In vivo imaging refers to the scientific discipline of qualitatively and quantitatively analyzing biological processes and temporal dynamics at the cellular and molecular levels using imaging techniques in living organisms. Technologies mainly include bioluminescence and fluorescence imaging, isotopic imaging, X-ray imaging, etc. Among these, bioluminescence utilizes the luciferase gene to label cells or DNA, while fluorescence techniques employ fluorescent proteins (GFP, EGFP, RFP, YFP), fluorescent dyes, etc., to label expression of reporter groups, followed by detection using instrumentation. Isotopic imaging uses radioactive isotopes as tracers to label research subjects and is a trace analysis method for in vivo imaging. Through in vivo imaging technology, biological processes such as tumor growth and metastasis, infectious disease development, and expression of specific genes in living animals can be observed. Bioluminescence is particularly practical in these applications.

Figure 1. Overlay Image of Bioluminescence, Fluorescence, and X-ray Imaging after Tumor Drug Injection (Image source: slidesplayer)

Table 1. Comparison of Different Imaging Technologies

	Bioluminescence	Fluorescence	Isotope imaging
Advantages	(1) High sensitivity   (2)Fast imaging speed with clear images   (3)Detectable in vivo up to 102 cells	(1)Multiple proteins and dyes are available   (2)Multiple labeling is possible with simple labeling   (3)Suitable for simultaneous use in FACS sorting	(1)Sensitivity   (2) Does not affect the behavior of the labeled object   (3) No background noise
Disadvantages	1)Weak signal requires sensitive CCD lens   2) High precision requirements for the instrument   3)Target cells or genes need to be labeled	1) Non-specific fluorescence limits its sensitivity   2) In vivo detection has a minimum limit of approximately 106 cells   3) Requires excitation light of different wavelengths, making in vivo quantification difficult	1) Relatively low spatial resolution   2)Prone to radiation damage   3) Equipment is relatively expensive

The scope of in vivo imaging applications

The principle of bioluminescence technology is as follows:

In vivo bioluminescence technology refers to the utilization of reporter genes (such as the luciferase gene) expressed in living organisms to produce luciferase protein, which reacts with the substrate luciferin in the presence of oxygen and Mg²⁺, consuming ATP to undergo an oxidation reaction. This reaction converts some chemical energy into light energy, which is then captured by sensitive CCD devices to form images ex vivo. The luciferase reporter gene plasmid can be inserted into various gene promoters, becoming a reporter gene for a specific gene. By detecting the reporter gene, monitoring of the target gene can be achieved.

Figure 2. Principle of Bioluminescence In Vivo Imaging Detection

Biological fluorescence is essentially chemical fluorescence. During the oxidation process of luciferin by luciferase, luciferin can release photons of visible light with a wide wavelength range, ranging from 460 to 630 nm (with an average wavelength of 560 nm). In mammals, hemoglobin is the main component that absorbs visible light, absorbing most of the visible light in the blue-green light range. Water and lipids mainly absorb infrared light, but both have poor absorption capacity for red light near-infrared light with wavelengths of 590-800 nm. Therefore, although some scattering consumes light with wavelengths exceeding 600 nm, most of it can penetrate mammalian tissues and be detected by highly sensitive CCD detectors.

Figure 3. Bioluminescent Imaging Detection Results

 

Applications of Bioluminescence Imaging

Disease Research	Oncology Research, Drug Metabolism-related Studies
Cell or Bacterial Labeling	Labeling of Tumor Cells, Stem Cells, etc., Dual Labeling with Bioluminescence and Fluorescent Proteins
Gene Expression	Labeling Endogenous Proteins via Fusion Proteins to Study Gene Expression
Protein Interaction	Splitting the Luciferase Gene into Two Fragments, Each Fused to the Two Proteins Under Study, Where Luminescence Occurs Upon Proximity of the Two Proteins Interacting

1. Disease Research

Oncology: The luciferase gene is inserted into random chromosomal sites of tumor cells, which are then transferred into animal models to establish various tumor models. This enables real-time observation of tumor cell proliferation, growth, and metastasis within the body, allowing researchers to observe and study under near non-invasive conditions. Its high sensitivity allows detection of tiny tumor lesions (as few as a few hundred cells), significantly improving detection sensitivity compared to traditional methods, avoiding inter-group differences caused by sacrificing mice, and saving on animal costs.

2. Drug Research

Anti-tumor Drug Research: By administering mice with tumor grafts different doses, durations, and routes of administration of anti-tumor drugs, suitable dosing regimens and administration times can be observed and formulated. Labeling tumor cells with luciferase to establish various visible tumor models allows real-time evaluation of the therapeutic effects of various treatments. It enables dynamic observation of changes in tumor cells after treatment, whether tumor cells die, and whether tumor volume decreases, making it the most important application area of bioluminescence in vivo imaging technology.

Drug Metabolism Research: Labeling genes related to drug metabolism to study the effects of different drugs on gene expression, indirectly understanding the metabolism of related drugs in the body. In pharmacological research, fluorescent enzyme reporter gene plasmids can be directly incorporated into carriers to observe the targeting organs and distribution patterns of drug carriers in vivo. In pharmacological research, genes of interest can be labeled with luciferase genes to observe the pathways of drug action.

3. Cell Labeling

Immunocyte Research: Labeling immune cells to observe the recognition and killing function of immune cells against tumor cells, evaluating immune specificity, proliferation, migration, and other functions of immune cells.

Stem Cell Research: Labeling genes expressed constitutively, marking stem cells at the transgenic animal level, and tracking the proliferation, differentiation, and migration of stem cells in vivo using in vivo bioluminescence imaging technology.

