Ion Imaging: Definition, Principles, Benefits, Dyes, and Uses

Inorganic ions within organisms are essential substances that play vital roles in life processes. Increasing research indicates that disruptions in ion balance are closely associated with health issues, with most disease development processes accompanied by inorganic ion disorders. This creates a microenvironment with ion discrepancies at disease sites. Thus, evaluating ion levels in vivo and monitoring their dynamic changes is particularly important for the early, precise diagnosis and treatment of diseases, as well as for exploring disease mechanisms.

What is Ion Imaging?

The ion composition in the cytoplasm determines many crucial functions, such as neuronal excitability, gene transcription, and cell movement. Therefore, the spatial and temporal regulation of intracellular ions is a major focus in life sciences research. Ion imaging (e.g., calcium, chloride, magnesium) is a commonly used method that employs fluorescent dyes or specially designed proteins that change emission behavior when binding to calcium. This enables researchers to observe dynamic changes in cellular ion concentrations. Additionally, special fluorescent dyes allow for imaging intracellular pH or voltage. One specialized technique for imaging ion levels, pH, or voltage changes is ratio imaging. These methods can precisely determine intracellular ion concentrations, unlike non-ratio methods, which monitor relative changes. Such techniques are essential for examining cellular responses to external stimuli and understanding ion-dependent cellular processes under various physiological and pathological conditions. For example, tumor microenvironments often exhibit imbalanced ion homeostasis. Utilizing these characteristics, tumor-responsive ion-sensitive probes can be constructed to enable early tumor diagnosis, differentiation between benign and malignant tumors, and monitoring during tumor treatment.

Fig. 1. Ion-sensitive fluorescence imaging probes (Chem Commun (Camb). 2023, 59(39): 5807-5822).

Principles of Ion Imaging

The principles of cellular ion fluorescence imaging primarily rely on the ion sensitivity of fluorescent probe molecules. Fluorescent probes are molecules that interact with specific ions and emit measurable fluorescence signals, typically consisting of a fluorophore and an ion-binding site. The fluorescence intensity or emission spectrum of a fluorescent probe changes based on the concentration of the target ion in the environment, allowing researchers to detect concentration changes of specific ions. Key steps in cellular ion fluorescence imaging include probe selection, cell labeling, fluorescent microscopy observation, and image analysis.

1. Probe Selection: Fluorescent probes are selected based on the type of target ion, required sensitivity, and specificity. For example, Fura-2 and Fluo-4 are commonly used for calcium ion detection, SBFI for sodium, PBFI for potassium, and BCECF for measuring hydrogen ion (pH). Selecting the appropriate probe enhances detection accuracy, avoids interference, and yields more precise results.
2. Cell Labeling: Introducing fluorescent probes into cells requires specific methods, such as microinjection, electroporation, or using cell-permeable molecules to ensure probes effectively enter cells and remain functional. Once inside, probes usually bind to the target ions, resulting in fluorescence signals that vary with ion concentration.
3. Fluorescent Microscopy Imaging: Fluorescence imaging systems are typically equipped with an excitation light source, filter sets, and a CCD camera. Different fluorescent probes have specific excitation and emission wavelengths, requiring suitable filter sets. Under a microscope, the target ion's fluorescence signal is captured and further analyzed through image processing software.
4. Image Analysis: The final data from fluorescence imaging is quantitatively processed using image analysis software to determine the spatial distribution and concentration changes of target ions. For example, fluorescence intensity ratio or spectral shift methods are used to estimate absolute ion concentration and map intracellular ion distribution.

Benefits of Ion Imaging

Cellular ion fluorescence imaging technology has found broad applications in fields such as biology, medicine, and pharmacology due to its unique advantages. Its non-invasive imaging approach enables real-time monitoring of ion concentrations within live cells, unveiling dynamic cellular changes. Additionally, the high sensitivity and selectivity of fluorescence probes allow this technique to detect trace ion fluctuations, which is challenging to achieve with conventional detection methods. Compared to other imaging techniques, fluorescence imaging also offers extremely high temporal and spatial resolution, allowing the tracking of localized ion concentration changes. This capability helps researchers understand the complex, dynamic roles of ions in biological functions. These technical strengths make cellular ion fluorescence imaging an indispensable tool in cell and molecular biology research.

