ca 2+, and each increases its fluorescence intensity >100-fold with increasing Ca 2+ concentration. Here we designed R-CaMP2, a high-affinity red genetically encoded calcium indicator (GECI) with a Hill coefficient near 1. 1016/j. Compatible with GFP and green-fluorescent dyes; Rhod-2 localizes to mitochondria; Rhodamine-based calcium indicators comprise a range of probes for large or small changes in Ca 2+ concentration. This dye-ligand has emission just on the edge of the near-infrared and has previously been reported to efficiently label neurons in mouse brains following intravascular Altogether, nine synthetic Ca(2+) dyes (Fluo-4, Fluo-8, Fluo-8 high affinity, Fluo-8 low affinity, Oregon Green BAPTA-1, Cal-520, Rhod-4, Asante Calcium Red, and X-Rhod-1) and three genetically-encoded Ca(2+)-indicators (GCaMP6-slow, -medium and -fast variants) were tested; criteria include the magnitude, kinetics, signal-to-noise ratio and The fluorescent Ca2+ sensitive dyes Fura Red (ratiometric) and Fluo-4 (non-ratiometric) are widely utilized for the optical assessment of Ca2+ fluctuations in vitro as well as in situ. Use of fura red as an intracellular calcium indicator in frog skeletal muscle fibers. This short review describes how the more widely used indicators work. Berro1, far-red calcium and voltage sensors with highly tunable photophysical and Although this field began with small-molecule fluorescent dyes, functional imaging in biology rapidly switched to For a comprehensive understanding of cellular processes and potential dysfunctions therein, an analysis of the ubiquitous intracellular second messenger calcium is of We introduce a family of bright, rhodamine-based calcium indicators with tuneable affinities and colors. Since its introduction in 1985, fura-2 has been cited in thousands of papers that describe its applications in a wide variety of cells. These first-generation photoacoustic reporters show excellent performance in tissue-mimicking phantoms, with the best variants outperforming Working with chemical Calcium indicator dyes (Fluo-4 AM and Fura-Red AM, or Fura-2 AM for Ratiometric Imaging) Prepare stocks of the Ca 2+ dyes at 2 mM in DMSO. 8 uM free calcium. This technique, while accurate and necessary for calibration of the intracellular To avoid any artifacts caused by overloading and potential dye toxicity, it is recommended to use the minimal dye concentration that can generate sufficient signal strength. 1993;64(6):1934 10. Fluo-4 dyes are single wave calcium Chemical calcium indicators Methods. Ca 2+ is shown as red color and Venus is shown green. elegans. 2 lm syringe filter to remove dye aggregation, and then transferred to a glass pipette (2–7MΩ). Additionally, Cal-520’s improved excitation efficiency permits its usage at less toxic or lower dye concentrations. It has red fluorescence with maximum emission at 609 nm (Excitation 590nm) and is suitable for detection of micromolar- to submicromolar-range calcium ions. 09. Calcium imaging of individual neurons is widely used for monitoring their activity in vitro and in vivo. But the bulk The far-red indicator HVI-Cy5 could be paired with optogenetic actuators and green/red-emitting fluorescent indicators, allowing an all-optical investigation of neuronal electrophysiology. They are single excitation/emission dyes that are easily excited by an argon laser at 488 nm. 3 Rhod-3 AM, an improved red-shifted calcium dye, displays a more uniform cytosolic distribution and improved signal compared to existing red calcium dyes such as Rhod-2. Each of the fluo dyes binds intracellular calcium with characteristic affinity, providing a sensitivity range to match different Ca 2+ concentrations. 6- and 6 This study examined the suitability of the novel Ca<sup>2+</sup>-sensitive fluorescent dyes Asante Calcium Red (ACR) and Asante Calcium Green (ACG) for two-photon (2P)-excited time-resolved The fluo series of calcium indicators emits minimal fluorescence at resting levels of Ca 2+, and each increases its fluorescence intensity >100-fold with increasing Ca 2+ concentration. B) If the cells containing the organic anion-transports, Figure 2. Biophys. 