Immunofluorescence: Dyes and Other Haptens Conjugated with Antibodies

Abstract The ‘immunoimaging’ is indebted to the emergence of immunofluorescence from fluorescence. The immunofluorescence technique is a mixed method where fluorescence reflects the amount of target structure(s) measured by molecular probes. This measurement can be done in direct and indirect manners, and various biomolecules, including fluorescent‐labelled antibodies, biotin‐labelled single‐stranded deoxyribonucleic acid and hapten‐labelled proteins, can be employed as molecular probe. Phycobiliproteins, the brightest fluorescent probes applicable to the immunofluorescence assays, can become conjugated with biologically active molecules and proteins, such as antibodies. Quantum dots have provided a highly sensitive method for detection of biomolecules, favourably antibodies and key antigens. However, the main limitation of using quantum dots that restricts their application to clinical and in vivo settings is the potential toxicity of particles used in quantum dot synthesis. The fluorescent dyes in the near‐infrared region can be used very effectively for monitoring intracellular processes, and there has been much done to improve their quality (importantly quantum yield, water solubility and photobleaching). The family of Brilliant Violet fluorophores is a recently introduced product aimed at providing fluorescent dyes suitable for multilabel fluorescence. The conventional method of immunofluorescence is too costly in terms of the amounts of reagents and time required. In addition, its efficacy is influenced by many factors related to reagents and specimen. Therefore, although it provides us a noninvasive method for single‐cell tracking and monitoring subcellular molecules, the cost‐ effectiveness of conventional immunofluorescence is not satisfying and efforts must be intensified to improve its efficacy and economy. Key Concepts Photoluminescence, the process through which atoms emit light following light absorption, includes fluorescence and phosphorescence. Immunofluorescence, making major advances in the area of immunoimaging, is based on the application of immunological mediators to the fluorescence assay. Immunofluorescence is used to detect and identify the investigated biomolecules (receptor molecules) by the predefined biomolecules (receptor probes). The factors that affect the efficiency of fluorophores include peak excitation wavelength, peak emission wavelength, quantum yield, brightness, water solubility, pH insensitivity and photostability, the spillover effect and autofluorescence effects. Fluorophores can be categorised according to their nature into three: molecular compounds with certain structures (small organic dyes, metal–ligand complexes, phycobiliproteins and genetically encoded fluorescent proteins), nanocrystals with size‐dependent optical features (quantum dots) and particles with size‐independent optical features. There exist two major factors limiting the use of small organic dyes: (1) they require optimisation in order to obtain an ideal fluorescent dye and (2) they rely entirely on the ability of antibodies for recognition of target proteins. Great care should be taken when applying quantum dots to clinical and in vivo settings because of the potential toxicity of particles (such as cadmium) used in quantum dot synthesis. The main obstacles that limit the use of immunofluorescence techniques are as follows: (1) the molecular probes must be entered into the cell when probing intracellular structures and (2) the multivalent probes have great potential to oligomerise the target proteins. The near‐infrared (NIR) fluorescent probes have provided valuable insights into intracellular existence and related biomarkers and molecular processes including reactive species, thiol‐containing molecules, hydrogen sulfide, metal ions, anions and pH homeostasis. There has been a lot of effort to improve the quality of NIR fluorescent dyes (importantly quantum yield, water solubility and photobleaching), including development of NIR‐magnetic nanoparticles (greater emissive spectrum), modification of NIR probes using Si–rhodamine (better quantum efficiency) and development of brilliant violet fluorophores (multilabel fluorescence).