• Real-time Monitoring: This technique enables dynamic tracking of ion fluxes in real time, allowing researchers to observe changes as they happen. This is essential for studying rapid, transient ion signaling events in response to stimuli.
• High Spatial Resolution: Advanced imaging methods, such as confocal and super-resolution microscopy, provide high spatial resolution, capturing detailed ion distribution within cells. This enables the localization of ion activity to specific organelles or cellular compartments.
• Quantitative Measurement: Cellular ion imaging allows for quantitative analysis of ion concentrations. By calibrating imaging sensors, researchers can measure absolute ion concentrations, providing essential data for cell signaling studies.
• Non-invasive: Many cellular ion imaging techniques are minimally invasive, enabling ion measurements in live cells without compromising cell integrity. This ensures that biological processes can be studied under natural conditions.
• Insights into Pathophysiology: By studying ion dynamics, researchers can gain insights into diseases caused by ion imbalances, such as neurological disorders, cardiovascular diseases, and metabolic syndromes. The technique helps identify potential therapeutic targets.
• High Sensitivity and Selectivity: Specific fluorescent probes can detect target ions at extremely low concentrations, aiding in the study of the biological roles of trace ions.
• Multichannel Imaging: With various probes and different excitation wavelengths, it is possible to observe simultaneous changes in multiple ions or molecules, enabling multicomponent analysis.

Ion Imaging Probes

The successful application of cellular ion fluorescence imaging relies on the development and optimization of selective probes for different ion types. Each ion plays a unique role in cellular physiology, so probe selection and design must be precisely adjusted for ion specificity and concentration range. Fluorescent probes generally fall into two categories: ratio-based probes, where fluorescence intensity changes with ion concentration, and non-ratio probes, which emit a single-wavelength fluorescence upon binding the target ion. Both types of fluorescent probes undergo significant fluorescence property changes upon ion binding, providing scientists with high-resolution data to observe changes in intracellular ion concentrations. Specific fluorescent probes have become central tools for studying the role of ions in cellular activities. The following are some widely used probe types and their applications:

• Calcium Ion Fluorescent Dyes

Calcium (Ca²⁺) is one of the most critical elements in the human body, involved in all life activities and maintaining cellular physiological functions. Calcium primarily acts as ions, serving as a second messenger akin to hormones, often referred to as a "biological messenger." Although plasma calcium ion concentration is thousands of times higher than intracellular concentration, it is still low compared to bones and other tissues. These calcium ions balance intracellular calcium levels, playing vital roles within cells. Calcium ion imaging technology (Calcium imaging) utilizes calcium indicators to monitor intracellular calcium concentration, allowing direct measurement of dynamic calcium flows in neurons, which can indicate neuronal activity. Common fluorescent dyes include:

• Magnesium Ion Fluorescent Dyes

Magnesium ions (Mg²⁺) play diverse biological roles within cells, serving as cofactors for numerous enzymes and are extensively involved in critical biochemical processes like protein synthesis, energy metabolism, and signal transduction. Intracellular magnesium concentrations are typically high, and these levels are linked with changes in other divalent cations, such as calcium ions. Therefore, detecting magnesium ions requires highly selective probes like Mag-Fura-2 and Mag-Indo-1. These probes bind to magnesium ions and can be detected using ratiometric imaging, with fluorescence changes that directly reflect magnesium ion concentration, allowing for the study of cellular magnesium metabolism regulation mechanisms. Additionally, probes like Magnesium Green and Mag-Fluo-4, known for their cell permeability and sensitivity, are widely used to observe the dynamic changes in magnesium ions.