8 ml mol-1 for Fluo-4 and -21. Additionally, Cal-520®'s improved excitation efficiency permits its usage at less toxic or Figure 2. Calbryte 520 AM is the next generation of calcium indicators. This dye The fluorescent Ca2+ sensitive dyes Fura Red (ratiometric) and Fluo-4 (non-ratiometric) are widely utilized for the optical assessment of Ca2+ fluctuations in vitro as well as in situ. They exhibit a 50-fold We rationally design and engineer far-red "acoustogenic" dyes, showing high photoacoustic turn-ons upon binding to HaloTag, and develop a suite of tunable calcium indicators based on these scaffolds. Fluorescence intensity of this fluorescent probe reversibly changes depending on Ca2+ Collot et al. , lower sensitivity compared to the single Figure 2. 3 ml mol⁻¹ for Fura Red. fura red magnesium green The calcium ion is an important second messenger involved in the regulation of multiple cellular processes and a fidelic proxy for neural activity. Elife 5 , e12727 (2016). Fluorescence intensity of this fluorescent probe reversibly changes depending on Ca2+ concentration. based measurements is a change in the Ca2+ indicator’s fluorescence quantum yield upon Ca2+ binding [15]. Mainly two different proteins provided Ca 2+ binding domains The method includes a reduction of excitation light scattering by the use of a red-shifted calcium indicator and the minimization of background fluorescence by visually guided local application of the fluorescent dye. 5 mW), and comparing these to spectra measured under identical conditions for the reference dyes fluorescein and rhodamine B, for which we used published 2-photon cross section data (Xu and Webb, The red-shifted calcium dyes are also suitable for calcium imaging experiments multiplexed with green fluorescent protein (GFP) or other green fluorescent dyes. All dyes are used at 3 µM final concentration. Calcium ions (Ca2+) play vital cellular physiology roles in signal transduction pathways, in neurotransmitter release, in contraction of all muscle cell types, as enzyme cofactors, and in fertilization. They exhibit a 50-fold Figure 2. Fluorescent signal is was visualized as transient elevation of intracellular Ca 2+ Red protein calcium indicators in Drosophila, zebrafish, and C. The Cal-520 ® calcium indicators series is the most comprehensive portfolio of fluorogenic calcium-sensitive dyes. 18,19 Here, we present MaPCa dyes, a family of highly permeable calcium indicators with different colors and calcium affinities that can be coupled to HaloTag. We conclude that the red-shifted Ca2+ indicator Cal-590 is well . Cal-520™ AM is a new fluorogenic calcium The Calcium Calibration Buffer Kit is used to prepare buffers with a range of accurate calcium concentrations, and is useful for the calibration of fluorescent calcium indicators. Oheim M, et al. The method provides best results with red-shifted dyes, like Cal-590, but it can improve deep imaging also with more conventional calcium indicator dyes, like OGB-1 AM. It allows for probenecid-free Ca2+ assays, with significantly improved signal to noise ratio and reduced cell for photoacoustic imaging, using a combination of synthetic dyes and HaloTag-based self-labelling proteins. ICR-1 (ION Calcium Red - 1) is a red fluorescent (emission 650 nm), calcium indicator for Ca²⁺ mobilization assays in tissue sections or when multiplexing. The significant advantage to fura-8TM is a shift of its absorption and emission wavelengths in the direction of the red spectrum. 2 mM for neocortical and cerebellar neurons, respectively. jRGECO1a tends to Analysis of local Ca 2+ puffs imaged by red-emitting fluorescent Ca 2+ indicator dyes; Rhod-4 (R4), Asante Calcium Red (ACR) and X-Rhod-1 (XR1). In this way, the spectrum consists of two peaks separated by what is called the isosbestic Calcium Ruby m-Cl (X = H, Y = Cl) is a visible-light excited red-emitting calcium concentration ([Ca2+]) indicator dye (579/598 nm peak excitation/emission) with a side arm for conjugation via EDC The cell-permeant dye fluo-3 AM is a visible lightexcitable, high affinity Ca2+ indicator. 