Cell Imaging: Definitions, Systems, Protocols, Dyes, and Applications

Modern biomedical research relies fundamentally on cell imaging technology as an essential investigative instrument. The technology enables scientists to visually study cell morphology and structure, together with their functions and interactions between cells, which offers strong capabilities to reveal biological life processes. Live cell imaging continues to attract substantial attention from researchers because it enables real-time observation of cellular dynamics while preserving cell health.

Cell Imaging

Cell imaging refers to the process of visualizing cells using various imaging technologies. It not only involves observing cell morphology but also includes changes at the cellular internal structure, function, and molecular levels. The purpose of cell imaging is to help researchers gain a deeper understanding of physiological and pathological processes within cells, providing essential insights for disease diagnosis, treatment, and drug development. The advancement of cell imaging technology has significantly propelled progress in disciplines such as cell biology, molecular biology, and pathology, offering unprecedented opportunities for life science research.

Fig. 1. Live cell imaging techniques.

Cell imaging technology encompasses a range of core tools used to study cell structure and function, including optical imaging, electron microscope imaging, and live cell imaging, among others. Optical imaging techniques primarily use microscopes, with fluorescence microscopy stimulating fluorescent molecules at specific wavelengths to visually display internal cell structures. Confocal microscopy, with its high-resolution three-dimensional imaging, reveals intricate cellular structures. Phase contrast microscopy and dark field microscopy each have unique features, suitable for observing transparent structures or cell surface details. Electron microscopy, replacing light beams with electron beams, provides ultra-high resolution images of cell surface morphology (SEM) and internal ultrastructures (TEM). Live cell imaging technology captures dynamic processes like cell division and migration in real-time while maintaining cell viability. Super-resolution imaging techniques further surpass the optical resolution limits, offering new perspectives for studying nano-scale structures and molecular mechanisms within cells. These technologies provide robust support for cell biology research, advancing our understanding of cellular life activities.

Live Cell Imaging

Live cell imaging technology is a technique used to study the structure and function of living cells through microscopy. Commonly used live cell imaging technologies include fluorescence imaging, confocal imaging, phase contrast imaging, and brightfield imaging. Fluorescence imaging utilizes fluorescent dyes or proteins to label specific structures or molecules within cells. Fluorescence microscopes observe their fluorescence signals, enabling real-time monitoring of dynamic changes inside the cell. Confocal imaging, employing confocal technology, provides higher resolution and clearer internal cell structure images, making it especially suitable for observing subcellular structures and molecular localization within cells. Phase contrast imaging and brightfield imaging are primarily used to observe changes in cell morphology and structure without the need for fluorescence labeling, allowing for non-invasive observation of cells. Additionally, emerging imaging technologies such as light-sheet imaging and super-resolution imaging are gradually being applied in the field of live cell imaging. These technologies offer faster imaging speeds and higher resolution, providing researchers with more detailed and accurate cellular information. Live cell imaging technology is a core method for visualizing and quantitatively analyzing dynamic cellular processes in real-time. This technology enables researchers to study cells and subcellular structures, functions, and organization under conditions closer to the in vivo environment, aiding in the development of more biologically relevant experimental methods and better predicting human responses to new drug candidates.

Live Cell Imaging Systems

Live cell imaging systems are comprehensive platforms that integrate imaging equipment, environmental control devices, software analysis tools, and other components. A complete live cell imaging system provides researchers with a one-stop imaging solution, enabling them to perform everything from cell cultivation to data collection and analysis within the same system. Live cell imaging systems allow scientists to observe various dynamic processes of cells, such as cell division, migration, and signal transduction, over extended periods while maintaining the cells in their natural state. Key components of the system include:

• Microscopes: Fluorescence microscopes, confocal microscopes, and other types are commonly used in live cell imaging systems. These microscopes provide high-resolution images, allowing researchers to clearly observe subtle structures and dynamic changes inside the cell.
• Fluorescent Labels: Fluorescent proteins and dyes are widely used to label specific molecules or structures inside cells, making them clearly visible under the microscope.
• Environmental Control Equipment: To maintain cell viability during experiments, live cell imaging systems are typically equipped with temperature control, humidity control, and CO₂ concentration control devices, which simulate the physiological environment of the cells.
• Imaging Equipment: High-sensitivity cameras and automated stages are essential components of the system. These devices can quickly and accurately capture images and allow for imaging from different positions and angles.
• Software: Specialized analysis software is used to process and analyze the captured images, such as tracking cell movement, analyzing cell growth curves, and more.

