Near-infrared (NIR) labeling of live cells is rapidly expanding into diverse areas of biotechnology research, where deep-tissue imaging and minimal background interference are key. Among NIR lipophilic dyes, DiR stands out for its ability to incorporate into plasma membranes. Below are several cutting-edge fields where DiR is yielding compelling results and driving new breakthroughs in translational medicine.  

Stem Cell Therapy and Regenerative Medicine

One of the most active research areas using DiR is tracking stem cell fate and biodistribution. In studies involving rat or mouse models of spinal cord, myocardial, or stroke injury, DiR-labeled mesenchymal stem cells (MSCs) or bone marrow–derived progenitors were injected systemically or locally (Macks et al., 2018; Li et al., 2018). With in vivo NIR imaging, investigators could pinpoint how many labeled cells remained in the target tissue over time. This type of “stem cell homing” analysis is proving instrumental in developing regenerative therapies for previously intractable diseases—such as myocardial infarction or neurodegeneration—while confirming cell viability and retention post-administration.  

Immunotherapy and Immune Cell Visualization

In the realm of cancer immunotherapy, DiR labeling enables precise monitoring of immune cells such as T lymphocytes and dendritic cells (Krzastek et al., 2018; Ji et al., 2020). For example, T-cells labeled with DiR can be followed after adoptive transfer to confirm their migration and infiltration into tumors, providing insights into how well they exert their cytotoxic function. Furthermore, dendritic cells labeled with DiR can reveal the dynamics of antigen presentation in lymph nodes and the success of vaccine strategies. This is critical for optimizing protocols in adoptive cell therapy and checkpoint blockade approaches.  

Drug Delivery and Nanoparticle Tracking

Another lucrative area centers on nanocarrier systems—for instance, polymeric micelles or liposomes—labeled with DiR for improved visualization (Noack et al., 2020; Zhao et al., 2024). Such NIR imaging clarifies how these carriers distribute throughout the body, whether they accumulate in tumor tissue, and how long they remain in circulation. In parallel, DiR’s hydrophobic chains anchor strongly to phospholipid bilayers, enabling stable association with nanosized vehicles. Researchers leverage this to fine-tune carrier composition and dosing schedules for enhanced therapeutic outcomes. The possibility of real-time, noninvasive imaging of these carriers is helping usher in more effective chemotherapy regimens, targeted tissue repair systems, and advanced gene delivery techniques.  

Lymphatic and Vascular Imaging

DiR also finds utility in mapping lymphatic drainage (Wang et al., 2018). This is especially relevant for evaluating metastasis risk or post-surgical complications such as lymphedema. Because DiR-labeled cells or particles can be tracked from peripheral injection sites to lymph nodes, investigators gain crucial spatial and temporal data that influence both cancer staging methods and reconstructive interventions.  

Translation to Clinical-Grade Optical Systems

The strong NIR signal from DiR is compatible with many preclinical optical imaging platforms and has the potential to integrate with emerging photoacoustic and hybrid FMT-XCT systems (Berninger et al., 2018). These combined modalities offer deeper tissue penetration, expanded tumor mapping, and precise quantitation of how DiR-labeled cells or nanoparticles distribute within complex 3D tissues.  

In summary, DiR’s near-infrared fluorescence, robust membrane integration, and proven safety record make it a transformative tool for cell tracking, immunotherapy optimization, regenerative medicine, nanocarrier evaluation, and beyond. As image-guided technologies advance, DiR continues to foster breakthroughs in biotech and pharmaceutical fields, fueling more reliable research outcomes and guiding the next wave of clinical innovations.

1. What is DiR dye?

DiR dye is a near-infrared, lipophilic carbocyanine used primarily to label the plasma membrane of live cells for fluorescence imaging.  

2. What is the wavelength of DiR dye?

Typical excitation peaks around 750–760 nm; emission is ~775–785 nm, spanning the near-infrared spectrum.  

3. How do lipophilic dyes work?

Lipophilic dyes incorporate into lipid bilayers through hydrophobic alkyl chains, stably tagging cellular membranes without binding to DNA or cytoplasmic proteins.  

4. What is the protocol of DiO staining?

DiO is a green-emitting carbocyanine (~484 nm excitation, ~501 nm emission) used similarly:

1.  Dilute DiO (1–5 µM) in serum-free media.
2.  Incubate cells (5–20 min, 37 °C).
3.  Wash away excess dye.
4.  Proceed with imaging (Honig and Hume, 1989).

5. What is DiO used for?

DiO is used for plasma membrane labeling in multi-color or neuronal tracing studies; its green emission channel pairs well with other fluorophores.  

6. What fluorescent dye binds to DNA?

Common nuclear stains (e.g., DAPI, Hoechst 33342, propidium iodide) target DNA, but DiI, DiO, DiD, and DiR remain membrane-selective.  

7. What is DiO dye?

DiO is a green lipophilic carbocyanine with octadecyl tails, anchoring into membranes for short- or long-term labeling in fixed or live-cell contexts.  

8. How does DiI work?

Similar to DiO, DiI integrates into cell membranes with fluorescence in the orange-red range (~550–565 nm excitation, ~565–595 nm emission) (Chazotte, 2011).  

9. What is the best stain for mitochondria?

For mitochondria, dedicated probes such as MitoLite™ or DiOC6(3) are recommended, as general carbocyanines (DiO, DiI, DiR) are less specific (Chen and Coons, 1987).  

10. How does DiOC6 stain mitochondria?

DiOC6(3) accumulates in negatively charged inner mitochondrial membranes at low concentrations, selectively highlighting mitochondria (Haugland, 2005).  

What is the principle of DiI staining?

DiI relies on the hydrophobic interaction of its alkyl tails with phospholipid bilayers, ensuring stable, uniform membrane labeling (Honig and Hume, 1989).  

What is the excitation and emission of DiR dye?

Exact values can vary slightly depending on experimental conditions, but generally: •   Excitation: ~750–760 nm •   Emission: ~775–785 nm  

What is the most commonly used fluorescent dye?

Popular visible-spectrum dyes include FITC, TRITC, and Alexa Fluor. For deep-tissue or low-autofluorescence imaging, near-infrared lipophilic dyes (DiI, DiO, DiD, DiR) are indispensable (Chazotte, 2011; Lakowicz, 2006).

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.

All carbocyanine dyes (DiI, DiO, DiD, DiR) employ a similar principle of lipophilic insertion into plasma membranes.