Indocyanine green (ICG) is a negatively charged, tricarbocyanine dye that is widely exploited for its low toxicity and infrared fluorescence emission. ICG's minimal toxicity is attributed to its strict binding with plasma proteins. This confines it to the vascular compartment where it is rapidly metabolized by the liver and excreted exclusively by the liver and bile ducts. ICG and ICG derivatives can be used as in vivo molecular imaging probes. Their infrared fluorescence emission penetrates tissues several millimeters to facilitate deep tissue and small animal in vivo imaging. ICG is used in medical diagnostics for determining cardiac output, hepatic function, and liver blood flow and for ophthalmic angiography. In addition, ICG has been conjugated to several antibodies for cancer cell and tumor targeting.
Spectral Properties of Indocyanine Green
Absorbance and emission maxima of indocyanine green-OSU (Cat No. 182)
|Laser Line:||785 nm laser|
|Extinction Coefficient (ε):||230,000 cm-1M-1|
|Quantum Yield (Φ):||0.04|
|Correction Factor at 260 nm:||0.112|
|Correction Factor at 280 nm:||0.072|
|Half-life:||150 to 180 seconds|
|LD50:||50-80 mg/kg for animals|
iFluor® 790 - superior alternative to ICG & Alexa Fluor® 790
iFluor® 790 is the brightest near-IR dye available that is spectrally similar to indocyanine green (ICG), Alexa Fluor® 790, and IRDye® 800 dye. The excellent water-solubility and salt tolerance, pH-insensitive fluorescence and photostable signal generation of iFluor® 790 dye is superior for high density labeling applications, Western blotting and immunocytochemical (ICC) assays, as well as for protein arrays, microscopy, in vivo imaging and IR optical probe development. The emission of iFluor® 790 is well separated from commonly used far-red fluorophores such as Cy5 (Cat No. 151), Cy7 (Cat No. 161) or allophycocyanin (ReadiUse™ APC, Cat No. 2503), facilitating multicolor analysis. iFluor® 790 is favorable for small animal in vivo imaging applications or for other imaging applications that require NIR detections such as the two-color western applications with the LI-COR® Odyssey® infrared imaging system.
HL-60 cells were incubated with (Red, +) or without (Green, -) Anti-human HLA-ABC (W6/32 mAb), followed by iFluor® 790 goat anti-mouse IgG conjugate (Cat No. 16790). The fluorescence signal was monitored using ACEA NovoCyte flow cytometer in APC-Cy7 channel.
iFluor® 790 is available in different reactive dye formats and in a ReadiLink™ labeling kit that can be used to label proteins, antibodies, peptides and nucleotides with bright IR fluorescence. Also available is the iFluor® 790 dye conjugated to a variety of antibodies, streptavidin and amplification substrates optimized for cellular labeling and detection.
Table 1. iFluor® 790 - superior alternative to ICG & Alexa Fluor® 790
|iFluor® 790, SE||786 nm||811 nm||250,000||0.13||0.1||0.09|
|ICG-OSu||788 nm||813 nm||230,000||0.04||0.112||0.072|
|Alexa Fluor® 790, NHS Ester||784 nm||814 nm||260,000||Not Listed||0.09||0.08|
|IRDye® 800 CW NHS Ester||774 nm||789 nm||240,000||Not Listed||0.03||0.03|
- ε = extinction coefficient at their maximum absorption wavelength. The units of extinction coefficient are cm-1M-1.
- Φ = fluorescence quantum yield in aqueous buffer (pH 7.2).
- CF260 nm is the correction factor used for eliminating the dye contribution to the absorbance at 260 nm.
- CF280 nm is the correction factor used for eliminating the dye contribution to the absorbance at 280 nm (for peptides and protein labeling).
Alternative Forms Available
Indocyanine green fluorescent dyes in combination with IR fluorescence imaging significantly enhances assay sensitivity. By utilizing fluorescent systems designed to operate within infrared wavelengths between 700 and 1000 nm, both light scattering and autofluorescence are drastically reduced due to the minimal absorption of biomolecules in this region. The resulting low background interference and high signal-to-noise ratios improves detection sensitivity.
We offer a broad selection of sulfonated and non-sulfonated, as well as, PEG-modified indocyanine green derivatives and reactive dyes for labeling proteins, antibodies, peptides and other biomolecules with IR fluorescence. Indocyanine green reactive forms available include:
- OSu: used for labeling primary and secondary amines (-NH2).
- Maleimide: used for labeling biomolecules that contain free sulfhydryl groups (-SH).
- Amine: used to label biomolecules that contain a carbonyl group (e.g. aldehyde or a carboxy group).
- Alkyne and azide: used to label biomolecules via click-chemistry.
- Hydrazide: used to label biomolecules via Schiff base chemistry.
Non-Sulfonated Indocyanine Green
Derivatives of non-sulfonated indocyanine green are well-suited for applications in organic media due to their very low aqueous solubility. These derivatives require the use of organic co-solvents, such as 5-20% DMF or DMSO, for efficient biomolecule labeling. When labeling biomolecules with indocyanine green derivatives, first dissolve dye in organic solvent and then added to a solution of biomolecule in appropriate aqueous buffer.
Sulfonated Indocyanine Green
Sulfonated indocyanine green derivatives contain sulfo-groups to facilitated solubility of dye in aqueous solution. Addition of charged sulfonate groups reduces the aggregation of free dye molecules and heavily labeled conjugates. Sulfonated indocyanine green dyes are highly water-soluble and do not require the use of organic co-solvents when labeling.
PEG-Modified Indocyanine Green
PEG-modified (or PEGylated) indocyanine green derivatives contain spacer arms comprised of poly(ethylene glycol), or PEG. This spacer arm increases the solubility by making indocyanine green derivatives with uncharged reactive groups soluble or those with charged reactive groups more soluble. Additionally PEG-modification of indocyanine green dyes can increase the hydrophobicity of the indocyanine green dye and significantly reduce unspecific binding.
Table 2. Chemical and physical properties of indocyanine green and indocyanine green derivatives.
|Indocyanine Green||788/813||774.96||Plasma Proteins||Non-Sulfonated|
|ICG acid||788/813||844.98||Amine (-NH2) and -OH||Non-Sulfonated|
|ICG alkyne||788/813||768.03||Click Chemistry||Sulfonated|
|ICG amine||788/813||1001.08||Carbonyl (-COOH)||Non-Sulfonated|
|ICG azide||788/813||799.05||Click Chemistry||Sulfonated|
|ICG hydrazide||788/813||973.04||Schiff Base Chemistry||Sulfonated|
|ICG Maleimide||788/813||853.09||Sulfhydryl or Thiol (-SH)||Non-Sulfonated|
Product Ordering Information
Table 3. Ordering Info for ICG Dyes Products
|91||Indocyanine Green *CAS 3599-32-4*||10 mg|
|96||Indocyanine Green *CAS 3599-32-4*||10 g|
|181||ICG-ATT [3-ICG-acyl-1,3-thiazolidine-2-thione]||1 mg|
|186||ICG Xtra-OSu||1 mg|
|187||ICG Maleimide||1 mg|