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DiR iodide [1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide]
DiR dyes are near-infrared (NIR) fluorescent probes engineered for live cell plasma membrane labeling in imaging applications such as confocal microscopy or in vivo small-animal studies. By combining two C18 hydrocarbon tails for stable membrane anchoring with a tricarbocyanine backbone emitting ~775–785 nm, these dyes decrease background autofluorescence and enhances morphological fidelity. Because NIR excitation energy is lower than visible wavelengths, it reduces photobleaching and cellular stress. Carbocyanine analogs with extended hydrocarbon tails can remain in membranes for hours or days, allowing multi-hour time-lapse experiments without re-dyeing. DiR’s emission range also prevents overlap with standard GFP/RFP channels, making multiparametric experiments possible where near-infrared labeling is paired with fluorescent cell cycle reporters. Furthermore, deeper tissue penetration and reduced scattering have been demonstrated in NIR imaging of organs like the colon epithelium, enabling clearer 3D organoid or embryonic system visualizations (Okada, 2013). By bringing together stable bilayer association, lower phototoxicity, and minimal spectral interference with visible fluorophores, DiR stands out as an optimal solution for prolonged, low-background labeling in delicate or complex tissue contexts. Instrument-wise, it is compatible with modern systems that include NIR channels, promoting better tissue penetration and fewer spectral conflicts.
Comparison of DiI, DiO, DiD and DiR
|
Dye |
Excitation (nm) |
Emission (nm) |
Color |
Primary Use |
|
DiO |
~484 |
~501 |
Green |
Plasma membrane, neuronal tracing |
|
DiI |
~550–565 |
~565–595 |
Orange-Red |
Long-term cell labeling, tracing |
|
DiD |
~646 |
~663 |
Far-Red |
Deeper tissue imaging |
|
DiR |
~750–760 |
~775–785 |
Near-Infrared |
Low autofluorescence, deep imaging |
All carbocyanine dyes (DiI, DiO, DiD, DiR) employ a similar principle of lipophilic insertion into plasma membranes.
Core Features of DiR
- Structure
- Carbocyanine Family: DiR contains a tricarbocyanine chromophore responsible for its near-infrared fluorescence.
- Long Alkyl Tails: Two C18 hydrocarbon chains promote insertion into lipid bilayers and enhance dye stability.
- Molecular Weight
- Iodide Salt: 1013.39 g/mol. This relatively high molecular weight underpins robust hydrophobic interactions that keep DiR locked in membranes rather than diffusing intracellularly.
- Color and Spectral Properties
- Appearance: Colorless in solution; near-infrared fluorescence observed upon excitation.
- Extinction coefficient: 270000 cm -1 M -1
- Excitation: 754 nm
- Emission: 778 nm
(Exact spectral peaks vary slightly with solvent, concentration, or environment.)
- Lipophilic Dye Mechanism
- Membrane Partitioning: When added to cells or tissues, DiR diffuses from the medium into the lipid bilayer, anchoring via its hydrophobic tails.
- Stable Labeling: Once embedded, it remains in the plasma membrane for hours to days, enabling time-lapse and long-term studies without extensive signal loss.
- Experimental Advantages
- Reduced Phototoxicity: NIR excitation is lower in energy than visible wavelengths, reducing cellular stress and photodamage—beneficial for delicate or primary cells.
- Multiplex Compatibility: Because DiR emits in the near-infrared, it can be paired with visible fluorophores such as GFP or TRITC with minimal overlap. This feature supports multi-parameter experiments in confocal or wide-field setups.
- Low Detection Limit: 2×104 cells labeled with 5–10 µg/mL DiR were successfully detected ex vivo (Berninger et al., 2017).
Applications
Additional Notes
Co-labeling with DNA Synthesis Markers
Although DiR does not monitor proliferation itself, it can be combined with BrdU or EdU to discriminate dividing cells while preserving membrane morphology (Mort et al., 2014).
Deep-Tissue and In Vivo Applications
NIR excitation/emission fosters improved tissue penetration. Studies leveraging near-infrared dyes have shown success in murine models for live tracking of cell migration or tumor homing (Frangioni, 2003).
Multi-channel Imaging
As DiR rarely overlaps with fluorophores that excite <700 nm, you can freely combine it with standard green or red labels to gather multi-parametric data.
