Fluorescence: a little sensitivity goes a long way
The world, as we see it, is completely dependent on the way mater interacts with light. Light is the most ubiquitous form of electromagnetic radiation - it is a defining part of our existence. Despite its penetrating effects into every part of our world as we know it, it exhibits almost mysterious behavior at times. Light behaves as both a particle and a wave; the duality of light is crucial to our understanding of it as it relates to absorbance and fluorescence spectroscopy.
We perceive color as the reflected portion of the visible spectrum, and we observe this in the presence of broadband light. A solid understand of the electronic mechanism of absorbance is crucial to our understanding of the complementary phenomenon, fluorescence.
Light is a form of electromagnetic radiation; all electromagnetic radiation consists of perpendicular and in phase oscillating magnetic fields and electric fields. Electromagnetic radiation has the ability to transfer energy through matter and through a vacuum.
Let's start by considering the ground state of the matter and its relationship to what we visually observe and can quantitively measure through absorbance. In the absence of light, does a material possess color? Arguably, no. A material in the absence of light possess no color, but it does have properties on a chemical level that would produce color in the presence of light. Electronically, the fingerprint of matter determines the color perceived by the human eye.
On the atomic level, when light is absorbed, energy is transferred from photons to atomic electrons when specific conditions are met. The physical properties of matter determine the substances ability to "absorb" the energy from a particular wavelength of light; only photons carrying energy equal to the gap between energy levels will be absorbed.
Absorbance and excitation
Absorbance, electronically, is the promotion of an electron from one state, usually ground, to a higher, less stable state. The ground state of a material is the lowest possible energy configuration. When matter is in a state that is higher in energy (less stable) than the ground state, it is referred to as excited, sometimes denoted by an asterisk.
If we think about the electron's orbitals (the region of space that a particular electron has the highest probability of being found) as probable locations, we can use this region of high probability for atoms and extend that to molecules and determine the regions where electrons will reside and the energy levels associated with different configurations (linear combinations of atomic orbitals form what we know as molecular orbitals). These regions of highest probably are mathematically represented as electron orbitals, or wave functions.
Aleksander Jab?oński first illustrated the electronic transitions graphically in 1933 by plotting the relative positions of the orbital wave functions to illustrate potential pathways for excitation and emission. The Jab?oński diagram illustrates the various excited states of a molecule. It is used to visually represent the possible transitions between states after excitation by electromagnetic radiation.
The y-axis represents energy, and the horizontal lines correspond to the energetic levels of a molecule. Bold horizontal lines designate the lowest vibration level of each electronic state, and higher finer lines are the vibrational levels. Singlet states have an angular momentum of zero, denoted S, and triplet states (responsible for phosphorescence), with angular momentum of one, are T (not shown).
The energy required to promote (excite) an electron to that state is equal to the difference between the desired state and the ground state.?
?In the presence of light (hv) of a specific wavelength, electrons can be promoted from the ground state, the lowest energy configuration, or lowest occupied molecular orbital (LUMO) to higher unoccupied molecular orbitals (HOMO). The available excited states correspond to the unoccupied orbitals further from the nucleus and higher in energy on the Jab?oński diagram. The energy required to promote the electron to the excited state must equal the energy of the photons absorbed. The Planck hypothesis states that electromagnetic radiation is quantized and exists as finite packets of energy, photons, with energy equal to the product of Planck's constant, h = 6.626x10^-34 joule*sec, and the frequency, v.??When the energy absorbed is in excess of the transition between states, the electron will reside in the excited state but have excess vibrational energy; the excess energy is lost as rotational motion and heat (red arrows).
In the case of absorbance, a photon transfers energy in and the matter absorbs the energy from the photon, transmitting the remaining energy that is not absorbed through the material. Absorbance measurements are subtractive. The intensity of the incident light is measured and the intensity after passing through the material is measured. The instrumentation is measuring for small differences in very large numbers, in the case of low concentrations, the values for incident and transmitted light will be large numbers. We've discussed how the reaction time solely provides enough signal to develop, since the assay is typically intended for low concentration samples. (Spotlight on Absorbance: a closer look at the conventional detection method for BET | LinkedIn)
History and Science of Fluorescence
A solid understanding of the electronic mechanisms of absorbance sets the stage for the discoveries that occurred over a century later.
