In order to understand the structures that make up a cell, it is vital that we can visualise and measure the life existing within cells. This is done through staining and labelling cells. By using fluorescent imaging or microscopy, for example, we are able to view a whole new world that exists all around us despite being invisible to the naked eye. So let's zoom in and have a look at cell labelling!
What is Cell Labelling?
Cells are the smallest units of life. They contain subcellular structures called organelles that carry out one or more specific jobs, similar to organs in a body.
In its simplest form, cell labelling refers to the visualisation of cells and the identification of cellular structures such as organelles.
More sophisticated types of cell labelling allow for the tracking of nucleic acids and proteins in living cells to study the various biochemical processes that occur in cells.
Cells can be visualized using microscopes. There are different types of microscopes, the most popular ones being the light microscope and the electron microscope. These microscopes have different magnifications and resolving powers and are used with different goals in mind.
The term 'resolution' or 'resolving power' in microscopy refers to a microscope's capacity to see detail. In different words, the resolution is the smallest distance between two different points of a specimen that may still be viewed as independent entities when seen under the microscope.
The capacity of a microscope to generate a picture of an item at a scale bigger than its true size is referred to as magnification.
For instance, organelles visible under a light microscope include the nucleus, cytoplasm, cell membrane, chloroplasts, and the cell wall. Other organelles are smaller than the resolution of light microscopes, so they appear blurry and not well-defined under light microscopes.
Electron microscopes, on the other hand, are much stronger in magnification and resolution. This allows them to capture clearer images from the cells with much higher definition. Electron microscopes are essential for observing smaller organelles such as ribosomes, vesicles, granules, and filaments that cannot be seen directly with light microscopy. Therefore, electron micrographs of cells are detailed images of the cells in which organelles can be identified and labelled.
Cell Labelling Methods
Cells can be labelled in multiple ways. These include some of the most basic laboratory techniques like Gram staining, or more complex techniques like using fluorescent dyes, immunolabeling or fluorescent fusion proteins. These all give us ways to see the structures of a cell more easily.
Some dyes used in cells can be toxic. Depending on the research question and the type of technique, cells might be "fixed" and killed during the staining process. Therefore, when wanting to track a cellular process in real-time through the use of dyes, it's important to know if the process will kill the cells or keep them alive.
We will have a look at the more advanced methods of cell labelling in this article, but you can check out our Gram-Staining article to learn more about this method!
Fluorescent Dyes as Cell Labelling Methods
Fluorescent dyes are biological molecules composed of at least one fluorophore. A fluorophore is a molecule that can emit light after getting excited by light energy. In other words, light reaches the fluorophore, which absorbs it and increases its energy. The fluorophore then emits light to liberate the extra energy it absorbed.
Fluorophores can be designed to bind to specific cell structures or components, and to absorb or emit light at a specific wavelength, allowing different types of fluorescent dyes to be combined in the same staining.
Fluorescent dyes can occur naturally in the living world, like the case of the Green Fluorescent Protein (GFP), or can be made synthetically. Synthetic dyes can be used to label biomolecules such as proteins, antibodies, peptides, nucleic acid, yeast and bacteria. These dyes include:
- Fluorescein IsoThioCyanate (FITC)
- Derivatives of rhodamine (TRITC)
- Coumarin
- Cyanine
Green Fluorescent Protein, or GFP for short, is a protein that has revolutionized the field of molecular biology. Originally discovered in the bioluminescent jellyfish Aequorea Victoria, GFP has since been isolated and extensively studied. GFP has allowed researchers to track biological processes in real-time, providing insights into the behaviour of cells that were previously impossible or really hard to observe.
| Table 1. Advantages and disadvantages of fluorescent dyes |
|---|
| Advantages of fluorescent dyes | Disadvantages of fluorescent dyes |
| High sensitivity and specificity | Requires specialized equipment |
| Can be used for both fixed and live cells | Limited number of colours available |
| Long-lasting and photostable | May affect cell viability and function |
| Can be multiplexed for simultaneous labelling of multiple targets | May show non-specific binding |
| Allows for quantitative analysis | May require optimization for different cell types and applications |
| Compatible with various imaging techniques | May require optimization for different experimental conditions |
| Allows for localization and tracking of specific cellular components | May require additional steps for sample preparation and staining |
Immunolabelling as a Cell Labelling Method
Immunofluorescence or immunolabelling involves labelling a biological target using an antibody. It can also be referred to as immunocytochemistry (ICC) and immunohistochemistry (IHC) or antibody labelling.
