Unlocking the Power of Fluorescence Imaging: A Comprehensive Guide

As an advanced imaging method, fluorescence imaging technology proves essential across biomedical science, materials science, and environmental monitoring. Fluorescence imaging technology generates high-resolution images through non-invasive means, which assists scientists in examining biochemical processes and structural details of cells and tissues as well as complete living organisms. The rapid progress of optical technology combined with advancements in materials science and computer image processing has enabled fluorescence imaging technology to evolve significantly from basic microscopy methods to advanced multimodal systems, which now support in vitro cell imaging as well as in vivo animal and human imaging.

Fluorescence Imaging

Fluorescence imaging is an imaging technique based on the phenomenon of fluorescence. In physics, fluorescence refers to the emission of light by a substance after it absorbs photons, causing its electrons to transition to a higher energy state. As the substance returns to its ground state, it releases excess energy in the form of photons. In biomedical science, fluorescence imaging focuses on using molecules or nanomaterials with specific fluorescence properties as imaging agents to label biomolecules, cells, or tissues. The fluorescence signals emitted are detected by optical instruments to obtain structural and functional information inside the body. The core features of fluorescence imaging as an imaging technique include high sensitivity, high specificity, and non-invasiveness. It can detect very low concentrations of fluorescent markers, achieve specific labeling of particular biological targets by selecting appropriate fluorescence imaging agents, and, during the imaging process, avoid destructive treatment of biological samples, enabling real-time dynamic monitoring of living cells and organisms.

Fig. 1. Fibroblast cell imaging.

Fluorescence Imaging Technique

The biomedical research and diagnostics fields now recognize fluorescence imaging as a robust and adaptable instrument. This method takes advantage of fluorescent molecules to achieve precise visualization of biological structures and processes. Fluorescence imaging allows scientists to peer into living systems by detailing structures from cellular organelles up to complex tissue architectures.

• By Imaging Principle

Fluorescence Resonance Energy Transfer (FRET)

FRET imaging is based on the principle of fluorescence resonance energy transfer. When two fluorescent molecules (donor and acceptor) are within a certain distance, the excited-state energy of the donor molecule can be transferred non-radiatively to the acceptor molecule, resulting in a decrease in the donor's fluorescence intensity and an increase in the acceptor's fluorescence intensity. This imaging technique is mainly used to study interactions between biomolecules, such as protein-protein interactions and nucleic acid-protein interactions. It provides information on molecular distances and dynamic changes, with significant applications in cell signal transduction and gene expression regulation research.

Fluorescence Lifetime Imaging (FLIM)

Fluorescence lifetime refers to the time interval during which a fluorescent molecule emits light as it returns from the excited state to the ground state. FLIM measures the fluorescence lifetime of molecules to obtain image information. Since fluorescence lifetime is highly sensitive to changes in the microenvironment of fluorescent molecules, such as local concentration, pH, and ion concentration, FLIM can be used to study the changes in the microenvironment of biomolecules and their interactions, such as detecting changes in intracellular calcium ion concentrations or protein conformational changes. It offers high spatiotemporal resolution and specificity.

Multiphoton Fluorescence Imaging

Multiphoton fluorescence imaging uses multiple low-energy photons to simultaneously excite fluorescent molecules, causing them to emit fluorescence. Compared to conventional single-photon fluorescence imaging, multiphoton fluorescence imaging has deeper tissue penetration, higher spatial resolution, and lower phototoxicity. This is because the multiphoton excitation process requires multiple photons to interact with the fluorescent molecule simultaneously, and only at the focal point can the photon density reach a sufficient level for excitation, reducing photodamage and background fluorescence interference in non-focal regions. This technology has significant advantages in high-resolution and deep tissue imaging areas such as in vivo brain imaging and tumor tissue imaging.