Advantages of Live Cell Imaging Technology

• Long-Term Monitoring: It allows for continuous observation of cells, capturing transient events and avoiding information that may be missed in endpoint experiments, while also determining the optimal time point for endpoint experiments.
• Environmental Control: By strictly controlling environmental conditions (e.g., gases, temperature, and humidity), the system ensures that cells remain in their natural physiological state.
• Reduced Artifacts: Compared to other imaging techniques such as cell fixation and immunostaining, live cell imaging reduces artifacts.
• Multi-Parameter Simultaneous Detection: It allows for the observation of the localization and transport of cellular biomolecules and the progression of multiple pathways at the same time.
• Four-Dimensional Imaging: By imaging along the horizontal, axial, and time axes, it generates four-dimensional images and data.
• Qualitative and Quantitative Data: It provides qualitative and quantitative data that cannot be obtained through other biochemical methods.

Live Cell Imaging Dyes

Dyes play a critical role in live cell imaging, as they are used to label specific structures or molecules inside cells, enabling researchers to observe dynamic changes within the cell using equipment like fluorescence microscopes. Fluorescent dyes are compounds that absorb light of a specific wavelength and emit fluorescence at a longer wavelength. These dyes have different excitation and emission spectra, allowing for the selection of appropriate dyes based on experimental needs. The characteristics of fluorescent dyes include fluorescence intensity, photostability, and water solubility, which determine their suitability for cell imaging. Commonly used live cell imaging dyes include DAPI, Hoechst dyes, FITC (fluorescein isothiocyanate), and Rhodamine, among others.

Fluorescent Dyes for Cell Imaging

Application of Fluorescent Dyes in Cell Imaging

Fluorescent dyes provide a powerful tool for biological research in cell imaging, enabling precise labeling and tracking of specific molecules and structures within cells. These dyes bind to target molecules and emit fluorescence under specific excitation light, allowing for real-time monitoring of complex biological processes inside cells. Fluorescent dyes not only enhance the resolution and sensitivity of imaging but also enable dynamic observation of cellular activities, such as protein localization, cell division, and signal transduction pathways.

Live Cell Imaging Protocol

Live cell imaging technology provides a powerful tool for cell biology research. By designing experiments properly and strictly following experimental protocols, dynamic processes in cells can be effectively observed and analyzed, providing important data support for life sciences research. In the live cell imaging process, the cell's activity is crucial. Therefore, it is necessary to strictly control the growth environment of cells, such as temperature, humidity, CO₂ concentration, etc., during the experiment. At the same time, it is important to minimize interference with the cells to avoid reducing their activity due to human factors. Additionally, selecting appropriate markers is critical for the success of the experiment. Markers should have good specificity, stability, and low toxicity. The appropriate concentration and labeling time should be chosen based on the experimental goal and cell characteristics to ensure labeling effectiveness and cell viability.

• Pre-Experiment Preparation

• Cell Culture: Select the appropriate cell type based on the experimental needs and culture under suitable conditions to ensure cells are in good growth status. For example, adherent cells are typically cultured in media containing specific nutrients, and the culture medium should be replaced regularly to maintain the growth environment.
• Imaging Equipment Selection: Choose the appropriate microscope based on the experimental purpose and cell characteristics. Common live cell imaging microscopes include confocal microscopes, wide-field microscopes, and light-sheet microscopes. For example, confocal microscopes provide high resolution and contrast, suitable for observing fine structures inside the cell, while light-sheet microscopes have lower light toxicity, making them suitable for long-term observation of dynamic cell changes.
• Marker Preparation: To better observe specific cell structures or molecules, fluorescent markers are often used. For example, MitoTracker can be used to label mitochondria, and CellMask can be used to label cell membranes. Select the appropriate markers according to experimental requirements and follow the instructions for labeling.