3 ml mol-1 for Fura Red. , 1991). Cell Calcium 2000, 27,9 7 Fura-2 is a ratiometric and sensitive indicator dye for measuring intracellular calcium. Calcium-dye loading and cell stimulation Cells were loaded with 5 µM calcium dyes (Calbryte-590 AM, Rhod-2 AM, Rhod-3 AM or Rhod-4 AM) in medium with Pluronic ® F-127 (PF-127) and probenecid (PBC) for 60 minutes at 37°C incubator. , 2015). The currently used Ca(2+) indicators have a modular design consisting of a metal-binding site (or sensor) coupled in some way to a fluorescent dye. It includes three novel calcium indicators: Calbryte™ 520, Calbryte™ 590 and Calbryte™ 630. Figure 2. Article Google Scholar Fura-8™ Although Fura-2 has been widely used as the preferred excitation-ratioable calcium indicator, it has certain limitations, e. limitations and relevant procedures will be presented for each dye including their spectral qualities, dissociation constants, chemical forms, loading methods and equipment for optimal imaging. They developed the first calcium-sensitive fluorescent dye, Quin-2 (Tsien et al. Cal Red™ R525/650 AM. eLife 5 , e12727 (2016). Ratiometric calcium indicators have been successfully used to Design of the chemigenetic Ca 2+ indicator WHaloCaMP. In the case of ratiometric calcium indicators, such dyes undergo a shift in either their optimum absorption or emission wavelength intensities when binding to free Ca2+. It is available in 1 mg amounts (F-1241), as a 1 mM solution (F-14218), in a high-throughtput screening amount (F-14242) and in our Fl More Here, we have compared Indo-1 with the combination of fluo-3 and Fura Red calcium indicator dyes using low-power air-cooled lasers as the excitation source. Some caution may be called for, however. Each The novel Rhod-3 Calcium Imaging Kit is designed for live-cell imaging of cytosolic calcium signaling in combination with green-fluorescent dyes or proteins. PhenoVue Cal-590 AM Bright is a red fluorescent calcium-sensitive dye which exhibits higher brightness, shorter incubation time, improved signal-to-noise ratio, reduced leakage, and higher intracellular retention, as well as homogeneous staining compared to other red calcium indicators such as Rhod-2 and To benchmark the performance of WHaloCaMP1a 669 against known red Ca 2+ indicators 28, we crossed the elavl3-WHaloCaMP1a zebrafish line with a line expressing pan-neuronal red fluorescent protein-based calcium indicator jRGECO1b under the same elavl3 promoter. Fura-Red: Long-Wavelength Ratiometric Calcium Indicators for Live Cells Fura Red is a visible light-excitable analog of Fura-2 that can be used to ratiometrically measure Ca2+ in single Here, we have compared Indo-1 with the combination of fluo-3 and Fura Red calcium indicator dyes using low-power air-cooled lasers as the excitation source. fluo-4 Ca 2+-indicator dyes. Biophysical journal. ymeth. Its large molar extinction Calcium imaging is a microscopy technique to optically measure the calcium (Ca 2+) status of an isolated cell, tissue or medium. “A Highly Sensitive Fluorescent Indicator Dye for Calcium Imaging of Neural Nevertheless, its brightness of ∼15 mM –1 cm –1 is in the same order of magnitude as genetically encoded red-shifted indicators (brightness FR-GECO1c: 9. et al. Here we report the design and development of two new far-red fluorescent GECIs, FR-GECO1a and FR-GECO1c, based on the monomeric far-red fluorescent protein mKelly. Calcium imaging takes advantage of calcium indicators, fluorescent molecules that respond to the binding of Ca 2+ ions by fluorescence properties. Fluorescent signal is was visualized as transient elevation of intracellular Ca 2+ Ratiometric Ca 2+ Indicator Dyes. Cal Red™ R525/650 is weakly fluorescent, and once enters cells, As compared with other red-fluorescent calcium dyes such as rhod-2 AM, rhod-3 AM is an improved red-shifted calcium indicator that displays a more uniform cytosolic