Cell Apoptosis: Using molecular biology methods to attach protein inhibitors that inhibit luminescence (such as caspase) at both ends of the luciferase, but adding caspase at the connection. When cells undergo apoptosis, caspase is expressed, cleaving the protein that inhibits luciferase luminescence, causing luciferase to start luminescing, and observing the apoptosis of cells.

4. Gene Expression and Function Research

Gene Expression and Function: Inserting the luciferase gene downstream of the target gene promoter and stably integrating it into the experimental animal chromosome to form a transgenic animal model. This method enables parallel expression of the target gene and luciferase, allowing direct observation of the expression pattern of the target gene, including quantity, timing, location, and factors influencing its expression and function.

5. Protein Interactions

 Protein Interaction: Connecting the C-terminus and N-terminus of two different proteins with fluorescent enzymes that do not emit light separately when separated. If these two proteins interact, the C-terminus and N-terminus of the fluorescent enzyme will be connected, activating the transcription expression of the luciferase, resulting in bioluminescence in the presence of a substrate. Studying the effect of drugs on protein interactions under in vivo conditions can observe the effect of in vivo environments on protein interactions, which cannot be simulated in vitro experiments.

6. Others

Other applications of bioluminescence include RNAi, protein nuclear transport, etc. At one end of the luciferase gene is the gene of the protein to be studied, and at the other end is the gene of the protein that is known to be expressed in the nucleus. When the protein outside the nucleus is transported into the nucleus, the N-terminus and C-terminus of the luciferase come close together, restoring luminescence.

 

Factors Affecting Biological Imaging

1. Performance of CCD

2. Expression of cells and genes used in experiments

3. Selection of fluorescent markers

4. Influence of substrate concentration and temperature during luciferase imaging

5. Interference of autofluorescence

 

Technical Applications Outlook

Live imaging technology enables the transfer of molecular biology techniques from in vitro to in vivo studies. Therefore, this technology allows the observation of gene expression and cell activities in live animals. With advantages such as high detection sensitivity and simplicity of operation, it is increasingly being used in the fields of medicine and biology. Its applications can be summarized as follows:

1. Understanding Disease Mechanisms: Live imaging technology transforms complex processes such as gene expression and signal transduction into intuitive images, enabling better understanding of disease mechanisms and characteristics at the molecular and cellular levels.

2. Early Detection of Diseases: It can detect molecular and cellular variations and pathological changes in the early stages of diseases.

3. Evaluation of Therapeutic Effects: It enables continuous observation of the mechanisms and effects of drug or gene therapy in vivo.

As a non-invasive method for in vivo detection, the advantages of live imaging technology lie in its ability to obtain three-dimensional images of molecular and cellular components of the human body continuously, rapidly, over long distances, and without damage. It can reveal early molecular biological characteristics of lesions, promote early diagnosis and treatment of diseases, and introduce new concepts into clinical diagnosis.

 

FAQ

Q1: Difference between Potassium Salt, Sodium Salt, and Free Acid of Luciferin?

A: There are three substrates for firefly luciferase: luciferin free acid and its salt forms, including potassium salt and sodium salt.The main differences among them are:

Solubility: Salt forms are more soluble in water, with a solubility of 60 mg/ml for potassium salt and 100 mg/ml for sodium salt. The free acid is less soluble in water and can be weakly alkalized with sodium bicarbonate solution.

Toxicity: Salt forms are more convenient to use, especially in vivo imaging experiments, as they can dissolve in water, resulting in lower reaction toxicity.

Usage Effect: No significant difference. In in vivo experimental studies, potassium salt is often preferred for use.

Q2: How can Visible Light Emitted from the Body be Detected?

A: The high sensitivity of the cooled CCD lens, reaching -105°C, ensures that even very few photons emitted from the body can be detected. The absolutely sealed dark box device can shield all light, including radiation.

Q3: Does the Low Temperature of the CCD Lens Affect Small Animals?

A: No, the low temperature is limited to a small range around the CCD lens, and the rest is at room temperature.

Q4: What are the Advantages of Using Luciferin for Live Imaging Compared to Detecting Luminescence from Green Fluorescent Protein (GFP) in the Body?

A: The red-shifted light emitted by luciferase is approximately 100 times stronger in tissue penetration than the green light emitted by GFP. Luciferase luminescence is based on the interaction with luciferin, providing high specificity and signal-to-noise ratio. GFP requires excitation light to generate reflected light, but during detection, non-specific fluorescence from mouse fur and skin can reduce the signal-to-noise ratio. GFP detection is more suitable for in vitro detection, while luciferase detection is more suitable for in vivo detection.

Q5: In What Aspects is Bioluminescence Imaging Technology Superior to Traditional Techniques?

A: Compared to traditional techniques, this technology is more sensitive for studying tumor metastasis, gene therapy, epidemiological pathogenesis research, stem cell tracing, leukemia-related research, etc. It is more sensitive in tumor efficacy research than traditional methods and can quickly and intuitively conduct pathogenesis and drug screening research for related diseases through a series of transgenic animal disease models.

Q6: How to Label Stem Cells with Luciferase Genes?

A: Constitutively expressed genes can be labeled to create transgenic mice, marking the stem cells. Hematopoietic stem cells can be taken from the bone marrow of this mouse and transplanted into the bone marrow of another mouse, enabling tracking of hematopoietic stem cell proliferation, differentiation, migration, and the process of migration throughout the body. Another method is to label neural stem cells with lentiviruses.

 

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