Near Infrared iFluor® Dyes for In Vivo Imaging

In vivo fluorescence imaging uses specialized cameras to detect the fluorescence emission from fluorophores in whole-body living small animals. To overcome the photon attenuation in living tissue, fluorophores with long emissions at the near-infrared (NIR) region are generally preferred, such as small indocyanine dyes. Recent advances in imaging strategies and reporter techniques for in vivo fluorescence imaging include novel approaches to improve the probes' specificity and affinity and modulate and amplify the signal at target sites for enhanced sensitivity. Further developments aim to achieve high-resolution, multimodality, and lifetime-based in vivo fluorescence imaging.

Near-Infrared iFluor^® Dyes

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iFluor^® 740, iFluor^® 750, iFluor^® 770, iFluor^® 780, iFluor^® 790, iFluor^® 800, iFluor^® 810, iFluor^® 820, iFluor^® 840, and iFluor^® 860 - marks the next generation of long-wavelength dyes for deep-tissue imaging, small animal imaging, NIR Western blotting and multicolor flow cytometry. These exceptionally bright and photostable dyes feature absorption and emission wavelengths in the NIR region, between 700 and 1000 nm. In this scope, cellular and tissue components exhibit lower absorption of NIR light, significantly reducing photon scattering and autofluorescence and enabling deeper tissue penetration and higher signal-to-noise ratios. Owing to its excellent sensitivity and spatiotemporal resolution, optical imaging in the NIR window provides enormous potential for detecting disease and monitoring therapy non-invasively. Moreover, it allows for assessing treatment efficacy and thereby facilitates the potential to adjust and customize treatment in vivo.

iFluor^® near-infrared fluorescent dyes are used for protein and cellular assays, flow cytometry, Western blot, whole organ or tissue section assays, and in vivo molecular imaging in animals for research purposes. These dyes are available as stand-alone reagents in a variety of reactive formats and as components of our ReadiLink™ labeling kits for labeling proteins, antibodies, nucleic acids, and other biomolecules. Benefits of near-infrared iFluor^® dyes and their conjugates include:

• Excellent spectral match to common long-wavelength excitation sources
• Brighter fluorescence and more photostability than Alexa Fluor^®, Cy7^®, and other spectrally similar fluorophores
• High water solubility permitting conjugations to be performed without organic solvents resulting in conjugates that are resistant to precipitation and aggregation
• pH-insensitive fluorescence over a broad pH range
• Low fluorescence quenching upon conjugation to proteins, even at high degrees of substitution
• Compatible with popular instruments, including LI-COR Odyssey^®, PerkinElmer^® IVIS^®, Olympus OV-100, and FMT^® systems
• NIR iFluor^® dye applications include in vivo imaging, near-IR Western blotting, In-Cell Western™ assays, and multicolor flow cytometry

iFluor^® 700

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iFluor^® 700 is a near-infrared emitting synthetic fluorophore similar to Alexa Fluor^® 700. With an absorption maximum at 690 nm, iFluor^® 700 can be excited with a xenon-arc lamp, far-red diode lasers, or dye-pumped lasers operating in the 630 to 700 nm range. The dim fluorescence of iFluor^® 700 makes it useful in flow cytometry for staining highly expressed antigens, allowing brighter dyes to be used for less-abundant targets. In multicolor flow cytometry panel design, iFluor^® 700 fits between APC and APC-iFluor^® 750 (i.e., all three fluorophores can be simultaneously excited by the 633 nm spectral line).

Spectral Properties

• Abs/Ex maxima: 690/713 nm
• Molecular Weight: 977.16
• Extinction Coefficient: 220,000 cm^-1M^-1
• Quantum Yield: 0.23
• Laser Lines: 633 to 700 nm
• Correction Factor_{280 nm}: 0.04
• Correction Factor_{260 nm}: 0.09
• Spectrally Similar Dyes: Alexa Fluor^® 700 and DyLight^® 700

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iFluor^® 710

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The near-infrared iFluor^® 710 dye has an absorption maximum at 717 nm and an emission maximum at 739 nm. Because of its broad absorption profile, iFluor^® 710 can be excited with a xenon-arc lamp, far-red diode lasers, or dye-pumped lasers operating in the 630 to 720 nm range. The fluorescence emission of iFluor^® 710 is well separated from commonly used red fluorophores, such as TRITC, Texas Red^®, Cy5, phycoerythrin, iFluor^® 594, and iFluor^® 647. Tandem conjugates of iFluor^® 710 with phycoerythrin further expand the utility of this dye in multicolor flow cytometry. The multicolor panel of FITC, PE, PE-Cy5, and PE-iFluor^® 710 permits the simultaneous detection of four parameters using the argon-ion laser (488 nm).