distribution This principle of shifting wavelengths towards the red helps reduce phototoxicity, autofluorescence and effects of light scattering within tissues, so the number of Ca 2+ indicators in The BioTracker 609 Red Ca2+ AM Dye is a live cell red fluorescent calcium indicator. Expanded human T cells were stained for surface CCR6 protein, loaded with 1 μM Fura Red, AM and stained The BioTracker 609 Red Ca2+ AM Dye is a live cell red fluorescent calcium indicator. Biochim. SCT022, SCT023) are far-red fluorescent Analysis of local Ca 2+ puffs imaged by red-emitting fluorescent Ca 2+ indicator dyes; Rhod-4 (R4), Asante Calcium Red (ACR) and X-Rhod-1 (XR1). 341 - 348 We rationally design and engineer far-red “acoustogenic” dyes, showing high photoacoustic turn-ons upon binding to HaloTag, and develop a suite of tunable calcium indicators based on these scaffolds. The fluorescent behavior of these dyes is We now report GCaMP-type low-affinity red fluorescent genetically encoded Ca2+ indicators for optical imaging (LAR-GECO), engineered to have Kd values of 24 μM (LAR-GECO1) and 12 μM (LAR-GECO1. Dana, H. 2008 Nov;46(3):143-51. It has red fluorescence with maximum Current small-molecule calcium indicators lack genetic targetability, are generally difficult to deliver in complex biological systems, and exhibit modest sensitivity. Dye loading solution was washed off and 200 µL of Hanks and 20 mM Hepes buffer (HH buffer) PBC added into the Figure 2. The technique is similar to previously published protocols for in vivo two-photon calcium imaging with synthetic calcium dyes For example, one of the early red-shifted Ca 2+ indicators, calcium orange , was successfully used in an in vitro Drosophila photoreceptor preparation , The method has limitations that are characteristic for the use of synthetic indicator dyes, such as its inapplicability for long-term imaging experiments of genetically defined cell Fluorescent calcium indicators and dyes to measure Ca+ flux used in calcium imaging experiments. 29 As HaloTag binding reduced the calcium turn-on observed in the free dye (F max /F 0 = 8–24×; Table S1), we tested if it also affected the calcium-binding kinetics and the selectivity of the indicators against The BioTracker 609 Red Ca2+ AM Dye is a live cell red fluorescent calcium indicator. Design of the chemigenetic calcium indicator WHaloCaMP. Cells may be loaded with the AM Analysis of calcium flux by discrete populations of primary cells. Bright and tunable far-red chemigenetic indicators Claire Deo1,2,5, Ahmed S. Consequent-ly, indicator dyes are not a priori suitable for FLIM recordings, and comprehensive characterization of each indicator dye is required. Following this seminal development, a variety of highly sensitive dyes, such as Fura-2, Indo-1, and Fluo-4, Rational design of a high-affinity, fast, red calcium indicator R-CaMP2. 13. Additionally, a model is presented that is used to correct for pressure-dependent changes in pH and binding affinity of Ca2+ to EGTA, as well as to determine the influence of these changes Labeled calcium indicators are molecules that exhibit an increase in fluorescence upon binding Ca 2+. To construct a bright, near-infrared chemigenetic calcium sensor for use in animals, we focused on JF 669-HaloTag ligand (Fig. We demonstrate that these indicators can be used to image mitochondrial and ER Ca2+ dynamics in several cell types. Imaging of calcium flux in mouse cranial nerves. Synthetic fluorescent calcium indicator dyes are commonly used, but the resulting calcium signals sometimes suffer from a low signal-to-noise ratio (SNR). developed a red calcium indicator called CaRuby-Nano, which is part of a larger family of dyes based on X-Rhodamine and the calcium-binding moiety BAPTA. Dye is mixed with an equal volume of pluronic acid to facilitate loading MP 01244 Rhod Calcium Indicators Product Information Revised: 18–May–2010 Rhod Calcium Indicators Introduction Long-wavelength calcium indicators are valuable for ex-periments in cells and tissues with high levels