Fluorescence is a phenomenon where certain molecules emit light after being excited by electromagnetic radiation. Fluorescein isothiocyanate, also known as FITC, is a popular fluorescent marker used in biological and biomedical research.
Overview of Fluorescence
Fluorescence occurs when a molecule absorbs light at a specific wavelength and then emits light at a longer wavelength. The absorbed and emitted wavelengths are known as the excitation and emission wavelengths, respectively.
The process occurs in three steps:
- Excitation – The molecule absorbs a photon and an electron is promoted to an excited state
- Intersystem crossing – The excited electron transitions to a lower energy state
- Emission – The electron returns to the ground state, emitting a photon in the process
The difference between the absorbed and emitted wavelengths is known as the Stokes shift. This shift allows the emitted light to be detected separately from the excitation light.
Properties of FITC
FITC (C21H11NO5S) is a derivative of fluorescein, a naturally occurring fluorescent molecule found in many plants, animals, and microorganisms. FITC has the following key properties:
- Excitation maximum = 495 nm
- Emission maximum = 519 nm
- High molar extinction coefficient, enabling bright fluorescence
- Good quantum yield of 0.5-0.9
- pH insensitive over physiological pH range
The isothiocyanate (-N=C=S) reactive group allows FITC to be coupled to proteins and other biomolecules. This makes it useful for fluorescent labeling and tracking in biological systems.
Applications of FITC
Some major applications of FITC include:
Immunofluorescence Microscopy
FITC is commonly conjugated to antibodies to visualize their binding to specific target antigens by fluorescence microscopy. This technique is known as immunofluorescence. It allows researchers to determine the expression and localization of proteins within fixed cells and tissues.
Flow Cytometry
In flow cytometry, cells in suspension are passed through a laser beam. FITC-labeled antibodies bound to cell surface antigens fluoresce as they pass through the beam, providing quantitative data about antigen expression. This is useful for phenotyping cell populations.
Protein Labeling
FITC enables fluorescent detection and tracking of proteins during biochemical assays and analysis. It can also be used to label intracellular proteins in live cells through microinjection or electroporation.
Nucleic Acid Labeling
FITC intercalates between DNA base pairs and binds to RNA. It is used to label and visualize nucleic acids in applications like fluorescence in situ hybridization (FISH).
Cell Proliferation Assays
FITC is used to monitor cell proliferation by covalently coupling it to components of the cell membrane. As cells divide, the FITC is partitioned between daughter cells and can be quantified by flow cytometry.
Chemistry of FITC Conjugation
The isothiocyanate (-N=C=S) group of FITC readily reacts with primary amines (-NH2) on proteins and other biomolecules to form stable thiourea bonds. This provides a targeted way to fluorescently label specific molecules. The reaction occurs optimally at pH 9-10.
For example, FITC is commonly conjugated to antibodies by reacting with lysine residues or antibody fragments like F(ab’)2:
Each FITC molecule contains a single isothiocyanate group, limiting substitution to ~1-2 FITC molecules per antibody. Higher levels of labeling can decrease solubility and specificity.
Comparison to Other Fluorophores
FITC is one of many fluorophores used in biological research and biotechnology. Some commonly used alternatives include:
| Fluorophore | Excitation Max (nm) | Emission Max (nm) | Key Features |
|---|---|---|---|
| FITC | 495 | 519 | – High absorbance – Good quantum yield – Versatile labeling |
| TRITC | 548 | 572 | – Red-shifted – Lower background |
| Alexa Fluor 488 | 495 | 519 | – Photostable – High quantum yield |
| GFP | 395 | 509 | – Genetically encodable – Live cell imaging |
While other fluorophores may have advantages, FITC remains widely used due to its versatility, affordability, and long history in biological research.
Working with FITC – conjugate and labeling considerations
When working with FITC for fluorescent labeling experiments, there are some important considerations:
Conjugate Quality
- Use high quality FITC and reactive molecules to produce stable, brightly fluorescent conjugates
- Remove unreacted or hydrolyzed FITC to minimize background fluorescence
- Characterize degree of labeling by spectrophotometry or other assays
Labeling Conditions
- Use proper buffers, pH, temperature, and incubation times for efficient labeling reactions
- Avoid excess exposure to light during labeling to minimize FITC degradation
- Remove unbound conjugate by gel filtration or dialysis
Storage
- Store FITC conjugates away from light at 4°C or -20°C
- Avoid repeat freeze-thaw cycles
- Add stabilizing agents like BSA or serum proteins as needed
Controls
- Include positive and negative labeling controls
- Use unlabeled samples to control for autofluorescence
- Optimize labeling conditions for each target molecule
Challenges of working with FITC
Some challenges associated with using FITC include:
- Photobleaching – FITC is susceptible to photobleaching during extended illumination
- Environmental sensitivity – Fluorescence is impacted by pH, polarity, and quenchers
- Nonspecific binding – FITC can interact nonspecifically with components in complex samples
- Self-quenching – FITC fluorescence decreases at high degree of labeling
- Thiol reactivity – Thiols can react with FITC conjugates, releasing free FITC
These potential drawbacks must be controlled for in carefully designed experiments. Newer alternative dyes like Alexa Fluor overcome some of the limitations of FITC.
Conclusion
In summary, FITC is a versatile and commonly used fluorescent label in biological research and testing. Its reactive isothiocyanate group provides an easy way to fluorescently tag proteins, antibodies, nucleic acids, and other biomolecules. When conjugated and used properly, it enables sensitive fluorescence-based detection and imaging of biological samples through microscopy, flow cytometry, and other modalities. Care must be taken to optimize labeling conditions and account for the environmental sensitivity of FITC fluorescence. Overall, FITC remains a useful tool for fluorescence applications despite some limitations.