Spectral Properties

• Abs/Ex maxima: 717/739 nm
• Molecular Weight: 1206.39
• Extinction coefficient: 190,000 cm^-1M^-1
• Laser Lines: 633 to 725 nm
• CF_{280 nm}: 0.07
• CF_{260 nm}: 0.12

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iFluor^® 720

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iFluor^® 720 dye is a near-IR dye with an absorption maximum at 716 nm and an emission maximum at 740. Although its spectra are virtually identical to iFluor^® 710 (maxima vary by 1 nm), the structure and performance of iFluor^® 720 differ quite significantly, especially when developing tandem dyes. The amine-reactive succinimidyl ester of iFluor^® 720 dye is available in a 1 mg unit size (Cat No. 1046).

Spectral Properties

• Abs/Ex maxima: 716/740 nm
• Molecular Weight: 1089.29
• Extinction coefficient: 140,000 cm^-1M^-1
• Quantum Yield: 0.14
• Laser Lines: 633 to 725 nm

 

iFluor^® 740

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With a 742/764 nm excitation/emission maxima, the near-IR fluorescent iFluor^® 740 dye is ideally excited by a xenon-arc lamp or dye-pumped lasers operating in the 720 to 750 nm range and well-separated from far-red dyes, such as Alexa Fluor^® 647 and iFluor^® 647, for multicolor flow cytometry. iFluor^® 740 dye exhibits a high FRET efficiency with PE and APC, producing brighter, more photostable tandems than Alexa Fluor^® 700. The amine-reactive succinimidyl ester of iFluor^® 740 dye is available in a 1 mg unit size (Cat No. 1047).

Spectral Properties

• Abs/Ex maxima: 742/764 nm
• Molecular Weight: 1551.19
• Extinction coefficient: 125,000 cm^-1M^-1

 

iFluor^® 750

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iFluor^® 750 is a bright and photostable near-IR fluorescent dye spectrally identical to Alexa Fluor^® 750 and Cy7. With a peak excitation at 757 nm and maximum emission at 779 nm, iFluor^® 750 is optimally excited by a xenon-arc lamp or dye-pumped laser operating in the 720 to 750 nm range. Its long-wavelength emission is well separated from commonly used far-red fluorophores, such as Alexa Fluor^® 647,iFluor^® 647, and APC, facilitating multicolor analysis. iFluor^® 750 is available as a stand-alone reagent in a variety of reactive formats (e.g., succinimidyl ester, maleimide, hydrazide, and amine), as a component in our ReadiLink™ antibody labeling kit, and conjugated to a variety of antibodies, proteins, tracers, and amplification substrates. iFluor^® 750 tandems conjugated to primary and secondary antibodies are commonly used in multicolor flow cytometry.

Spectral Properties

• Abs/Ex maxima: 757/779 nm
• Molecular Weight: 1416.83
• Extinction coefficient: 275,000 cm^-1M^-1
• Quantum Yield: 0.12
• Laser Lines: 633 to 685 nm, 720 to 750 nm
• CF_{280 nm}: 0.039
• CF_{260 nm}: 0.044
• Replaces: Alexa Fluor^® 750 and Cy7

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iFluor^® 770

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iFluor^® 770 is an exceptionally bright and photostable NIR dye with a peak excitation at 777 nm and maximum emission at 797 nm. It is well-excited by the 785 nm laser line used in flow cytometers, fluorescence microscopes, and gel imaging systems, such as the LI-COR^® Odyssey^®. The long-wavelength emission of iFluor^® 770 is ideally suited for in vivo imaging, multicolor Western blotting with iFluor^® 680 dye, In-Cell Western^®, immunohistochemistry (IHC), and flow cytometry. The amine-reactive succinimidyl ester and thiol-reactive maleimide iFluor^® 770 dyes are available as stand-alone reagents in 1 mg unit sizes.