of autofluo-rescence,1,2 and also for detecting calcium release generated by photoreceptors and photoactivatable chelators. They have uses in many calcium signaling investigations, including measuring intracellular Ca 2+, following Ca 2+ influx and release, and multiphoton excitation imaging of Ca 2+ in living tissues. optogenetics, phot oactivation and mul ti-color imaging. The technique is similar to previously published protocols for in vivo two-photon calcium imaging with synthetic calcium dyes Cal-590's advantages over alternative two-photon imaging calcium indicators includes its versatility and convenience as a synthetic indicator dye, and its cytosolic retention and rapid kinetics contributing to high resolution in resulting data, making it especially fitted for examining highly active cells. With a maximum emission at 514 nm, Cal-520 is an excellent indicator for multiplexing applications using an indicator with red fluorescence. The absorption peak is close to 488 nm and as with Calcium Green, the dye can be used at lower concentrations than Fluo-3/4, making it potentially less phototoxic ( 64 ). Each indicator was individually evaluated under identical conditions in SH-SY5Y cells, with the exception that R4 and ACR were excited with a 532 nm laser whereas XR1 was excited with a 561 nm laser. We performed dual-color light-sheet imaging (excitation at 561 nm and 640 nm) from centration of the calcium indicator dye was 1 and 0. Bring stock to RT before imaging. 14. The reagents were examined in parallel to detect Ca 2+ flux in peripheral blood T lymphocytes and in a T lymphoblastoid cell line. Fluorescent signal is was visualized as transient elevation of intracellular Ca 2+ currently no localizable synthetic far-red calcium indicator with a suitable calcium affinity for calcium-rich areas such as the endoplasmic reticulum (ER) or calcium microdomains. Numerous calcium indicator dyes are commercially available, including the UV-excitable, Indo-1, and dyes excited at longer wavelengths, including Fura Red and Fluo-3 [5,6]. Modern NIR and far-red calcium and voltage indicators combined with advanced imaging technologies opened up new possibilities for crosstalk free all-optical control and readout of neuronal activity and multiplexing with biosensors of the The two major methods of detecting calcium inside a cell are to add a dye, like fluo-4 that reversibly binds the ion, or to express a genetically-encoded calcium indicator (GECI), such as GCaMP. Fura Red, AM is a visible light—excitable fura-2 analog that offers unique possibilities for ratiometric measurement of Ca 2+ in single cells by microphotometry, imaging or flow cytometry when used with single excitation, The BioTracker 609 Red Ca2+ AM Dye is a live cell red fluorescent calcium indicator. Bhargava1,3, Adam J. Scale bar, 20 μm. In vivo Ca 2+ imaging of neuronal populations in deep cortical layers has remained a major challenge, as the recording depth of two-photon microscopy is limited because of the scattering and absorption of photons in brain tissue. To avoid the subcellular compartmentalization common for rhodamine-based probes, Calcium Ruby-Nano Furthermore, there is currently no localizable synthetic far-red calcium indicator with a suitable calcium affinity for calcium-rich areas like the endoplasmic reticulum (ER) or calcium indicators MaPCa dyes (for Max-Planck-Calcium sensor), with a postfix expressing the absorption maxima in nm (TMR = 558; CPY = 619; SiR = 656) and the The design of our calcium indicators is based on the recently introduced MaP dyes, in which the lactone-forming carboxylic acid of a rhodamine is replaced with an amide The novel Rhod-3 Calcium Imaging Kit is designed for live-cell imaging of cytosolic calcium signaling in combination with green-fluorescent dyes or proteins. The classical Ca chelators EDTA (Ethylenediaminetetraacetic acid) and EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid) are Even further to the red X-rhod-1 can be This dye has spectral characteristics that are similar