Fluorescent Labeling

Definition

Fluorescent labeling is the process of binding fluorescent dyes to functional groups contained in biomolecules so that they can be visualized by fluorescence imaging (nature.com). The availability of new fluorophores has dramatically changed the possibilities for the sensitive detection of biomolecules and the analysis of their interactions. Improved fluorescent dyes are now enabling previously impossible studies of cellular structures and cellular processes. Fluorescent labels offer many advantages, as they are highly sensitive even at low concentrations, are stable over long periods of time, and do not interfere with the function of the target molecules. The targeted imaging of labeled cells enables tracking them in vitro and in vivo. Using different fluorophores in the same sample can also allow the simultaneous observation of several molecules at the same time. The most commonly used fluorophores are Fluorescein IsoThioCyanate (FITC), derivatives of rhodamine (TRITC), coumarin and cyanine. These synthetic organic dyes are used to label biomolecules as proteins, peptides, antibodies, nucleic acids, bacteria or yeast. Naturally occurring fluorochromes, such as Green Fluorescent Protein (GFP), can also be used to label living cells genetically.

  • Fluorescent protein labeling
    Fluorescent labeling allows researchers to investigate the conformational dynamics and molecular interactions of proteins or to track their movements in order to better understand their biological functions (Modesti M, 2011). To get better insight into receptor-ligand-binding, protein structures and enzyme activity, single peptides can be also labeled. Fluorochrome labeled antibodies are widely used in biomedical research to detect antigens in immunofluorescence assays and are also essential tools in immunodiagnostics. To ensure reliable results, the fluorophore conjugation should not interfere with the antigen-binding characteristics of the antibody (Nath N et al, 2016).
  • Fluorescent labeling of nucleic acids
    Fluorescence-based assays play a major role in biophysical studies of the structure, function and dynamics of nucleic acids. Recent advances in labeling methods and imaging systems have allowed the direct in vivo observation of DNA and RNA, and of their interactions with other cell components. Live-cell imaging of nucleic acids has opened new paths for better understanding chromatin organization and gene expression regulation. (Dirks RW et al, 2018).
  • Fluorescent labeling of polysaccharides
    Complex polysaccharides, such as heparin are structural components of the extracellular matrix. These polysaccharides are essential for cellular adhesion, migration and growth (Prigent-Richard S. et al, 1998). Some compounds are also known for their anticoagulant, antithrombotic, anti-inflammatory, antiviral and antiangiogenic properties. Fluorescence-based methods make it easier to identify new bioactive polysaccharides and to characterize their biological functions (Roger O et al, 2002).
  • Fluorescent labeling of lipids
    Cellular lipids play a crucial role in the cell for energy storage, for the formation of cellular membranes and for intracellular signaling processes (Maekawa M. and Fairn G, 2014). The lipophilic dye Nile red is widely used to stain intracellular lipids in order to analyze their location and organization (Greenspan P et al, 1985). Moreover, for studying lipid dynamics, specific labeling in living cells is also possible (Schultz C et al, 2010).

Fluorescent Labeling Techniques

Commonly used fluorescent labeling methods use chemical, enzymatic, peptide/protein tag and genetic labeling techniques (Sahoo H, 2012 , Toseland CP, 2013).

  • Chemical labeling techniques: Fluorophores dock to the target molecules through chemical modification (covalent or non-covalent binding). Chemical labeling methods have several advantages as they are robust, easy to perform and very efficient with a wide range of fluorophores. They are more suitable for in vitro studies rather than in vivo.
  • Enzymatic labeling techniques: Enzymatic reactions allow fast, highly-efficient and selective labeling in vivo and in vitro and can be used to target proteins or whole cells. Yet due to the large size of the labels, interferences with the function of the target molecules can occur.
  • Peptide/protein tag: A recently developed, and very promising, technique allows the specific and selective labeling of proteins through the incorporation of short fluorescent tags which do not disrupt the folding or the function of the molecule. This technique is also easy to perform and can be used to investigate different sites of single proteins depending on the peptide tag specificity.
  • Genetic labeling: Genetic labeling can be achieved using protein domains, small peptides or single amino acids which are marked with fluorescent dyes and can specifically attach to sites along chromosomes in vivo. This approach allows the detection of chromosomal abnormalities such as deletions or duplication.
  • Multicolor labeling: A common requirement for live cell imaging and for flow cytometry applications is the ability to follow or detect multiple fluorescently tagged proteins at the same time. For this purpose, specifically designed dyes with a very large Stokes shift can be used, which allow the simultaneous monitoring of different biochemical processes.