• Imaging Process

• Cell Processing: Process the cultured cells according to the experimental design. For example, in experiments observing drug effects, cells need to be incubated with the drug for a period of time. During processing, it is important to maintain the cell's activity and stability, avoiding cell damage due to improper handling.
• Imaging Operation: Place the treated cells under the microscope for imaging. Set appropriate imaging parameters, such as exposure time, aperture size, and magnification, according to the experimental needs. During imaging, minimize light exposure to the cells to avoid cell death caused by prolonged illumination.
• Image Acquisition: During the imaging process, capture real-time images of the cells and save them in an appropriate format. For experiments requiring long-term observation, an automatic acquisition function can be set to capture images at regular intervals to record the dynamic changes of the cells.

• Image Analysis

• Image Preprocessing: Preprocess the acquired images, such as removing noise and correcting the background. This step improves the image quality and provides a better foundation for subsequent analysis.
• Quantitative Analysis: Perform quantitative analysis of the images based on the experimental objectives. For example, measure cell size, morphology, fluorescence intensity, etc. Professional image analysis software, such as Fiji-ImageJ, can be used for analysis.
• Result Interpretation: Combine the analysis results with the experimental goals to interpret the results. For example, by observing changes in cell morphology, researchers can understand processes such as cell growth, division, and apoptosis; by measuring changes in fluorescence intensity, they can learn about the expression levels and dynamic changes of molecules inside the cells.

Live Cell Imaging Media

Live cell imaging media is a physiological medium developed for live cell imaging applications. It is optically transparent, buffered with HEPES, and has a pH of 7.4, enabling cells to remain healthy for up to 4 hours at room temperature and pressure. Compared to traditional culture media, it provides better imaging clarity, signal-to-noise ratio, and cell viability. This medium is suitable for a variety of cell experiments, including live cell imaging, dye loading, and washing steps. It is applicable to multiple imaging techniques, such as fluorescence microscopy and confocal microscopy. The medium is also ideal for dye loading and washing steps, helping researchers load fluorescent dyes into cells and remove excess dye through washing. This feature makes live cell imaging media important in cell labeling and fluorescence imaging experiments.

What is Live Cell Imaging Used For?

• Cell Structure and Function Research

Cell imaging technology provides intuitive visual support for studying cell structures. Using optical and electron microscopy, researchers can clearly observe various cell structures such as the cell membrane, nucleus, mitochondria, endoplasmic reticulum, and more. The morphology and distribution of these structures are closely related to cell functions. For example, changes in the shape of mitochondria are closely linked to cellular energy metabolism. By labeling and imaging mitochondria with specific dyes, researchers can study changes in mitochondria during cellular stress responses. Additionally, cell imaging can be used to observe protein distribution and dynamic changes within cells, thus revealing the role of proteins in cellular functions.

• Cell Cycle and Cell Death Research

The cell cycle is the process of cell growth and division, and its regulation is crucial for maintaining normal physiological functions in organisms. Cell imaging technology allows for real-time observation of various stages of the cell cycle by labeling proteins or chromosomes associated with the cell cycle. For example, by using fluorescent protein markers for cyclins, dynamic changes in cyclins during the cell cycle can be observed, thus revealing mechanisms of cell cycle regulation. Cell death is another important phase of the cell lifecycle, including apoptosis and necrosis. Cell imaging technology can observe morphological changes during cell death, such as membrane bubbling and chromatin condensation, to distinguish between different types of cell death.