In 1852, Sir George Gabriel Stokes published the now infamous (in very small circles) "On the Refrangibility of Light" wherein he describes dispersive reflection. Stokes used a prism to refract sunlight to demonstrate a peculiar characteristic of quinine sulfate solution. When held incident to the visible part of the spectrum, the solution remains colorless, the portion of the spectrum is fully reflected back, but when the sample was in the ultraviolet region, a blue color was emitted. “ It was certainly a curious sight to see the tube instantaneously light up when plunged into the invisible rays; it was literally darkness visible.” Stokes's experiment demonstrated that the solution absorbed light but emitted a different wavelength, as the blue light coming out of "the darkness" was in the blue region, a longer wavelength than the incident ultraviolet light. Stokes coined the term fluorescence the following year, correcting dispersive reflection:
“ I confess I do not like this term. I am almost inclined to coin a word, and call the appearance fluorescence, from fluorspar, as the analogous term opalescence is derived from the name of a mineral. ”
Fluorescence, as observed by Stokes, exhibits a unique characteristic - the Stokes shift. The wavelength of light absorbed is always higher than the wavelength emitted. Mathematically, the stokes shift is the difference between the absorbance peak and the emission peak for a fluorophore. The Jab?oński diagram explains this visually; due to non-radiative transitions, the vector from the excited state to the ground state is smaller (lower energy) and the calculated wavelength of light emitted is longer (E = hc/λ).
The Stokes shift allows for the separation of the high intensity excitation signal and the much lower intensity emission signal. This principle results in enhanced sensitivity that is usually three orders of magnitude greater than absorbance measurements due solely to detection method.
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Instrumentation
A theoretical fluorometer contains
The light source is often upgraded from a halogen bulb to a xenon flash lamp. Xenon bulbs have a longer service life. These high energy sources can excite molecules that absorb from 200 to 2500 nm with a high output intensity.
The selectors for fluorometers are used to prevent unwanted signal from hitting the sample. By choosing the peak of the excitation spectrum, the instrument is able to be highly selective in measurement. Only photons at the emission wavelength, generated from the excitation wavelength, will be measured at the detector. Unlike absorbance, where stray light can hit the detector, or sample color can absorb at the same wavelength, fluorescence measurements are highly specific to only the fluorophore of interest.
The photomultiplier tube detector on a fluorometer converts the light to an electronic signal using the photo electric effect. The gain can be adjusted on fluorometers to amplify low signals, allowing for increased sensitivity on the low end of the assay. Newer instrumentation can mathematically scale the signal over a range of gain settings, effectively increasing the dynamic range of an instrument.
Advantages
Fluorescence detection is up to three orders of magnitude more sensitive, simply because the method does not require comparison to a reference beam. Measuring such low signal is not possible with a subtractive method like absorbance. With this low level of sensitivity, there is potential to reduce sample volume and assay run time, with carefully designed assay parameters and future development.
Fluorescence detection is also highly specific to the assay's fluorophore; only molecules that fluoresce with the paired excitation and emission wavelengths will be measured.
Although there are distinct advantages to fluorescence detection, it is still subject to limitations. Fluorescence measurements are affected by pH, so for assays like the bacterial endotoxin test, this limitation is minimal - pH impacts the traditional method as well.
Conventional methodology is adequate and fulfills the requirements for the BET, but the future is bright for fluorescence. The technology alone provides a platform to push the sensitivity lower, allowing for higher MVD, potentially reducing sample volume requirements, and highly specific measurement of fluorophore concentration.
With increased adoption, investigation, and exploration, the recombinant factor c method of endotoxin testing can be expanded to fully reap the benefits of the detection method.
References and continued reading
On the change of refrangibility of light; G. G. Stokes, M. A., F. R. S. Phil. Trans. R. Soc. Lond. 1852 142, 463-562, published 1 January 1852;?https://rstl.royalsocietypublishing.org/content/142/463.full.pdf+html
Business Development Manager Drug Discovery
3 年Great work Caitlin!!
Thank you for writing this up, Caitlin!
Sr. Director at Eli Lilly and Company - USP Microbiology Expert Committee
3 年Thanks for the deep dive Caitlin - education will make us all better!
Director Global Marketing, KOL & Scientific Strategy
3 年What a great read ! Very thorough and I hope there is more to come