Spectral Properties

• Abs/Ex maxima: 777/797 nm
• Molecular Weight: 1082.27
• Extinction coefficient: 250,000 cm^-1M^-1
• Laser Lines: 785 nm
• Replaces: IRDye800 and DyLight^® 800
• Instruments: LI-COR^® Odyssey^®

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iFluor^® 780

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iFluor^® 780 is an exceptionally bright and photostable NIR dye with a peak excitation at 784 nm and maximum emission at 808 nm. It is optimally excited by the 785 nm laser line used in flow cytometers, fluorescence microscopes, and gel imaging systems, such as the LI-COR^® Odyssey^®. The long-wavelength emission of iFluor^® 780 is ideally suited for in vivo imaging, multicolor Western blotting with iFluor^® 680 dye, In-Cell Western^®, immunohistochemistry (IHC), and flow cytometry. The amine-reactive succinimidyl ester and thiol-reactive maleimide iFluor^® 770 dyes are available as stand-alone reagents in 1 mg unit sizes.

Spectral Properties

• Abs/Ex maxima: 784/808 nm
• Molecular Weight: 1526.90
• Extinction coefficient: 250,000 cm^-1M^-1
• Laser Lines: 785 nm
• Replaces: IRDye800 and DyLight^® 800
• Instruments: LI-COR^® Odyssey^®

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iFluor^® 790

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iFluor^® 790 is a near-infrared fluorescence dye. Conjugates prepared with iFluor^® 790 have an excitation and emission spectra similar to indocyanine green (ICG) and the IRDye^® 800 dye, with excitation/emission maxima at 783/814 nm. The emission of iFluor^® 790 is well separated from commonly used far-red fluorophores such as Cy5, Cy7, or allophycocyanin (APC), facilitating multicolor analysis. This fluorophore is also useful for small animal in vivo imaging applications or other imaging applications requiring NIR detections, such as two-color Western blotting using the LI-COR^® Odyssey^® infrared imaging system. The carbonyl-reactive iFluor^® 790 hydrazide (Cat No. 1364) can be conveniently used for labeling antibodies and other amine-containing biomolecules.

Spectral Properties

• Abs/Ex maxima: 787/812 nm
• Molecular Weight: 1768.30
• Extinction coefficient: 250,000 cm^-1M^-1
• Quantum Yield: 0.13
• CF_{280 nm}: 0.09
• CF_{260 nm}: 0.1
• Laser Lines: 785 nm
• Replaces: IRDye800 and DyLight^® 800
• Instruments: LI-COR^® Odyssey^®

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iFluor^® 800

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With an absorption and emission maxima at 801 and 820 nm, iFluor^® 800 is an ideal NIR fluorophore for FACS sorting, confocal microscopy, Western blotting, In-Cell Western™ assays, and, more importantly, in vivo imaging of biological targets and diseases. Because autofluorescence, absorption, and light scattering in cells and tissues are dramatically reduced in the NIR-I window (700-950 nm), iFluor^® 800 affords higher spatial resolution, greater image contrast, and deeper tissue penetration than visible fluorescence imaging. Conjugates of iFluor^® 800 are optimally excited by the 785 nm laser and well excited by a xenon-arc lamp or dye-pumped lasers operating in the 720-750 nm range. The long-wavelength emission of iFluor^® 800 is well separated from the red and far-red fluorophores, such as iFluor^® 647, iFluor^® 660, Alexa Fluor^® 647, Alexa Fluor^® 680, and APC, facilitating multicolor analysis. The amine-reactive succinimidyl ester and thiol-reactive maleimide iFluor^® 800 dyes are available as stand-alone reagents for labeling proteins, antibodies, and other biomolecules with NIR fluorescence. Also available are iFluor™ 800 conjugates of secondary antibodies, streptavidin, and CD antibodies.

Spectral Properties

• Abs/Ex maxima: 801/820 nm
• Molecular Weight: 1541.91
• Extinction coefficient: 250,000 cm^-1M^-1
• Quantum Yield: 0.11
• CF_{280 nm}: 0.08
• CF_{260 nm}: 0.03/li>

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