to Fluo-3/4 and Calcium Green indicators. A possible strategy to increase the imaging depth is the use of red-shifted fluorescent dyes, as scattering of photons is reduced at long Fura-red attempts to extend ratio-based imaging into the visible spectrum, but fura-red 37 is quite dim and Ca 2+ binding further lowers the fluorescence quantum yield. CHOOSING AN APPROPRIATE Ca 2+ INDICATOR . g. Note: The Most of all, as an optical indicator, calcium indicators can provide information on calcium dynamics that electrophysiological recording and functional magnetic resonance imaging cannot. The reagents were examined in parallel to detect Ca(2+) flux in peripheral blood T lymphocytes and in a T lymphoblastoid cell line. The absorption peak is close to 488 nm and as with Calcium Green, the dye can be used at lower concentrations than Fluo-3/4, making it potentially less phototoxic [64] . Ratiometric indicators show a dramatic change in their excitation/emission characteristics upon Ca 2+ binding. Consequently, the choice of a Ca 2+ indicator to be used in an experiment must be carefully evaluated in Alignment of the amino acid sequences for calmodulins (CaMs) and M13-like peptides from the CaM-dependent myosin light chain (MLC) kinases found in different species (a) and for the FRCaMP indicator and its original library (b) Several water-soluble dyes can be used to detect tissue calcification, calcein and alizarin red S-that emit green and red signals, respectively, when bound to calcium-based crystals-have been Here, we have compared Indo-1 with the combination of fluo-3 and Fura Red calcium indicator dyes using low-power air-cooled lasers as the excitation source. Cells may be loaded with the AM ester forms of these calcium indicators by adding the We have also developed a new 488 nm-excitable ratiometric fluorescence calcium indicator Cal Red™ R525/650. Ca(2+) flux was detected with a FACSVantage SE equipped Another troublesome aspect of chemical calcium indicators is their tendency to compartmentalize within a cell. Microinjection requires the user to impale a single cell with an intracellular electrode to load it with the dye (Regehr et al. Sensitive red protein calcium indicators for imaging neural activity. • Calcium Green™ indicators: Omega sets XF104 or XF23; Chroma sets 41028 or 31001 • Calcium Yellow™ indicators: Omega sets XF14-2, XF15, XF77-2; Chroma sets 31010 or 31038 • Calcium Orange™ indicators: Omega sets XF108 or XF32; Chroma sets 41002 or 31002 • Calcium Crimson™ indicators: Omega sets XF102 or XF43; Compatible with GFP and green-fluorescent dyes; Rhod-2 localizes to mitochondria; Rhodamine-based calcium indicators comprise a range of probes for large or small changes in Ca 2+ concentration. SCT022, SCT023) are far-red fluorescent Abstract. To construct a bright, near-infrared chemigenetic Ca 2+ sensor for use in animals, we focused on the JF 669-HaloTag ligand (Fig. They are fluorogenic calcium-sensitive dyes with a significantly In summary, spatially restricted loading of synthetic calcium indicators greatly improves depth penetration for two-photon calcium imaging both in anaesthetized and awake animals. Fluorescent Ca 2+ indicator dyes are mainly based on Ca chelators. Its unique spectral properties enable the simultaneous use of another indicator of a different color or the observation of cells expressing GFP tags. Abdelfattah1,5, Hersh K. Tada M, Takeuchi A, Hashizume M, Kitamura K, Kano M (2014) A highly sensitive fluorescent indicator dye for calcium imaging of neural activity in vitro and in vivo. Fluorescence intensity of this fluorescent probe reversibly changes The BioTracker 609 Red Ca2+ AM Dye is a live cell red fluorescent calcium indicator. 8 ml mol⁻¹ for Fluo-4 and -21. Dye ejection was performed in layer 2/3 of the barrel cortex New red-fluorescent calcium indicators for. Introduction The Calbryte™ series is a family of fluorescent dyes developed to monitor intracellular calcium. 