• Sodium Ion Fluorescent Dyes

Sodium ions (Na⁺), essential for physiological functions, make up about 0.1% of body weight and help regulate acid-base balance, maintain fluid volume, support membrane potential, and activate numerous signal transduction pathways. Sodium ions are crucial in maintaining osmotic pressure, and dramatic shifts in blood sodium levels can alter cell morphology, causing swelling (hyponatremia) or shrinkage (hypernatremia), impacting cellular function. Fluorescent probes allow researchers to monitor sodium ion flow and distribution in real-time during cellular stimulation, helping investigate cellular excitation and conduction mechanisms. Common sodium ion fluorescent dyes include:

• Potassium Ion Fluorescent Dyes

Potassium ions (K⁺) are essential for various physiological functions, including muscle contraction, nerve conduction, and kidney function. In most resting cells, intracellular sodium (Na⁺) concentration is about 10 mmol/L, while potassium (K⁺) concentration is approximately 120 mmol/L. In the extracellular space, sodium is around 140 mmol/L, and potassium is about 4 mmol/L. Selective recognition of potassium in the presence of sodium is particularly important. Commonly used potassium probes like PBFI can bind potassium ions and emit specific fluorescence signals, allowing researchers to observe potassium flux in neurons and cardiomyocytes to study their excitability regulation mechanisms.

• Zinc Ion Fluorescent Dyes

Zinc (Zn²⁺) is a crucial trace element in living organisms and the second most abundant transition metal after iron. Zinc ions play vital roles in cellular function, gene regulation, and neuronal signaling. Zinc ion concentrations range from nanomolar to millimolar levels, and imbalances can lead to diseases like growth retardation, anorexia, neurodegenerative diseases, Parkinson's, and Alzheimer's. Common dyes include:

• Iron Ion Fluorescent Dyes

Iron is essential in organisms, being the most abundant transition metal in the body and involved in various physiological activities. Disruption in cellular iron homeostasis can cause harmful oxidation and stress damage, with abnormal levels linked to cancer, cardiovascular disease, Alzheimer's, and Parkinson's disease. Free iron ions in cells exist as ferrous (Fe²⁺) and ferric (Fe³⁺) ions. A new class of fluorescent probes enables Fe²⁺ imaging in live cells, offering high sensitivity and compatibility with other dyes.

• Copper Ion Fluorescent Dyes

Copper ions (Cu²⁺) rank third in abundance among essential trace elements in organisms after iron and zinc, playing crucial roles in numerous physiological processes. Deficiency can lead to growth and metabolic disorders, while excess copper produces reactive oxygen species that disrupt cellular metabolism. Copper ion detection is thus a focal point in metal ion imaging, valuable for basic biological research and studies on drug screening and pathology in copper-related diseases.

• Chloride Ion Fluorescent Dyes

Chloride ions, as the primary anions in the human body, play a critical role in maintaining osmotic pressure, acid-base balance, and water volume inside and outside cells. Chloride ions are the most abundant anions in extracellular fluid, accounting for about 80% of total ions that maintain osmotic pressure together with sodium ions, helping to regulate extracellular fluid volume and stabilize osmotic balance. Additionally, chloride is involved in the formation of gastric acid, which activates pepsinogen, promotes the absorption of vitamin B12 and iron, and aids in food digestion. Chloride also stimulates liver function, promoting the excretion of toxic waste from the liver. Chloride ion fluorescence imaging commonly uses the MQAE (N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide) probe, which specifically detects changes in chloride ion concentration. MQAE exhibits fluorescence quenching in response to changes in chloride ion concentration, enabling researchers to study the distribution and dynamic regulation of chloride ions inside and outside cells by measuring changes in fluorescence intensity.

What is Ion Imaging Used For?

Cell ion fluorescence imaging technology has been widely used in many scientific and medical research fields due to its high sensitivity and real-time performance. By accurately detecting the dynamic changes of intracellular and extracellular ions, this technique reveals the core role of ions in cell function regulation, physiological state maintenance and pathological mechanism. Whether in basic scientific research or in the field of biomedicine, cell ion fluorescence imaging plays an irreplaceable role and provides powerful tool support for ion-related disease diagnosis, treatment and drug development.

