Labelling Tools used in Fluorescence Microscopy

Proteins are known to play a major part in maintaining cellular integrity, including processes such as catalytic activity, molecular transport, signaling cascades, metabolism and cell adhesion.

Consequently various proteins of interest (POI) are in the focus of many researches employing fluorescence microscopy. Most proteins are not able to emit a fluorescent signal by themselves and thus need to be marked with a label to study their localization and organization in a cellular context.

Most commonly either a recombinant protein tag or an organic dye molecule coupled to a specific affinity probe is used to introduce fluorescence.

1.3.1 Detection of Proteins via Recombinant Tags

On DNA level different variants of the (enhanced) green fluorescent protein (GFP/EGFP) or other fluorescent proteins can be added as a recombinant fusion tag to the POI [35]. This way the POI can be observed or followed under a fluorescence microscope to study its cellular localizations. Today, after 23 years that the GFP was presented to the scientific community, thousands of laboratories around the world use it together with several dozens of modified fluorescent proteins that cover the whole visual spectrum.

As an alternative to already fluorescent proteins, also other recombinant tags such as the HALO, CLIP or SNAP-tag can be fused to the POI. These engineered enzymes acquired their fluorescence by adding a modified fluorophore that it can be recognized as the substrate for these enzymes, whereas the enzyme activity results in the covalent binding of the fluorophore to itself. Today, several modified fluorophores, both cell-permeable and cell-impermeable, as well as and different colors allow these system to be very flexible to follow fusion proteins in living cells with different colors [36].

To introduce such modified protein constructs into cells, transient or stable transfection is required, which is commonly associated with overexpression of the fusion protein. This may result in mislocalization of the protein induced by the recombinant tag or impair the actual function and interactions of the POI [37]. Additionally, if the cell-type used for the transfection also expresses the POI endogenously, this protein will not be fluorescent and thus not be detected under a fluorescent microscope, which might exacerbate the conclusions.

Recent gene technologies can overcome these limitations by using molecular toolkits to directly edit the cellular genome. Specific genes can for instance be modified using the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated 9 (Cas9) - system in cell cultures [38]. Due to direct modification of the endogenous protein, artifacts caused by overexpression can be omitted.

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In conclusion, the addition of recombinant tags to the POI is a straight forward approach and requires relatively little effort to study protein organization in fluorescence microscopy. The CRISPR/Cas9-system is very promising to reduce overexpression of the target protein, however it is still in development and few labs worldwide are using it routinely. Fusion chimeras expressed at endogenous levels with this technology might create transport, activation or localization problems.

Moreover, genome editing or overexpression of protein fusion constructs so far cannot be applied on human pathology samples or biopsies. Therefore, many applications and scientists still rely in affinity probes as an alternative detection method.

1.3.2 Affinity-Based Detection

The interaction and affinity of proteins is an alternative way to visualize the target POI, which can be used for indirect labeling based on detection with specific affinity probes.

For instance the specific binding of natural toxins to cellular proteins can be exploited for molecular marking. The cholera and pertussis toxins interfere with cellular signaling by binding to specific domains of G-protein coupled receptors. This interaction can be used to employ fluorescently labeled toxins in vitro for binding assays using fluorescence microscopy [39].

Another prominent example is the fungus toxin phalloidin, which binds and stabilizes filamentous actin molecules [40]. Fluorescently labeled derivatives of phalloidin are widely used in fluorescence microscopy to visualize intracellular actin filaments [40].

The specific binding between molecules is not limited to the detection of protein-protein interactions, but can also be used to detect lipids or even ion concentrations.

The pleckstrin homology domain binds to phosphatidylinositol in various cellular signaling pathways mediating signal transduction [41]. Fluorescently labeled pleckstrin homology domains have been used to monitor the intercellular pools of different phosphoinositide pools in microscopy [42].

Yet the number of known natural ligands binding strongly to specific proteins is limited, hence only a minor subset of POI can be studied by this method. Therefore antibodies, evolved to detect a plethora of different targets, are commonly raised to specifically bind target antigens for indirect detection in microscopy.

Upon immunization with a target antigen, animals present peptides of the antigen on their major histocompatibility complexes (MHC), which generates an immune response [43]. Due to genetic recombination and somatic hypermutation antibodies are created binding to the antigen. These molecules can be purified and subsequently used for the specific detection of POI in biological samples.

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The implementation of antibodies into fluorescence microscopy constituted the concept of immunofluorescence (IF) microscopy, which is up to now a common technique used in many laboratories. Commonly, the immunoglobulin gamma (IgG) antibodies used for IF are raised by animal immunization. When extracted from the serum and selected to bind a specific antigen, usually a plethora of IgG molecules is obtained that bind different epitopes of the same antigen. Such preparations are thus referred to as affinity-purified polyclonal antibodies.

In contrast, monoclonal antibodies are typically produced by single B-cells, producing a defined type of antibody, which were fused with an immortalized cell line. This procedure allows the creation of hybridoma cell lines that are able to be maintained growing while secreting one defined type of IgG molecules [44]. Consequently, monoclonal antibodies bind to their target antigen at one defined epitope, although the exact localization or sequence of the epitope is not always known. In IF microscopy, the POI is conventionally detected indirectly using a two-step antibody detection procedure. Antigen-specific primary antibodies are used to bind the POI and these are subsequently detected by secondary antibodies that carry enzymatic or fluorescent labels.

Secondary antibodies are mostly polyclonal, which results in signal amplification due to the binding of multiple secondary antibodies per primary antibody. This system provides high flexibility as differentially conjugated secondary antibodies can easily be substituted without requiring the use of a different primary antibody.

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