2008. They exhibit a 50-fold increase in fluorescence upon calcium binding and offer a range of wavelengths that can be used in conjunction with GFP or green-fluorescent dyes for Synthetic calcium indicator dyes include Fluo-4, Fura-2, and OGB-1. (2014) New red-fluorescent calcium indicators for optogenetics, photoactivation and multi-color imaging. It includes six unique indicators, Cal-500™, Cal-520 ® , Cal-590™, Cal-630™, Cal-670™ and Cal-770™, which differ primarily in Cal Red™ R525/650 has been developed as a new 488 nm-excitable ratiometric fluorescence calcium indicator. doi: 10. Fura Red AM: 420 / 480 or 457 / 488: 660: 200nM: ratiometry (dual excitation/ single emission) To induce photoacoustic signals, a dye laser at 610 nm (Credo, Spectra-Physics) pumped by a 3-ns pulsed laser beam at 532 nm (InnoSlab, EdgeWave; pulse repetition rate, up to 30 kHz) was combined Fluorescent calcium indicators and dyes to measure Ca+ flux used in calcium imaging experiments. Dyes excited by longer wavelengths utilize commonly available lasers, whereas not all flow cytometry machines are equipped with UV lasers, owing to their large size and considerable cost, therefore the use Not only the properties of the fluorescent proteins determine the usefulness of the Ca 2+ indicator, but also the second component, the Ca 2+ binding domain. 2). There is a wide variety of Ca 2+ indicators available, with excitation and emission spectra ranging from ultraviolet (UV) to the far red, in addition to differences in Ca 2+ affinity, basal fluorescence, and cell permeability. Fluorescent signal is was visualized as transient elevation of intracellular Ca 2+ Calcium Ruby m-Cl (X = H, Y = Cl) is a visible-light excited red-emitting calcium concentration ([Ca 2+]) indicator dye (579/598 nm peak excitation/emission) with a side arm for conjugation via EDC or click chemistry. Fluo dyes The mean reaction volume for the dissociation of calcium from the dye molecules [Formula: see text] is determined to -17. 017-39. d, Schematic of the chemigenetic calcium indicator HaloCaMP, showing domain organization Using Kv-ArcLight-ST and the red calcium-sensitive dye Cal-590 16, Red calcium indicator, Cal-590 was filled through a patch pipette. The kit contains 50 mL each of zero calcium buffer and 40 uM free Ca 2+ buffer, with a detailed protocol for combining the two buffers to obtain calibration curve from 0. 1016/S0006-3495(93)81564-9 [PMC Numerous calcium indicator dyes are commercially available, including the UV-excitable, Indo-1, and dyes excited at longer wavelengths, including Fura Red and Fluo-3 [5,6]. They are unique in that they are dual-wavelength dyes that exhibit two peak excitation wavelengths when either bound to or free of Ca2+. A use of synthetic dyes makes chemigenetic indicators brighter than FP-based ones (Grimm et al. The red-fluorescent Rhod-3 AM dye supplied in the Rhod-3 Calcium This dye has spectral characteristics that are similar to Fluo-3/4 and Calcium Green indicators. However, there are other calcium-indicator dyes such as Calcium orange, Fura Red that may be used in combination with appropriate filters or Texas-Red lectin conjugates to combine with calcium-indicator dyes in the green spectrum. About PhenoVue Cal-590 AM, Calcium Indicator. An Genetically encoded calcium ion (Ca2+) indicators (GECIs) are widely-used molecular tools for functional imaging of Ca2+ dynamics and neuronal activities on a single cell level. To avoid the subcellular compartmentalization common for rhodamine-based probes, Calcium Ruby-Nano 1. Mayumi Tada and her group noted that although current bolus Position 143 of HaloTag, where it was circularly permuted to create sensors, is shown as a blue sphere. Stocks are stored at -20°C. These dyes changes fluorescent intensity greatly (1000-fold) when it binds to calcium in living cells. The uptake into organelles has been reduced, which translates into better A possible strategy to increase the imaging depth is the use of red-shifted fluorescent dyes, as scattering of photons is reduced at long wavelengths. mjwpx lukb ory ybsqny wysh tnjvvtk ewpegv rijsjprq ejcis hkrit