• Neuroscience Research

Cellular ion fluorescence imaging can track the changes of these key ions in real time, providing insights into neuronal signal transduction, synaptic plasticity and neural circuit function. In particular, calcium imaging technology is widely used to study neuronal activity and help scientists analyze how neurons regulate synaptic transmission and memory formation through calcium ion flow. In addition, through zinc and copper ion imaging, researchers can further explore the function and pathological mechanism of these metal ions in the nervous system, such as neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.

• Oncology and Cancer Research

In the tumor microenvironment, the concentration changes of calcium, hydrogen and zinc ions are closely related to the proliferation, migration and metabolic abnormalities of tumor cells. Cell ion fluorescence imaging can help researchers monitor changes in ion concentration in cancer cells to analyze the role of these ions in tumor progression. For example, the dynamic imaging technology of calcium ions can reveal the abnormal changes of calcium signals in cancer cells, which is of great significance for understanding the proliferation and invasion characteristics of cancer cells. In addition, the application of hydrogen ion probes provides a powerful tool for studying the acidification and metabolic characteristics of tumor tissues, which is helpful for the further development of targeted therapy based on acidic microenvironment.

• Cardiovascular Research

The electrical activity of cardiomyocytes, myocardial contraction and the function of vascular smooth muscle are dynamically regulated by calcium, sodium and potassium ions. Therefore, cellular ion fluorescence imaging technology has been widely used in cardiovascular research to explore the core functions of ions in the cardiovascular system. By monitoring the concentration of calcium ions in cardiomyocytes in real time, researchers can gain a deeper understanding of the pathogenesis of myocardial excitation-contraction coupling and arrhythmia. At the same time, the dysfunction of sodium-potassium pump is closely related to diseases such as hypertension and heart failure. Ion imaging technology provides new ideas and detection methods for the study of these diseases.

• Drug Screening and Development

Cellular ion fluorescence imaging has played an important role in drug screening and toxicology research. Many drugs achieve therapeutic effects by regulating intracellular and extracellular ion concentrations or affecting ion channels. Through imaging technology, researchers can quickly screen out drugs with specific ion channel regulation capabilities and evaluate their effects on intracellular ion levels. For example, drug screening for calcium and potassium channels has been successfully applied in drug development for diseases such as hypertension and epilepsy. This technique can also be used to detect the effect of drugs on the ion concentration of target cells such as liver cells and kidney cells, so as to evaluate their potential toxic effects and ensure drug safety.

• Immunological Research

Ions play an important role in the activation, migration and function of immune cells. Cellular ion fluorescence imaging technology enables researchers to gain a deeper understanding of the role of calcium and potassium ions in T cells, B cells and macrophages. For example, changes in the concentration of calcium ions are considered to play a key role in T cell activation and functional regulation. Related fluorescence imaging studies have revealed the regulatory mechanism of calcium signals in immune responses. In addition, the chloride ion probe can be used to analyze the phagocytic activity and lysosomal function of macrophages, which further promotes the understanding of the working principle of the immune system.

• Metabolism and Nutrition Research

Cell metabolic activities require the participation of a variety of ions, such as magnesium, calcium, and potassium, which support the normal physiological functions of cells by regulating enzyme activity and maintaining metabolic homeostasis. Real-time detection of the concentration changes of these ions by fluorescence imaging can help to reveal the activity changes of key enzymes in the metabolic process and their regulatory mechanisms. Especially in the study of metabolic diseases such as diabetes, the imaging of sodium and calcium ions can reveal the ion dynamics of islet cells, so as to further understand the relationship between insulin secretion and cell ion balance. In addition, the imaging application of magnesium ions provides a new perspective for understanding nutritional balance and cellular energy metabolism, especially in nutrition and obesity research.

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Reference:

1. Wang, Q. et al. Inorganic ion-sensitive imaging probes for biomedical applications. Chem Commun (Camb). 2023, 59(39): 5807-5822.

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