• Gene Expression and Regulation Research

Gene expression refers to the process where genetic information from DNA is transcribed into RNA and then translated into proteins. Cell imaging technology allows for the real-time observation of gene expression and localization within cells by using fluorescent RNA probes or fluorescent protein fusion proteins. For example, fluorescent in situ hybridization (FISH) technology uses fluorescent RNA probes to bind specifically to mRNA within cells, allowing researchers to observe gene expression levels and localization. Additionally, fluorescent protein fusion techniques can be used to study gene expression and subcellular protein localization. By fusing a fluorescent protein gene with a target gene, the co-localization of the fluorescent protein and target protein can be observed, revealing mechanisms of gene expression regulation.

• Protein Interaction Research

Protein interactions are fundamental to various biological processes within cells. Cell imaging technology can be used to study protein-protein interactions through techniques like fluorescence resonance energy transfer (FRET). FRET is based on the energy transfer principle between fluorescent proteins, where energy transfer occurs when two fluorescent proteins are within a certain distance, generating a specific fluorescence signal. By observing changes in fluorescence signals, researchers can determine if protein-protein interactions exist and assess their interaction strength. Additionally, bimolecular fluorescence complementation (BiFC) can be used to study protein interactions. This technique involves fusing two fragments of a fluorescent protein with target proteins, and when the target proteins interact, the two fragments reassemble to emit fluorescence signals.

• Disease Biomarker Detection and Localization

Disease biomarkers are specific biomolecules that appear during the onset and progression of diseases. Cell imaging technology can be used to detect and locate disease biomarkers within cells by using fluorescently labeled antibodies or probes. For example, in cancer research, fluorescently labeled antibodies can be used to identify specific antigens on the surface of tumor cells, allowing for early detection and localization of tumor cells. Furthermore, cell imaging can be used to observe the distribution and dynamic changes of disease biomarkers within cells, helping to reveal disease mechanisms.

• Drug Screening and Efficacy Evaluation

Cell imaging technology has significant applications in drug screening and efficacy evaluation. By establishing cell models and using cell imaging technology, researchers can observe the effects of drugs on cell morphology, function, and molecular levels to quickly screen potential therapeutic drugs. For example, in anti-tumor drug screening, the effects of drugs on tumor cell growth, proliferation, and apoptosis can be assessed. Moreover, cell imaging can be used to study the mechanisms of drug action, providing theoretical support for drug development.

• Tissue Regeneration Monitoring

Tissue engineering and regenerative medicine aim to repair and regenerate damaged tissues and organs through cell transplantation and tissue engineering scaffolds. Cell imaging technology can be used to monitor changes in cell behavior and tissue structure during tissue regeneration. For example, fluorescently labeled cells transplanted into damaged tissues can be used to observe cell survival, proliferation, and differentiation in real-time. Cell imaging can also be used to observe processes like angiogenesis and nerve regeneration during tissue regeneration, providing important support for research in tissue engineering and regenerative medicine.

• Stem Cell Imaging and Tracking

Stem cells have the ability to self-renew and differentiate into various cell types, making them highly promising in tissue engineering and regenerative medicine. Cell imaging technology can be used to image and track stem cells by labeling them with fluorescent tags or magnetic nanoparticles. By observing the distribution and migration of stem cells in vivo, the effectiveness and safety of stem cell therapies can be evaluated. Additionally, cell imaging can be used to study the differentiation process of stem cells and the effects of the microenvironment on their differentiation, providing theoretical support for optimizing stem cell treatments.

Summary and Outlook

Cell imaging technology, as an essential tool in modern biomedical research, provides a powerful means for exploring the internal world of cells. From optical microscopy to electron microscopy, from fluorescent dyes to fluorescent proteins, cell imaging technology is continually advancing and refining, providing strong support for research in fields such as cell biology, molecular biology, and pathology. The widespread application of cell imaging technology in disease diagnosis, drug development, tissue engineering, and regenerative medicine brings new hope for medical research and clinical practice.

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