Antibodies are Y-shaped molecules that bind to other molecules called antigens.
Fig. Shape of an antibody.
Immunofluorescent dyes are designed so that the antibody part of the dye can bind to the specific molecule or organelle that we want to observe, which in this case plays the role of the antigen. The antibody is also bound to a fluorophore, which as we saw in the previous section, can emit light at a certain wavelength when it is excited with light in a different wavelength. In this way, the fluorescent label is specific due to the antibody being specific. The antibody is detected because it is bound to the fluorophore.
Now that we got through the first layer of complexity, let's have a look at how immunofluorescence really works. There are two types of ICC:
- Direct ICC: it works as we mentioned above. Only one antibody is used. This antibody can recognise the target molecule and can emit light when excited.
- Indirect ICC: it is rarely the case that the antibodies with the fluorophore are used directly to bind to the target molecule. Usually, two antibodies are used: one antibody with no fluorophore that can bind to the target molecule (primary antibody) and then a second antibody with the fluorophore, known as the secondary antibody, that can bind to the primary antibody specifically. The secondary antibody will be visible when excited under the microscope.
| Table 2. Advantages and disadvantages of direct vs indirect immunostaining |
|---|
| Direct immunostaining | Indirect immunostaining |
| Advantages | More sensitive than indirect staining | Higher specificity |
| No need for secondary antibodies | Signal amplification allows for detection at lower levels |
| Shorter protocol | Flexibility to use multiple secondary antibodies simultaneously |
| Disadvantages | Background staining can be a problem | Increased nonspecific staining |
| Limited availability of directly conjugated antibodies | Longer protocol |
| Possibility of epitope masking by conjugation | Increased cost due to the need for secondary antibodies |
Fluorescent Fusion Proteins
Fusion proteins can also be called chimeric proteins. They are formed by joining two or more genes which coded for separate proteins originally. Then, when the new "fusion gene" is transcribed and translated, there will be a new protein formed by the fusion of the sections that were joined together in the fusion gene. Fusion proteins are designed bearing in mind which sections of a protein are essential for its function and trying to disrupt the original conformation of the protein as little as possible.
The fusion of fluorescent tags to proteins is used to study their functions. Fusion proteins enable us to observe proteins in living cells and organisms. The first of these tags was done with GFP. It is still used today however photoconvertible fluorescent proteins (PCFPs), which were first isolated from Anthozoa are also used now. This method is very advantageous as it can be used to track proteins in real-time as the protein will change colour through the course of a mechanism.
| Table 3. Advantages and disadvantages of fluorescent fusion proteins |
|---|
| Advantages of fluorescent fusion proteins | Disadvantages of fluorescent fusion proteins |
| High specificity and sensitivity | Can interfere with protein functionality |
| Non-invasive | Can affect protein localization |
| Can be used for live imaging | Expression levels can vary |
| Multiplexing possible | Can be difficult to optimize |
| Can be used for protein-protein colocalization | Can be expensive |
| Does not require additional detection reagents | Can have low signal-to-noise ratio |
Types of Cell Labeling
There are many different types of cell labelling. These include:
- Fluorescence microscopy: this allows us to identify cells and cellular components with very high specificity. It uses optical filters to separate emitted light from excitation light and can allow observation of different biomolecules at the same time.
- Flow cytometry: this is used a lot in clinical practice. Cells and particles are passed through a light beam of detectors which can then analyse the labelled antibodies. Different cell types can be isolated and characterised as well as size and volume being measured.
- Fluorescence in situ hybridization (FISH): this allows specific DNA sequences to be localised on chromosomes. Fluorescent DNA or RNA probes are used to hybridize and identify target DNA sequences. FISH is most often used to map genes on chromosomes. One example of when this was used was the Human Genome Project. It is also used in things such as detection of chromosomal abnormalities in diagnostics.
- Fluorescence correlation spectroscopy (FCS): first introduced to investigate the interactions between drugs and DNA, FCS is useful for analysing changes in fluorochromes due to chemical, biological or physical influences.
- Microarrays: microarrays allow us to study gene expression. Many genes can be examined simultaneously. This data can then be used to create gene expression profiles.
Labelling parts of a cell
Cells are like cities. Similar to a city in which multiple sectors and departments work together for the city to thrive, cells also contain certain structures that carry out specific functions. These structures are called subcellular structures, and those subcellular structures that are membrane-bound are called organelles.