• By Imaging Object

Cellular Fluorescence Imaging

Cellular fluorescence imaging is primarily used to observe the distribution, dynamic changes, and interactions of various structures and biomolecules within cells. For example, fluorescently labeled antibodies can specifically recognize and label certain proteins on the cell surface or inside the cell. Fluorescent dyes can be used to specifically stain organelles like mitochondria, endoplasmic reticulum, and Golgi apparatus, making it possible to clearly observe cellular substructures under a fluorescence microscope. Moreover, genetic engineering techniques using fluorescent proteins (e.g., green fluorescent protein (GFP) and its variants) allow researchers to observe protein expression, localization, transport, and interactions with other molecules in live cells in real-time. The advantage of cellular fluorescence imaging is that it provides high-resolution image information at the cellular level, enabling observation and analysis of single cells or even single molecules. However, its drawback is that it often requires fixation, staining, and other treatments for cells, which may limit long-term observations of live cells.

Tissue Fluorescence Imaging

Tissue fluorescence imaging is mainly used for imaging tissue sections or blocks to study tissue structure, pathological changes, and drug distribution within tissues. In pathological research, fluorescently labeled antibodies or dyes can be used to detect and locate specific cell types, protein expression, cell proliferation, and apoptosis within tissue sections, providing important evidence for disease diagnosis and research. Furthermore, tissue fluorescence imaging can be combined with techniques such as optical coherence tomography (OCT) to achieve three-dimensional imaging and depth analysis of tissues. The advantage of tissue fluorescence imaging is that it provides structural and functional information at the tissue level, which is significant for studying physiological and pathological processes of tissues. However, its limitation lies in the depth and resolution of the imaging, with suboptimal imaging results for thick tissues.

In Vivo Fluorescence Imaging

In vivo fluorescence imaging is a non-invasive imaging technique that allows for real-time observation of biological processes and disease development in living animals or humans. By injecting fluorescence imaging agents into living organisms and detecting their fluorescence signals with external optical imaging devices, in vivo fluorescence imaging enables early detection and growth monitoring of tumors, in vivo distribution and metabolism studies of drugs, and real-time monitoring of gene expression. The advantage of in vivo fluorescence imaging is that it allows for real-time dynamic imaging in living organisms, reflecting true physiological and pathological processes within the body, making it valuable for disease research and treatment. However, in vivo fluorescence imaging also faces challenges such as limitations in imaging depth and resolution, background fluorescence interference within the organism, and concerns over the biocompatibility and safety of imaging agents.

Fluorescence Imaging Principle

• Physical Basis of Fluorescence Phenomenon

The physical process that generates fluorescence can be divided into the following steps: first, when a fluorescent molecule absorbs a photon, its electron transitions from the ground state (S0) to a higher energy excited state (S1 or S2). The electron in the excited state is in an unstable high-energy state and will return to the ground state through a non-radiative relaxation process, such as internal conversion or vibrational relaxation. During this process, the excess energy is released in the form of heat. Finally, when the electron transitions from the vibrationally excited state back to the lowest vibrational state of the ground state, it releases energy in the form of a fluorescence photon, thus generating fluorescence. The wavelength of the fluorescence is typically longer than that of the excitation light because some energy is lost during the process of the electron returning to the ground state. The intensity and wavelength characteristics of fluorescence depend on the structure of the fluorescent molecule and the microenvironment in which it resides. For example, factors such as the molecular conjugation structure, electron donor and acceptor groups, the polarity of the surrounding medium, and hydrogen bonding can all affect the fluorescence properties.

