In the field of in vivo imaging, fluorescent dyes are primarily categorized into water-soluble dyes, lipid-soluble dyes, near-infrared I (NIR-I) dyes, and near-infrared II (NIR-II) dyes. The choice of dye directly affects imaging depth, resolution, signal-to-noise ratio, and biocompatibility. A systematic comparison of these four types of fluorescent dyes is presented below across five dimensions: physicochemical properties, imaging characteristics, tissue penetration depth, biodistribution, and typical applications.
I. Core Characteristics Overview
| Dimension | Water-Soluble Dyes | Lipid-Soluble Dyes | NIR-I Dyes | NIR-II Dyes |
| Wavelength Range | Typically 400–700 nm (Visible) | Typically 400–700 nm (Visible) | 700–900 nm | 1000–1700 nm |
| Representative Dyes | FITC, Cy3, Cy5, Alexa Fluor series, SF6 | DiI, DiO, DiD, BODIPY, liposome-encapsulated dyes | ICG, Cy7, Cy7.5, IRDye 800CW | Quantum dots, Ag₂S, PbS/CdS, CH1055, Flav7 |
| Solubility | Hydrophilic, soluble in buffers and blood | Lipophilic, requires organic solvents | Mostly water-soluble or amphiphilic | Mostly water-soluble or nanoparticle-encapsulated |
| Cell/Tissue Penetration | Shallow (<1 mm), severe scattering | Shallow (<1 mm), prone to lipid absorption | Moderate (1–5 mm), low tissue autofluorescence | Deep (>5 mm), up to centimeter scale |
| Primary Imaging Modality | Confocal microscopy, widefield fluorescence | Membrane labeling, cell tracing, lipid droplet imaging | Live fluorescence imaging, angiography | Deep-tissue live imaging, surgical navigation |
| Biodistribution | Primarily in blood vessels, cytoplasm | Primarily in cell membranes, lipid droplets | Primarily in blood vessels, liver, tumors | Primarily in blood vessels, lymphatic system, tumors |
II. Water-Soluble Fluorescent Dyes
Physicochemical Properties
- Contain hydrophilic groups such as sulfonate and carboxyl groups
- Directly soluble in water, PBS, and cell culture media
- Typically conjugated to antibodies, proteins, and peptides via chemical coupling
Imaging Characteristics
- Emission wavelengths are concentrated in the visible region (400–700 nm)
- Extremely shallow tissue penetration (<1 mm), severely interfered by hemoglobin and melanin absorption
- High tissue autofluorescence, relatively low signal-to-noise ratio
Applications in Live Imaging
- Cell Tracing：Labeling stem cells or immune cells for in vivo injection
- Vascular Imaging：Extending circulation time via high-molecular-weight conjugates
- Targeted Imaging：Conjugation to antibodies or peptides for tumor targeting
Limitations
- Insufficient penetration depth, only suitable for superficial tissues or window chamber models
- Not suitable for whole-body imaging or deep organ imaging
III. Lipid-Soluble Fluorescent Dyes
Physicochemical Properties
- Contain long carbon chains or hydrophobic groups
- Insoluble in water, require DMSO, ethanol, or other solvents for dissolution
- Water solubility can be improved via liposome or nanoparticle encapsulation
Imaging Characteristics
- Emission wavelengths are also concentrated in the visible region
- Easily bind to cell membranes, lipid droplets, and lipoproteins
- Can reside on cell membranes for extended periods, suitable for long-term tracing
Applications in Live Imaging
- Membrane Labeling：DiI, DiO, DiD are commonly used for cell tracing, maintaining signal for weeks in vivo
- Lipid Metabolism Research：Labeling lipoproteins and lipid droplets
- Tumor Tracing：Loading lipid-soluble dyes onto nanoparticles for tumor targeting
Limitations
- Penetration depth is similarly limited
- Hydrophobicity leads to aggregation in blood, requiring specialized delivery systems
- Higher risk of non-specific binding
IV. Near-Infrared I Fluorescent Dyes (NIR-I, 700–900 nm)
Physicochemical Properties
- Mostly water-soluble or amphiphilic molecules
- Representative dyes: ICG (Indocyanine Green), Cy7, Cy7.5, IRDye 800CW
- Several have received FDA/EMA approval for clinical use
Imaging Characteristics
- Significantly improved tissue penetration depth (1–5 mm)
- Low tissue autofluorescence in the NIR-I region, favorable signal-to-noise ratio
- Some scattering and absorption remain (hemoglobin, water)
Applications in Live Imaging
- Vascular and Lymphatic Angiography：ICG widely used for intraoperative navigation and sentinel lymph node mapping
- Tumor Imaging：Tumor accumulation via targeted conjugation
- Liver Function Assessment：ICG metabolized by the liver, used for liver function testing
- Perfusion Imaging：Assessing tissue blood perfusion
Advantages
- Mature clinical translation; ICG and others are already in clinical use
- High availability of imaging instruments (IVIS, Pearl, and other live imaging systems)
- Significantly improved tissue penetration and signal-to-noise ratio compared to visible light
V. Near-Infrared II Fluorescent Dyes (NIR-II, 1000–1700 nm)
Physicochemical Properties
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Mostly inorganic quantum dots (Ag₂S, PbS), organic small molecules (CH1055, Flav7), or single-walled carbon nanotubes
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Require water-solubility modification or nanoparticle encapsulation to improve biocompatibility
Imaging Characteristics
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Tissue penetration depth can reach centimeter scale
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Extremely low scattering and tissue autofluorescence, optimal signal-to-noise ratio
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Enables high-resolution deep-tissue imaging (blood vessels, tumors, lymphatics)
Applications in Live Imaging
- Deep Vascular Imaging：Clear visualization of microvessels with diameters <100 μm
- Precise Tumor Localization：Enables identification of tumor boundaries in deep tissues
- Surgical Navigation：NIR-II imaging assists in tumor resection
- Lymphatic System Imaging：High-resolution assessment of lymphatic vessel structure and function
Advantages
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Both penetration depth and signal-to-noise ratio are superior to NIR-I
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Enables both macroscopic and microscopic imaging
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Has become a frontier hotspot in live imaging research in recent years
Limitations
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Most NIR-II dyes have not yet entered clinical use
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Inorganic nanoparticles have long-term toxicity concerns
- Lower availability of imaging instruments; require specialized InGaAs cameras
VI. Selection Guide
| Application Scenario | Recommended Dye Type | Rationale |
| In vitro cell staining, microscopy | Water-soluble/Lipid-soluble visible dyes | Low cost, widely available instruments, easy operation |
| Cell transplantation tracing (days to weeks) | Lipid-soluble dyes (DiI/DiD) | Stable membrane integration, long-lasting labeling |
| Superficial tumor imaging, lymphography | NIR-I dyes (ICG/Cy7) | Sufficient penetration depth, mature clinical translation |
| Deep organ imaging, whole-body imaging | NIR-II dyes | Optimal penetration depth, highest signal-to-noise ratio |
| Multimodal imaging (fluorescence + others) | Selected based on pairing requirements | Both NIR-I and NIR-II can be combined with CT, MRI, ultrasound |
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艳 李
