Introduction
Bioimaging refers to the set of techniques that allow visualisation, characterisation, and quantification of biological structures and processes in living systems. It bridges biology, chemistry, physics, and engineering to enable observation from the molecular scale to whole organisms. Modern bioimaging has become essential for understanding cellular dynamics, disease mechanisms, and therapeutic responses.
Advances in optics, fluorescent probes, computational analysis, and nanotechnology have transformed bioimaging into a quantitative and high-resolution discipline. From light microscopy to super-resolution imaging and multimodal in vivo imaging, these technologies now provide spatial and temporal information at unprecedented scales.
Historical Development of Bioimaging
01 Early Microscopy
The origins of bioimaging date back to the 17th century with pioneers such as Robert Hooke and Antonie van Leeuwenhoek. Hooke’s Micrographia (1665) introduced the term “cell,” while Leeuwenhoek observed bacteria and protozoa using handcrafted lenses.
In the 19th and early 20th centuries, improvements in optics, staining methods, and contrast techniques enabled clearer visualisation of cellular components.
02 Electron Microscopy Era
The invention of electron microscopy in the 1930s dramatically increased resolution beyond the diffraction limit of light. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) allowed visualization of organelles and viral particles.
03 Fluorescence and Molecular Imaging
The discovery and application of green fluorescent protein (GFP), originally isolated from Aequorea victoria, revolutionized live cell imaging.
04 Super-Resolution Breakthrough
Techniques such as STED, PALM, and STORM overcame the diffraction limit (~200 nm), achieving nanometer-scale resolution.
3 Magnetic Resonance Imaging (MRI)
Is a non-invasive, radiation-free diagnostic tool that uses powerful magnets and radio waves MRI is based on nuclear magnetic resonance of hydrogen atoms in water molecules. It enables , high-contrast imaging of soft tissues organs, and bones.
Principles and Mechanisms of Bioimaging
Bioimaging techniques rely on the interaction between energy (light, electrons, magnetic fields, or sound waves) and biological matter.
1 Optical Imaging
Optical bioimaging is a non-invasive diagnostic technique includes:
- Bright-field microscopy
- Fluorescence microscopy
- Confocal microscopy
- Multiphoton microscopy
Fluorescence imaging uses fluorophores that absorb photons and emit light at longer wavelengths. Signal specificity is achieved via fluorescent proteins, organic dyes or quantum dots.
2 Electron Imaging
uses high-energy electron beams, rather than light, to magnify, visualize, and analyze the nanometer-scale structure of materials and biological specimens
Electron beams provide high-resolution ultrastructural information due to shorter wavelengths compared to visible light.
4 Molecular and Functional Imaging
visualizes in vivo biological, biochemical, and physiological processes at the cellular level, metabolic and biochemical activity in vivo.
Techniques such as PET and optical molecular imaging .
In fluorescence imaging, several nanomaterials are used to enhance sensitivity, specificity, and optical performance. Quantum dots (QDs) are semiconductor nanocrystals (1–10 nm) with size-dependent band gaps that enable tunable emission, broad absorption spectra, high molar extinction coefficients, narrow symmetric emission peaks, strong photostability, and large Stokes shifts, making them highly bright probes suitable for deep-tissue imaging and FRET-based applications; however, their potential cytotoxicity due to heavy metal release and their hydrophobic nature requiring surface functionalization remain limitations. Quenching nanomaterials such as gold nanoparticles, graphene oxide, and carbon nanotubes regulate fluorescence through energy or electron transfer mechanisms, enabling controlled “on–off” signal modulation in biosensing, gene and protein detection, and enzyme activity assays, while dark quenchers provide non-emissive energy absorption for precise molecular diagnostics. Upconversion nanoparticles (UCNPs), typically lanthanide-doped systems incorporating ions such as gadolinium, absorb near-infrared light and emit higher-energy visible or ultraviolet photons via anti-Stokes processes, offering deep tissue penetration, low autofluorescence, high photostability, and reduced phototoxicity, thus supporting applications in molecular imaging, cellular tracking, multimodal imaging, and cancer diagnosis.
Examples used in CT imaging
In computed tomography (CT) imaging, gold nanoparticles (AuNPs) are advanced contrast agents due to the high atomic number of gold, which confers superior X-ray attenuation compared with iodine-based agents, bone, and soft tissue (mass attenuation coefficient at 100 keV: ~5.16 cm²/g for gold versus ~1.94 cm²/g for iodine). This enhanced attenuation improves contrast resolution and diagnostic sensitivity. AuNPs exhibit strong biocompatibility, structural tunability, and physicochemical stability, with optimal cellular specificity typically observed in the 4–30 nm size range. Unlike conventional iodinated agents, AuNPs present reduced risk of renal toxicity and osmotic imbalance. They provide stable X-ray attenuation independent of particle morphology and can function as intracellular tracers for tumor labeling, tumor growth monitoring, red blood cell tracking, and both passive (enhanced permeability and retention effect) and active tumor targeting, supporting applications in cancer detection and image-guided radiotherapy.
In parallel, iodine-based liposomal nanotechnology has been developed to reduce the limitations of free iodinated contrast agents. Liposomes are nanoscale lipid bilayer vesicles (typically 100–400 nm) widely used as biomedical delivery vectors and adapted for CT imaging. Compared with conventional iodine agents, liposomal iodine nanoparticles exhibit lower osmotic pressure, prolonged blood circulation time, and improved contrast enhancement. Their accumulation in pathological tissues occurs via cellular uptake, receptor-mediated surface binding, or passive targeting through the enhanced permeability and retention (EPR) effect, making them effective targeted CT contrast systems.
Examples used in ultrasound imaging
Gas-generating nanoparticles used in acoustic and ultrasound imaging can be engineered through three principal strategies. The first approach involves direct encapsulation of preformed gases within the nanoparticle core, including nitrogen, air, perfluorocarbons, or sulfur hexafluoride, enabling immediate acoustic reflectivity due to impedance mismatch. The second method relies on liquid–gas phase transition mechanisms, where volatile liquids (e.g., perfluorohexane) are encapsulated and subsequently vaporized into gas upon ultrasound stimulation after passive or active accumulation at the target site; this produces enhanced acoustic backscatter and has been demonstrated in gold nanoparticle-coated, PEGylated mesoporous silica nanocapsules. The third strategy employs in situ gas generation via chemical reactions, typically using carbonate or ammonium bicarbonate cores that remain stable at physiological pH but react in the acidic tumor microenvironment to release carbon dioxide, thereby amplifying ultrasound contrast. These pH-responsive systems exploit the enhanced permeability and retention (EPR) effect or ligand-mediated targeting, offering controlled, site-specific acoustic signal enhancement for tumor imaging applications.
