Fluorometry

Let’s talk about the blacklight poster of the laboratory world: fluorometry. We’ve seen that spectrophotometry is about measuring the light that gets blocked by a solution. Fluorometry is something else entirely — it’s about measuring the light that is given off by a solution after you’ve zapped it with energy. It’s the science of making things glow and then measuring how brightly they shine. This ability to measure a signal against a dark background makes fluorometry one of the most exquisitely sensitive techniques we have, allowing us to detect incredibly small amounts of an analyte

Think of it this way: Spectrophotometry is like trying to tell how cloudy it is outside by measuring the difference in brightness between a cloudy day and a sunny day. It works, but it’s a measurement of a small change in a very large signal. Fluorometry is like going into a pitch-black room with a single glow-in-the-dark star. Measuring the light from that star against a totally black background is much easier and far more sensitive

Core Principle: Excitation and Emission

The entire phenomenon of fluorescence relies on the behavior of electrons within certain special molecules called fluorophores. You can think of it as a simple, two-step dance:

1. Excitation (The Jump Up) First, we have to give the molecule a jolt of energy. We do this by shining a beam of light of a very specific wavelength (the excitation wavelength) at the sample. An electron in the fluorophore absorbs the energy from this light and jumps from its stable “ground state” up to a higher, unstable “excited state.” It’s like a kid on a trampoline hitting the mat and flying up into the air
2. Emission (The Fall Down) The molecule can’t stay in this high-energy state for long. Almost immediately, the electron loses a little bit of its energy as heat and then falls back down to its comfortable ground state. As it falls, it releases the remaining energy as a new photon of light. This new light that is given off is the fluorescence, and we measure its intensity. To continue the analogy, the kid falls back down to the trampoline, making a “thump” sound (the emitted light)

Stokes Shift: The Key to Sensitivity

This is the single most important concept in making fluorometry practical. Because the excited electron loses a little bit of energy as heat before it emits light, the emitted light is always of a lower energy—and therefore a longer wavelength—than the excitation light that started the process. This difference between the peak excitation wavelength and the peak emission wavelength is called the Stokes Shift

Why is this so critical? It means the light we are putting in has a different color than the light we are trying to measure out. This allows us to build an instrument that can easily separate the two, which is the secret to its incredible sensitivity

Anatomy of a Fluorometer: A Right-Angle Design

A fluorometer has some similar parts to a spectrophotometer, but with two key differences and one crucial geometric twist

1. Light Source Because we need to deliver a lot of energy to get a good signal, fluorometers use very bright lamps, like a xenon arc lamp
2. First Monochromator (Excitation) Just like in a spec, this device (a prism or grating) selects the single, precise wavelength of light needed to excite our specific fluorophore
3. The Sample Cuvette Holds our sample containing the fluorophore
4. The CRITICAL 90-Degree Angle This is the big structural difference. In a spectrophotometer, the detector is in a straight line with the light source. In a fluorometer, the detector is placed at a 90-degree angle to the path of the excitation light. Why? The excitation light beam is incredibly bright. If we put the detector in its path, it would be completely overwhelmed. But fluorescence is emitted in all directions. By moving our detector to the side, we can avoid the “noise” of the primary light beam and measure only the “glow” of the fluorescence against a dark background
5. Second Monochromator (Emission) Before the emitted light reaches the detector, it passes through a second monochromator. This one is set to select only the specific, longer wavelength of the emitted light, providing another layer of specificity and filtering out any scattered excitation light
6. Detector Because the fluorescent signal is relatively weak, we need a highly sensitive detector, like a photomultiplier tube (PMT), to capture and amplify the signal

Enemy of Fluorescence: Quenching

Fluorometry’s great strength—its sensitivity—is also linked to its biggest weakness. The fluorescence process is delicate. Anything in the sample that interferes with it and causes the emitted signal to be weaker than expected is called quenching. This is like molecular stage fright; it stops the fluorophore from performing correctly. Quenching can be caused by other compounds in the sample absorbing the energy, or by changes in temperature or pH that alter the structure of the fluorophore. Controlling for quenching is a major part of quality control in fluorescent assays

Applications

While not as common for routine chemistry analytes, fluorometry is a superstar in other areas:

• Immunoassays: This is its bread and butter. Antibodies are often tagged with a fluorescent dye. When the antibody binds to its target antigen, we can use a fluorometer to detect its presence. This is the basis for many automated immunoassay platforms
• Molecular Diagnostics: Fluorescent dyes are used to label DNA probes or to detect the amplification of DNA in real-time PCR (qPCR)
• Therapeutic Drug Monitoring (TDM): Some specialized assays for certain drugs use fluorescence

Key Terms

• Fluorometry: The measurement of the intensity of light emitted from a substance (a fluorophore) after it has absorbed light of a shorter wavelength
• Fluorescence: The phenomenon in which a molecule absorbs light at one wavelength and then rapidly emits light at a longer, lower-energy wavelength
• Fluorophore: A molecule or part of a molecule that is capable of fluorescence. It is the substance we are actually measuring
• Excitation Wavelength: The specific wavelength of light that is absorbed by a fluorophore to raise its electrons to a higher energy state
• Emission Wavelength: The specific, longer wavelength of light that is emitted by a fluorophore as its electrons return to the ground state
• Stokes Shift: The difference in wavelength (or energy) between the position of the excitation peak and the emission peak for a given fluorophore
• Quenching: Any process that decreases the intensity of a fluorescent signal. It is a major source of interference in fluorometry