Fluorescence-based Assays

Fluorescence-based assays rely on the ability of fluorophores to re-emit light after the exposure to light particles or photons. The difference in wave length between excitation light and emission light, the so-called Stokes shift, can be detected in microscopes and imaging-systems. Each fluorophore has a specific Stokes shift. Different assays allow researchers to localize biomolecules, to observe them in real-time, to investigate their interactions and to study enzymatic activities.

  • Fluorescence microscopy
    Fluorescence microscopy allows the identification of cells and cellular components and the monitoring of cell physiology with high specificity. Fluorescence microscopy separates emitted light from excitation light using optical filters. The use of two indicators also allows the simultaneous observation of different biomolecules at the same time. Whereas conventional imaging systems allow a resolution of 200 to 300 nm due to physical diffraction limits, new super-resolution fluorescence microscopes as STED (stimulated emission depletion) overcome these limits and provide insights into the nanoscale world of molecules (Sanderson MJ et al, 2014).
  • Flow cytometry
    Flow cytometry is widely used in basic research and clinical practice to measure the signal from specific fluorophores. Cells and particles are analyzed and eventually sorted in “real time” as they pass through the light beam of detectors which quantify the fluorescence produced by labeled antibodies or ligands. These markers bind to specific molecules on the cell surface or inside the cell allowing their detection and quantification. Several parameters as size and volume can be measured on single cells, and different cell types can be isolated and characterized (Nolan JT and Condello D, 2013). Flow cytometry finds broad applications in fields as immunology, hematology, transplantation medicine, oncology and genetics.
  • Fluorescence in situ hybridization (FISH)
    Fluorescence in situ hybridization (FISH) allows the localization of specific DNA sequences on chromosomes. Fluorescent DNA or RNA probes are used to hybridize and identify complementary target DNA sequences. FISH has been traditionally used to map genes on chromosomes, for example, during the Human Genome Project. Today fluorescence in situ hybridization is mainly used for diagnostic purposes in the detection of chromosomal abnormalities or in the analysis of cancer cells (O’Connor C, 2008).
  • Fluorescence correlation spectroscopy (FCS)
    Fluorescence correlation spectroscopy (FCS) allows the analysis of temporal changes in the fluorescence intensity of fluorochromes, which are caused by chemical, biological or physical influences. FCS was first introduced to investigate interaction between drugs and DNA and represents now a sensitive tool for determining concentration and aggregation levels of proteins, as well as for observing molecular interactions (Tian Y et al, 2011).
  • Microarrays
    Microarrays allow the study of gene expression under different conditions. Thousands of genes can be examined at the same time on DNA chips. These are microscopic slides printed with tiny spots containing known DNA sequences that can selectively bind to fluorescent-labeled mRNA/cDNA molecules. After hybridization, the DNA-chip is read out, and the data is used to create gene expression profiles (Hoen PAC et al, 2003).

Fluorescent Label: Live-Cell Imaging

The kinetic observation of cellular processes using time-lapse fluorescence microscopy has become a fundamental technique in cell biology, as live-cell imaging can provide very valuable insights into cellular growth and transport mechanisms. One major challenge in time-lapse microscopy is minimizing phototoxic effects resulting from photobleaching. The light exposure progressively destroys fluorescent molecules leading to a reduction of the fluorescence signal and to the formation of free radicals which can damage the cells. Therefore, it is crucial for the method of live-cell imaging to find a balance between reducing light exposure as much as possible and getting useful signals to observe the cells. Scientists also need to create a physiological environment which allow to closely replicate the in vivo dynamics. (Ettinger and Wittmann T, 2014).

PromoCell Fluorescent Labeling

The availability of new fluorophores has dramatically changed the possibilities for the sensitive detection of biomolecules and the analysis of their interactions. Improved fluorescent dyes are now enabling previously impossible studies of cellular structures and cellular processes. PromoCell provides a wide range of high-quality fluorophores for fluorescent labeling of diverse biomolecules: protein labeling, antibody labeling, nucleic acid labeling (DNA labeling, RNA labeling), as well as complete labeling kits and ready-to-use fluorescent conjugates.

Our PromoFluor dyes are cost-effective alternatives to well-known, leading fluorophores and span the wavelengths spectrum from blue to far red. They show an outstanding fluorescence intensity and photostability, a strong light absorption, high fluorescence quantum yield and good water solubility and can be used for fluorescence microscopy, fluorescent in situ hybridization (FISH), fluorescence correlation spectroscopy (FCS) and microarrays (protein, DNA). Some of them offer an extra large Stokes shift, which makes them ideally suited for multicolor labeling or flow cytometry applications. PromoFluor dyes are available e.g. as NHS esters, maleimides and amino-modified labels ready for covalent coupling or as conjugates with biotin, phalloidin and deoxynucleotides (dUTPs). Moreover, we also provide high-quality Protein & Antibody Labeling Kits with different PromoFluor dyes.