Fluorescence is a natural phenomenon where certain substances absorb light at one wavelength and emit light at a longer wavelength. In the context of microbial fermentation, fluorescence monitoring serves as means to monitor and study various cellular processes.
Fluorophores, or fluorochromes, are photoreactive chemicals. Under normal conditions, fluorophores exist in a low-energy, stable configuration known as the ground state.
When the fluorophore is exposed to light from an external source, it absorbs photons.
1. This absorption excites the fluorophore from its ground state to a high-energy, unstable state known as an excited state.
2. The fluorophore undergoes an internal conversion, and the electrons fall to a lower, more stable energy level.
3. As the fluorophore returns to the ground state, it emits photons. The emitted light has a longer wavelength (lower energy) than the absorbed light, resulting in a shift towards the red end of the electromagnetic spectrum. This shift is known as the Stokes shift. This emitted light is what we perceive as fluorescence.
Fluorophores absorb and emit light best at distinct, reported wavelength peaks.
Fluorescence is an almost instantaneous process, occurring within a very short time frame after the absorption of photons. The time scale of fluorescence is typically on the order of nanoseconds to microseconds, making it a rapid and dynamic phenomenon.
Fluorescence can be highly specific, with certain fluorophores exhibiting selectivity towards molecules or environments. This specificity enables fluorescence-based techniques to target and visualize specific cellular components or processes within complex biological systems.
Fluorescence is a highly sensitive technique, capable of detecting low concentrations of fluorophores and subtle changes in fluorescence intensity. This sensitivity allows for the detection and quantification of fluorescent signals even at trace levels, making fluorescence a valuable tool in analytical and diagnostic applications.
Fluorophores can be used to tag specific proteins or nutrients, allowing for real-time monitoring of nutrient availability and uptake kinetics.
With offline sampling and by employing fluorescence imaging (microscopy) techniques in which cellular proteins are tagged with a fluorophore, protein movements, dynamics, and subcellular localizations can be visualized.
Fluorescent proteins and dyes can be used to tag specific genes or gene products in microbial cells, enabling researchers to study the expression patterns and dynamics of genes involved in fermentation processes.
Fluorescence can be used to analyze and characterize microbial populations in fermentation by labeling specific microbial strains or species with fluorescent probes. This enables researchers to track the abundance and dynamics of different microbial populations during fermentation.
For other measurements of fluorescence, additional steps such as the application of fluorescent assays mean that sampling must be performed outside of the bioprocess. In enzyme assays, components are added that react with the metabolite, which in turn then emits fluorescence. This fluorescence is measured outside of the fermentation culture to measure how much product was produced. Such additional applications can include:
Fluorescence can be used to assess the viability and growth of microbial cells in fermentation by utilizing fluorescent dyes that specifically bind to certain cellular components, such as DNA or proteins.
Proteins engineered to function as pH sensors exhibit changes in fluorescence intensity in response to alterations in the surrounding pH, enabling researchers to track pH fluctuations within specific cell compartments over time. This approach has been particularly useful for studying pH regulation in organelles such as cellular vesicles.
Fluorescence can be used to detect and quantify the production of specific metabolites in microbial fermentation. Biosensors utilize fluorescent proteins or dyes coupled with recognition elements to produce a fluorescent signal in response to the presence or concentration of target molecules, such as ions, metabolites, or signaling molecules.
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Fluorescence is measured using a fluorometer, which is a type of spectrometer that measures the various parameters of fluorescence, including its intensity and wavelength distribution of the emission after excitation. Fluorescence measurements require an excitation light source, an emission monochromator, and a light detector.
The light source irradiates the sample. Depending on the instrumentation, illumination can be achieved via a small band light source (i.e. laser), broad band light source (i.e. Xenon (Xe) lamp), or with an intermediate peak width light source (i.e. LED).
A monochromator separates light into individual wavelengths, allowing a narrow band of those wavelengths to be selected. A monochromator may be composed of filters (high pass, band pass) or grating and additional optics.
Emitted light is received by a detector and used to calculate the florescence intensity. The detector may be made up of a photomultiplier, photodiode, or other detector array.
Multiparameter Sensor (MPS)
The MPS is an optical sensor technology for the monitoring of multiple parameters in shake flasks. Simply place the sensor beneath your shake flask and start measuring.