• Optical Principle of Fluorescence Imaging

The optical principle of fluorescence imaging primarily involves collecting and detecting fluorescence signals to form an image using an optical system. In a fluorescence imaging system, the excitation light source emits light of a specific wavelength to irradiate the sample, causing the fluorescent molecules in the sample to absorb light energy and emit fluorescence. The fluorescence signals are collected by the objective lens, and after passing through a series of optical elements (such as filters and beam splitters), they are separated and selected. Ultimately, the signals are detected by a detector (such as a photomultiplier tube or a CCD camera) and converted into an electrical signal. Understanding the relationship between excitation light and emission light is crucial for fluorescence imaging. The wavelength of the excitation light must match the absorption spectrum of the fluorescent molecule to ensure that the molecule effectively absorbs energy and generates fluorescence. The wavelength of the emitted light depends on the fluorescence spectral characteristics of the molecule, and by selecting the appropriate filter, excitation and emission light can be separated to only detect the fluorescence signal. Moreover, the imaging quality and resolution of the optical system directly affect the quality of the fluorescence image. High numerical aperture objectives can collect more fluorescence signals, enhancing the image brightness and resolution, while good optical focusing and calibration ensure the clarity and accuracy of the image.

Fluorescence Imaging System

A complete fluorescence imaging system typically consists of key components such as a light source, excitation filter, imaging objective lens, emission filter, and detector. The light source provides the specific wavelength of light required to excite the fluorescent substance, and its stability and spectral characteristics directly affect imaging quality. The excitation and emission filters are used to precisely select the wavelength range of excitation light and fluorescence, effectively filtering out unwanted light, thereby improving the signal-to-noise ratio of the image. The imaging objective lens focuses the fluorescence and forms an image on the detector; its resolution and numerical aperture determine the clarity and level of detail in the image. The detector is a key part of the imaging system, with common types including charge-coupled devices (CCD) or photomultiplier tubes (PMT), which can convert fluorescence signals into electrical signals and generate image data for analysis. The working process of the fluorescence imaging system can be divided into several steps:

• Selection of Excitation Light Source: The appropriate excitation light source is selected based on the absorption spectrum of the fluorescence imaging agent. Common excitation light sources include mercury lamps, xenon lamps, and lasers. Mercury and xenon lamps can provide a wide range of excitation wavelengths, suitable for exciting various fluorescence imaging agents, while lasers offer advantages such as high monochromaticity and high energy density, providing strong excitation light of a specific wavelength. These are suitable for fluorescence imaging that requires high resolution and sensitivity, such as confocal fluorescence imaging.
• Generation and Collection of Fluorescence Signals: The excitation light emitted by the light source passes through the optical system to irradiate the sample, causing the fluorescence imaging agent in the sample to absorb energy and emit fluorescence. After the fluorescence signal is collected by the objective lens, optical elements such as filters are used to separate out the fluorescence signals within a specific wavelength range to reduce background light interference.
• Image Formation and Processing: The collected fluorescence signals are detected by the detector and converted into electrical signals, which are then processed and analyzed by an image capture card and computer software to form a fluorescence image. During image processing, various operations such as noise reduction, enhancement, and segmentation can be performed to improve the image quality and readability. Additionally, quantitative analysis of fluorescence images can be conducted, such as measuring fluorescence intensity, localizing and mapping fluorescence signals, to obtain more comprehensive information.

Fluorescence Imaging Agents

• Types of Fluorescence Imaging Agents

• Selection of Fluorescence Imaging Agents

Choosing the appropriate fluorescence imaging agent depends on several factors related to the specific imaging requirements:

• Imaging Depth: For deep tissue imaging, such as in vivo imaging, fluorescence imaging agents with longer emission wavelengths should be selected. Longer wavelengths experience less scattering in biological tissues and penetrate deeper. For example, near-infrared fluorescence imaging agents (e.g., certain organic dyes and quantum dots) are particularly promising for in vivo imaging applications.
• Resolution: High-resolution imaging demands fluorescence imaging agents with high fluorescence brightness and good photostability to minimize photobleaching effects on image quality. The size of the imaging agent also affects resolution, with smaller agents being better suited for accessing fine cellular and tissue structures and providing more detailed resolution information.
• Biocompatibility: Fluorescence imaging agents should exhibit excellent biocompatibility and should not cause toxicity or immune responses in biological samples. Biocompatibility is especially critical for live-cell and in vivo imaging. Fluorescent proteins and biocompatibility-modified quantum dots are often preferred for such applications due to their favorable biocompatibility profiles.
• Specificity: The imaging agent's specificity is essential for targeting particular molecules or cells. For example, fluorescently labeled antibodies targeting specific cell surface markers or fluorescent dyes that bind selectively to specific nucleic acid sequences can achieve precise molecular or cellular labeling and imaging.
• Excitation and Emission Spectral Properties: The excitation and emission spectra of the fluorescence imaging agent should match the light source and detector of the imaging system. For multicolor fluorescence imaging, agents with distinct excitation and emission spectra should be chosen to avoid spectral overlap and interference.