Concentration and Incubation
Typical concentration range: 1–10 µM; short exposures (5–20 min) to mitigate nonspecific uptake (Chazotte, 2011).
Safety and Cytotoxicity
Lipophilic dyes like DiR are generally low-toxicity at standard usage levels (Baguley and Lehnert, 1991). Pilot tests are recommended for each cell line.
Frequently Asked Questions (FAQ)
Further Reading
Baguley, Bruce C., and Susan Lehnert. Cell Membrane Labelling with Lipophilic Dyes. Labelling Techniques in Cell Biology, vol. 2, Academic Press, 1991, pp. 75–91.
Barrasso, Anthony P., et al. “Live Imaging of Developing Mouse Retinal Slices.” Neural Development, vol. 13, no. 1, 2018, p. 23, doi:10.1186/s13064-018-0120-y. (Illustrates organotypic live imaging approaches).
Berninger, Markus T., et al. “Detection of Intramyocardially Injected DiR-Labeled Mesenchymal Stem Cells by Optical and Optoacoustic Tomography.” Photoacoustics, vol. 6, 2017, pp. 37–47, doi:10.1016/j.pacs.2017.04.002.
Campanale, Joseph P., et al. “Methods to Label, Isolate, and Image Sea Urchin Small Micromeres, the Primordial Germ Cells (PGCs).” Methods in Cell Biology, vol. 150, 2019, pp. 269–292, doi:10.1016/bs.mcb.2018.11.007. (Discusses various fluorescent labels and live imaging).
Chazotte, Bruce. “Lipophilic Dyes for Membrane Labeling.” Cold Spring Harbor Protocols, 2011, pdb.top66, doi:10.1101/pdb.top66.
Chen, R. F., and A. H. Coons. “Fluorescent Antibody, a New Tool for Immunology.” Experimental Cell Research, vol. 13, no. 2, 1957, pp. 276–278. (Earlier demonstration of fluorescent labeling, conceptual basis).
Frangioni, John V. “In Vivo Near-Infrared Fluorescence Imaging.” Current Opinion in Chemical Biology, vol. 7, no. 5, 2003, pp. 626–634, doi:10.1016/j.cbpa.2003.08.007.
Haugland, Richard P. Handbook of Fluorescent Probes and Research Chemicals. 10th ed., Invitrogen/Molecular Probes, 2005.
Honig, M. G., and R. I. Hume. “DiI and DiO: Versatile Fluorescent Dyes for Neuronal Labeling and Pathway Tracing.” Journal of Neuroscience Methods, vol. 10, no. 1, 1989, pp. 3–14, doi:10.1016/0165-0270(89)90059-8.
Ji, Yuanyuan, et al. “Near-Infrared Fluorescence Imaging in Immunotherapy.” Advanced Drug Delivery Reviews, vol. 158, 2020, pp. 62–82, doi:10.1016/j.addr.2020.06.008.
Kim, Jin Hee, et al. “Zinc Chelation Reduces Hippocampal Neurogenesis after Pilocarpine-induced Seizure.” PLoS ONE, vol. 7, no. 2, 2012, e30691, doi:10.1371/journal.pone.0048543. (Broad use of labeling in a neurological context).
Krzastek, Sarah C., et al. “Dendritic Cell Trafficking in Tumor-Bearing Mice.” Cancer Immunology, Immunotherapy, vol. 67, no. 12, 2018, pp. 1939–1947, doi:10.1007/s00262-018-2187-z.
Lakowicz, Joseph R. Principles of Fluorescence Spectroscopy. 3rd ed., Springer, 2006.
Li, Xiaoli, et al. “Targeted Migration of Bone Marrow Mesenchymal Stem Cells Inhibits Silica-Induced Pulmonary Fibrosis in Rats.” Stem Cell Research & Therapy, vol. 9, no. 1, 2018, p. 335, doi:10.1186/s13287-018-1083-y.
Macks, Christian, et al. “Rolipram-Loaded Polymeric Micelle Nanoparticle Reduces Secondary Injury after Rat Compression Spinal Cord Injury.” Journal of Neurotrauma, vol. 35, no. 3, 2018, pp. 582–592, doi:10.1089/neu.2017.5092.