There are two main types of cells, eukaryotes, and prokaryotes. Prokaryotes, also known as bacteria, do not have any membrane-bound organelles. Their essential subcellular structures include the cell wall, plasma membrane, nucleoid (a circular DNA chromosome), and 70S ribosomes. Some bacteria contain more subcellular structures that are non-essential, such as flagella (singular flagellum), pili (singular pilus), plasmids, and a capsule. The table below summarises the structure and functions of these structures.
Table 4. Essential and non-essential subcellular structures in prokaryotic cells |
|---|
Subcellular structure | Chemical composition | Function |
Cell wall | Peptidoglycans (sugars and proteins) | Rigid support and protection against osmotic pressure, antibiotics, and lysozyme (a degrading enzyme). |
Plasma membrane | Phospholipids and lipoprotein bilayer | Selective transport of molecules into and out of the cell. |
Nucleoid | Circular DNA | Genetic material |
70S ribosome | Proteins and rRNA. Made up of 50S and 30S subunits. | Protein synthesis |
Flagellum | Protein | Motility |
Pilus (or fimbrium) | Glycoprotein | Attachment to surfaces or other bacteria |
Plasmid | Small circular DNA | Contains genes for antibiotic resistance |
| Capsule | Polysaccharide (chains of sugars) | Protection from phagocytosis |
The rest of our discussion will be strictly on eukaryotes. These include animal, plant, and fungal cells. Eukaryotic cells are more advanced than prokaryotes and have more sophisticated subcellular structures. Going back to our previous analogy, comparing cells with cities, prokaryotes would be a small town while a eukaryote would be a large megacity. Eukaryotes are more than 100 to 10,000 times larger than prokaryotes and are much more complex.
All eukaryotes have a plasma membrane and some, such as plant cells, also have a cell wall surrounding them. The genetic material in eukaryotes is confined in the nucleus, which is a membrane-bound organelle. Eukaryotes also rely on ribosomes for protein synthesis, but the 80S eukaryotic ribosomes are larger than their 70S prokaryotic counterpart. Some other subcellular structures and organelles found in eukaryotic cells include mitochondria, chloroplasts, centrioles, rough and smooth endoplasmic reticulum, Golgi apparatus, and lysosome. The table below summarises the function of these structures.
Table 5. Organelle and subcellular structures in eukaryotes |
|---|
| Organelle/subcellular structure | Type of cell found in | Function |
| Mitochondrion - powerhouse of the cell' | Animal and plant cells | Aerobic respiration. Generating ATP from oxidation of sugars and fatty acids. |
| Chloroplast | Plant cell | Photosynthesis |
| Rough endoplasmic reticulum | Animal and plant cells | Protein synthesis for secretion, glycosylation, and assisting in protein folding (contain ribosomes anchored on the outer surface, hence called rough) |
| Smooth endoplasmic reticulum endoplasmic | Animal and plant cells | Lipid and steroid synthesis |
| Golgi apparatus | Animal and plant cells | Concentrating and packaging proteins, as well as modifying glycoprotein. |
| Lysosome | Animal and plant cells | Digestion and degradation of cellular waste products and damaged organelles. |
| Centriole | Animal and plant cells | Paired barrel-shaped organelles that organise microtubules and the cytoskeleton. |
| Vacuoles | Animal and plant cells | In animal cells, vacuoles help sequester waste products. In plant cells, vacuoles help maintain water balance. |
All of these organelles can be stained using one or more of the methods described above, or another staining method not included in this article, like Gram-staining or DAPI staining.
Cell labelling diagram
Below are some examples of what cells labelled with fluorescent dyes might look like.
Hopefully, you now have more understanding of cell labelling and how useful it is!
Cell Labeling - Key takeaways
- Cell labelling refers to the visualisation of cells and the identification of cellular structures such as organelles.
- Cells can be labelled in multiple ways. These include using fluorescent dyes, immunolabeling and using fluorescent fusion proteins.
- Fluorescence microscopy, Flow cytometry, Fluorescence in situ hybridization (FISH), Fluorescence correlation spectroscopy (FCS) and Microarrays are all different types of cell labelling.
- Fluorescent labels have the following advantages:
- They are very sensitive even at low concentrations
- They don't interfere with target molecules
- Over long periods of time, they are highly sensitive
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