Fluorescent Imaging Dyes

Fluorescent Imaging Probes

Fluorescent imaging probes are a class of fluorescent imaging agents designed to respond to specific biomolecules or physiological processes by altering their fluorescence signals. The design principle lies in tailoring their chemical structures to enable specific interactions with target molecules or processes, thereby inducing changes in fluorescence signals. For example, fluorescent probes for detecting intracellular ion concentration changes can be designed by incorporating ligand groups capable of binding specific ions (e.g., calcium ions or zinc ions). Similarly, by linking fluorescent molecules with peptides or small molecules that specifically recognize protein-binding sites, fluorescent probes for protein interactions can be developed. The design of fluorescent probes must consider factors such as their affinity and selectivity for the target molecule, sensitivity to fluorescence signal changes, and biocompatibility to ensure their accurate reflection of target molecule or physiological process variations in biological systems.

Fluorescence Image Analysis

• Image Processing Techniques

Fluorescence image processing techniques aim to enhance image quality to facilitate subsequent analysis and interpretation. Common image processing techniques include:

• Denoising: Fluorescence images often contain various types of noise, such as photon noise and electronic noise, which degrade image quality and signal-to-noise ratio. Denoising techniques use algorithms such as mean filtering, median filtering, or Gaussian filtering to remove noise while preserving useful information in the image.
• Enhancement: Image enhancement techniques improve image contrast and brightness, making details more visible. For example, methods such as histogram equalization or linear and nonlinear transformations can adjust the grayscale distribution of images to enhance their visual quality.
• Segmentation: Image segmentation separates regions of interest (e.g., cells, organelles, fluorescently labeled molecules) from the background. Segmentation can be achieved using algorithms based on grayscale, color, or texture features, such as thresholding, edge detection, or region-growing methods. Accurate segmentation is critical for subsequent quantitative analysis and biological interpretation.

• Quantitative Analysis of Images

Quantitative analysis of fluorescence images provides valuable information through various approaches:

• Fluorescence Intensity Measurement: Fluorescence intensity is a critical parameter in fluorescence imaging, reflecting the concentration of fluorescent imaging probes, the expression levels of biomolecules, or the activity of physiological processes. Measuring fluorescence intensity in specific regions of an image enables relative or absolute quantification. For instance, in cell experiments, fluorescence intensity measurements can compare the expression levels of fluorescently labeled molecules under different treatment conditions. In vivo imaging can monitor tumor growth or drug distribution by analyzing changes in fluorescence intensity.
• Localization and Distribution Analysis of Fluorescence Signals: Fluorescence images provide spatial distribution information of fluorescence signals within cells or tissues. By analyzing the localization and distribution patterns of fluorescence signals, researchers can gain insights into the intracellular localization and tissue distribution of biomolecules, as well as their dynamic changes during physiological processes. For example, fluorescence colocalization analysis can study whether two fluorescently labeled proteins colocalize within cells, indicating potential interactions or joint involvement in specific biological processes.
• Fluorescence Lifetime Analysis: Fluorescence lifetime refers to the time interval during which a fluorescent molecule returns from the excited state to the ground state while emitting fluorescence. It is highly sensitive to changes in the microenvironment of fluorescent molecules. Fluorescence lifetime imaging microscopy (FLIM) can measure fluorescence lifetimes to obtain information on the microenvironment of biomolecules, such as local concentrations, pH, or ion levels. Fluorescence lifetime analysis has significant applications in studying biomolecular interactions, intracellular signal transduction, and early disease diagnosis.