Mort, Richard L., et al. “Fucci2a: A Bicistronic Cell Cycle Reporter that Allows Cre Mediated Tissue Specific Expression in Mice.” Cell Cycle, vol. 13, no. 17, 2014, pp. 2681–2696, doi:10.4161/15384101.2015.945381. (Demonstrates combination of cell cycle probes with membrane labeling strategies).
Noack, Anne-Kathrin, et al. “Intratumoral Distribution and pH-Dependent Drug Release of High Molecular Weight HPMA Copolymer Drug Conjugates Strongly Depend on Specific Tumor Substructure and Microenvironment.” International Journal of Molecular Sciences, vol. 21, no. 17, 2020, p. 6029, doi:10.3390/ijms21176029.
Wang, Qi-Long, et al. “Blood Exosomes Regulate the Tissue Distribution of Grapefruit-Derived Nanovector via CD36 and IGFR1 Pathways.” Theranostics, vol. 8, no. 18, 2018, pp. 4912–4924, doi:10.7150/thno.27608.
Wu, Dayong, et al. “Determination of DNA Lesion Bypass Using a ChIP-based Assay.” DNA Repair, vol. 108, 2021, p. 103230, doi:10.1016/j.dnarep.2021.103230. (General advanced imaging reference, though not DiR-specific).
Zhao, Xueli, et al. “A Non-Invasive Osteopontin-Targeted Phase Changeable Fluorescent Nanoprobe for Molecular Imaging of Myocardial Fibrosis.” Nanoscale Advances, vol. 6, no. 14, 2024, pp. 3590–3601, doi:10.1039/d4na00042k.
Calculators
Common stock solution preparation
| 0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
| 1 mM | 98.679 µL | 493.393 µL | 986.787 µL | 4.934 mL | 9.868 mL |
| 5 mM | 19.736 µL | 98.679 µL | 197.357 µL | 986.787 µL | 1.974 mL |
| 10 mM | 9.868 µL | 49.339 µL | 98.679 µL | 493.393 µL | 986.787 µL |
Molarity calculator
| Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
| / | = | x | = |
Spectrum
Product family
| Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Quantum yield |
| Propidium iodide *CAS 25535-16-4* | 537 | 618 | 60001 | 0.21 |
| Propidium iodide *10 mM aqueous solution* | 537 | 618 | 60001 | 0.21 |
| DiI iodide [1,1-Dioctadecyl-3,3,3,3- tetramethylindocarbocyanine iodide] | 550 | 564 | 148000 | - |
| Propidium iodide *1 mg/mL aqueous solution* | 537 | 618 | 60001 | 0.21 |
Citations
Authors: Wang, Mengxian and Yu, Zeping and Li, Xinlong and Li, Junqiao and Li, Jianshu and Luo, Jun and Li, Jiyao and Xiong, Yan and Yang, Jiaojiao
Journal: Acta Biomaterialia (2025)
Authors: Meng, Jun-Lun and Dong, Zi-Xuan and Chen, Yan-Ru and Lin, Meng-Hsuan and Liu, Yu-Ching and Roffler, Steve R and Lin, Wen-Wei and Chang, Chin-Yuan and Tzou, Shey-Cherng and Cheng, Tian-Lu and others,
Journal: ACS nano (2025)
Authors: Chen, Yihan and Lin, Ling and Xu, Lingling and Jin, Qiaofeng and Fu, Wenpei and Bai, Ying and Huang, Tian and Gao, Tang and Wu, Wenqian and Xu, Chunyan and others,
Journal: Colloids and Surfaces B: Biointerfaces (2024): 114460
Authors: Luo, Ying and Xu, Zhongsheng and Du, Qianying and Xu, Lian and Wang, Yi and Xu, Jie and Wang, Junrui and Chen, Sijin and Zhang, Wenli and Liu, Bo and others,
Journal: Chemical Engineering Journal (2024): 155592
Authors: Ao, Hui and Fu, Yao and Wang, Xiangtao
Journal: Colloids and Surfaces B: Biointerfaces (2024): 114176
References
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Journal: Invest Ophthalmol Vis Sci (2006): 2943
Authors: Heinrich L, Freyria AM, Melin M, Tourneur Y, Maksoud R, Bernengo JC, Hartmann DJ.
Journal: J Biomed Mater Res B Appl Biomater. (2006)
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