1. Introduction to the Technical Principle
1) Bioluminescence imaging (BLI) in live animals involves the expression of a reporter gene (such as luciferase) in small mammals. The expressed luciferase catalyzes the oxidation of its substrate, luciferin, in the presence of oxygen, Mg²⁺, and ATP. This chemical reaction releases part of the chemical energy as visible light. The emitted light can then be captured by a highly sensitive CCD camera to generate an image. The intensity of the bioluminescent signal correlates linearly with the number of labeled cells. Luciferase reporter plasmids can be placed under various gene promoters to serve as reporters, allowing monitoring of the expression or regulation of target genes.
Figure 1: Bioluminescent reaction catalyzed by luciferase
2) Bioluminescence is a form of chemiluminescence. During the oxidation of luciferin by luciferase, light in a broad visible spectrum (460–630 nm, average ~560 nm) is emitted. In mammals, hemoglobin absorbs most of the visible light, particularly in the blue-green range. Water and lipids primarily absorb infrared light, while absorption in the red to near-infrared range (590–800 nm) is relatively low. As a result, red-shifted bioluminescence (>600 nm) can penetrate tissues more effectively and still be detected by sensitive CCD devices, despite some scattering loss.
Figure 2: Principle, instrumentation, and results of in vivo imaging
2. Applications of In Vivo Imaging
Oncology: Enables rapid, non-invasive quantification of tumor growth, metastasis, and therapeutic response in cancer models.
Drug Discovery: Applied in anti-tumor drug efficacy studies, pharmacokinetics, cell labeling, gene expression and function studies, and apoptosis tracking.
3. Comparison of Bioluminescence and Fluorescence Imaging
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Bioluminescence |
Fluorescence |
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Advantages |
(1) High sensitivity (2) Fast imaging with clear signal (3) Detects as few as 10² cells in vivo |
(1) Wide variety of dyes and proteins available (2) Multiplexing possible (3) Applicable to FACS sorting |
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Disadvantages |
(1) Weaker signal requiring sensitive CCDs (2) Requires precision instruments (3) Target cells or genes must be labeled |
(1) Non-specific background fluorescence reduces sensitivity (2) Detection limit ~10⁶ cells in vivo (3) Requires external excitation light (4) Quantification in vivo is challenging |
4. Differences Between Luciferin Potassium Salt, Sodium Salt, and Free Acid
Luciferin, the substrate of firefly luciferase, is available as a free acid or as potassium/sodium salts. Key differences include:
Solubility: Salt forms are water-soluble. Sodium salt dissolves at ~100 mg/mL, potassium salt at ~60 mg/mL. Free acid is poorly soluble in water but can be dissolved in weakly basic bicarbonate solutions.
Toxicity: Salt forms are more biocompatible and convenient for in vivo use due to their water solubility and lower toxicity.
Performance: No significant difference in imaging outcomes. Potassium salt is more commonly used in in vivo experiments.
5. How Is Weak Bioluminescent Light Detected from Within the Body?
Two main factors ensure detection of low bioluminescent signals:
Ultra-sensitive cooled CCD cameras, operating at temperatures as low as –105°C, capable of detecting minimal photon emission.
Light-tight imaging chambers, which eliminate ambient light and background radiation for clean signal capture.
6. Can Rats Be Used for In Vivo Imaging?
Yes. While differences in tissue penetration exist among adult mice, rats, and embryos, visible light can penetrate 3–4 cm of tissue. Numerous studies have successfully demonstrated in vivo imaging in rats.
7. Does the Cooled CCD Affect Animal Temperature?
No. The cooling system is localized to the CCD sensor and does not affect the ambient temperature within the imaging chamber, which remains at room temperature.
8. Advantages of Luciferin-Based Imaging Over GFP
Luciferase-based imaging emits red-shifted light, which penetrates tissue nearly 100 times better than green fluorescence from GFP. The enzymatic reaction provides high specificity and signal-to-noise ratio, whereas GFP requires external excitation, which can cause non-specific autofluorescence from skin or fur. Therefore, GFP is more suitable for in vitro detection, while luciferase is ideal for in vivo applications.
9. Advantages of Bioluminescence Imaging Over Traditional Methods
Bioluminescence imaging offers enhanced sensitivity and real-time tracking in studies of tumor metastasis, gene therapy, disease pathogenesis, stem cell tracing, and leukemia models. It outperforms traditional methods in drug efficacy evaluation and facilitates rapid screening using transgenic disease models.
10. How to Label Stem Cells with Luciferase?
Two main methods:
Use a constitutively active promoter driving luciferase in a transgenic mouse model. Harvest hematopoietic stem cells from bone marrow and transplant them into another mouse to monitor proliferation, differentiation, and migration in vivo.
Alternatively, lentiviral vectors can be used to transduce neural stem cells with luciferase for in vivo tracking.
11. When to Perform Imaging After Luciferin Injection? How Long Does the Signal Last?
After intraperitoneal injection, maximum bioluminescent signal appears at 10–15 minutes, stabilizes briefly, and begins to decline around 20–30 minutes.
By 3 hours, the luciferin is excreted and the signal is undetectable.
12. How to Inject Luciferin? What Are the Differences Between Methods?
Luciferin can be administered via intraperitoneal (IP) or tail vein (IV) injection:
IP injection: Slower diffusion, delayed signal onset, but prolonged signal duration.
IV injection: Rapid diffusion and immediate signal onset, but shorter signal duration.
A typical dose is 150 mg/kg; for a 20 g mouse, this equals ~3 mg of luciferin.
Product Overview
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Product Name |
Specification |
Cat No. |
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100 mg/500 mg/1 g |
40702ES01/02/03 |
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100 mg/500 mg/1 g |
40701ES01/02/03 |
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100 mg/500 mg/1 g |
40703ES01/02/03 |
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10 T/100 T/1000 T |
11402ES10/60/80 |
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100 T/1000 T/10×100 T/10 T |
11413ES60/81/80/10 |