What is Fluorescence Imaging Used For?

• Cellular Biology Research

Fluorescence imaging is widely used to study cellular morphology, organelle distribution and function, the expression and localization of biomolecules, as well as cell proliferation and apoptosis. For example, fluorescently labeled antibodies or fluorescent protein fusions can specifically tag proteins on the cell surface or within cells, enabling the study of protein expression, localization, transport, and interactions with other biomolecules. Fluorescent dyes can be used to stain organelles, allowing observation of their morphology, distribution, and dynamic changes, which provide insights into cellular physiological and pathological processes.

• Molecular Biology Research

Fluorescence imaging plays a crucial role in molecular biology, including monitoring gene expression, locating nucleic acids, and studying molecular interactions. For instance, by fusing a fluorescent protein gene to the target gene in an expression vector, researchers can monitor gene expression and protein synthesis in real time within live cells. Fluorescently labeled nucleic acid probes can locate and quantify specific nucleic acid sequences within cells, enabling the study of transcriptional regulation and RNA metabolism.

• Pathology Research

In pathology, fluorescence imaging can be used for staining and imaging tissue sections to detect pathological changes such as diseased cells, abnormal protein expression, and cell apoptosis. Fluorescently labeled antibodies or dyes can specifically identify and label biomarkers related to pathology, providing critical evidence for disease diagnosis and research. Additionally, fluorescence imaging can be combined with other imaging techniques such as optical coherence tomography (OCT) and magnetic resonance imaging (MRI) to achieve multimodal imaging and in-depth analysis of tissues, improving the accuracy and reliability of pathological diagnoses.

• Drug Development

Fluorescence imaging has diverse applications in drug development, including studying drug targeting, tracking drug distribution and in vivo metabolism, and evaluating drug efficacy. By conjugating fluorescent imaging agents to drugs, researchers can monitor drug distribution and metabolic processes in cells and tissues in real time, thereby assessing drug targeting and pharmacokinetics. Fluorescence imaging can also be used to observe the biological effects of drugs on cells and tissues, providing valuable insights for evaluating drug efficacy and safety during development and optimization.

• In Vivo Animal Model Research

In vivo fluorescence imaging is extensively applied in small animal models, such as tumor, cardiovascular, and neurological disease models. By injecting fluorescent imaging agents into live animals, researchers can monitor tumor growth, metastasis, and treatment effects in real time; observe hemodynamic changes, angiogenesis, and inflammatory responses in the cardiovascular system; and study neuronal activity, neurotransmitter release, and neurodegenerative changes in the nervous system. This technique provides powerful tools for investigating disease mechanisms, drug action, and the dynamic changes of biological processes, advancing medical research and drug development.

• Fluorescence Imaging in Cancer Research

Fluorescence imaging is pivotal in cancer research, supporting early diagnosis, therapeutic monitoring, and drug development. For early cancer diagnosis, fluorescence imaging can detect tumor-specific markers or changes in the tumor microenvironment. For instance, researchers have developed fluorescent probes that specifically bind to tumor cell surface receptors, enabling early detection and localization of tumor cells via fluorescence imaging. In therapeutic monitoring, fluorescence imaging can provide real-time observation of tumor size, angiogenesis, and drug distribution and efficacy in tumor tissues.

• Fluorescence Live Cell Imaging

In live-cell imaging, fluorescence imaging is used to observe the distribution and dynamic changes of various biomolecules within cells, such as the synthesis, transport, and degradation of proteins, changes in organelle morphology and function, and signal transduction processes. For example, fluorescently labeled proteins enable real-time tracking of protein localization and dynamics within cells, offering critical insights into protein functions and cellular physiology. Fluorescence imaging can also detect cell apoptosis, analyze cell cycles, and study intercellular interactions, providing diverse experimental tools for cellular biology research.

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