Fluorometry is the technique of measuring any parameter using fluorescent emission. In the context of modern bioscience, fluorometry is used to detect biological macromolecules such as proteins and measure their concentration in the experimental sample.

Fluoro – related to fluorescence
Metry – related to measurement

In modern bioscience, multiple experimental methods use fluorescence to qualitatively and quantitatively detect macromolecules such as nucleic acids and proteins, and cell organelles such as nuclei and mitochondria.

Such experiments are commonly classified as fluorescence detection assays.

In this article we will first look at the phenomenon of fluorescence and how it is used in biochemical detection. Next, we will discuss some common chemicals used in fluorometric detection assays, their working principle and the type of experiments they are used.

In this article:

What is fluorescence?

How is fluorescence exploited in biological experiments?

Fluorescence-based bioassays analogy

Methodology for fluorescence-based bioassays

Option 2: Probe with fluorescent tag

Option 3: Probe with enzyme tag

Biochemicals and reagents in fluorometric detection assays

Fluorescent proteins

Fluorophores and fluorescent stains

Antibodies for immunofluorescence assays

Fluorogenic substrates

Find Fluorogenic Substrates

What is fluorescence?

When a substance absorbs a light wave of a certain wavelength and then emits another light wave of a different and higher wavelength, this phenomenon is called fluorescence.

Consider a fluorescent item. It absorbs light in the UV range and emits light in the visible range. So, you can’t visually see the light that is triggering the fluorescence because humans cannot visually detect UV waves.

But we can see the material glow because the emission is in the visible spectrum. Fascinating, isn’t it?

This is shown in Figure 1.

Fluorescence mechanism. Excitation occurs within the UV range and light is emitted at a lower excitation range

Figure 1. Representation of how fluorescence works.

Fluorescence is commonly seen in nature. Monarch caterpillars are a good example of this. They fluoresce under ultraviolet light.

Caterpillar under ultraviolet light displays fluorescence

Figure 2. Monarch caterpillar under UV light shows incredible fluorescence and contrast. Image via Chris Clemens, Nature’s Rainbows.

Other examples of naturally fluorescent organisms include jellyfish, a range of other marine fish including some sharks, corals, some amphibians, scorpions etc.

How is fluorescence used in biological experiments?

Fluorescence is often utilized in the qualitative and quantitative detection of a single type of biological macromolecule. These include DNA or a specific protein, some cell organelles such as lysosomes, and interaction between two macromolecules – DNA-protein or protein-protein interactions.

Specific experimental techniques include fluorescence microscopy and Western blot. Nowadays fluorescence-based ELISA (F-ELISA) is also being reported in recent literature.

Fluorescence-based bioassays analogy

Consider a dark room where you need to detect a dark object. The challenge is that you cannot switch the lights on. However, the good thing is that the target object fluoresces red when you turn on the UV lamp – and the room indeed has a UV lamp that you may turn on. Of course, you have put on gear for UV protection.

So, you turn on the UV. You cannot see the UV wave but now the target glows red. And you seamlessly detect it.

This is illustrated in figure 3.

Fluorescent detection in a dark room - fluorescent object can be detected when ultraviolet light shines on it.

Figure 3. Basic example of fluorescent detection. The same concept can be applied to fluorescent assays.

When it comes to this example, consider the dark object as a cellular structure or component which you can visualize using fluorescence.

Likewise, this dark room is your sample – say a single cell or tissue section on a microscope slide.

The target object is a cellular structure that can fluoresce. So, you put the experimental sample on a microscope slide under a microscope that hits it with UV radiation and you see your target organelle glowing red under the microscope.

Methodology for fluorescence-based bioassays

In experiments where a biological structure such as a cell organelle, or a macromolecule such as DNA, or a specific protein is the target for detection, the experimental surface that has the sample (microscope slide/ Western blot membrane etc.) is first treated with a chemical that specifically detects and binds to that target.

As shown in the figures below, this chemical probe can be either a fluorescent probe, a probe with a fluorescent tag, or a probe with an enzyme tag.

We’ll look at each of these options in more detail.

Option 1: Fluorescent probe

The first option is where the probe itself is fluorescent – typically, a fluorescent dye or stain (figure 4-1).

The probe binds directly to the target, but still does not fluoresce yet because it is not excited with an incident light of appropriate wavelength.

This is depicted schematically in panel A (left) in figure 4-1.

Now when the target-bound fluorescent probe is excited with the incident light, there is a fluorescence that is detected and quantified. This is shown in panel B (right) of figure 4-1.

Fluorescent Dye as the probe

Figure 4-1. Fluorescent Dye as the probe that detects the target protein (as specifically shown here) or cellular target. A) probe binds to target but no visual signaling yet. B) probe fluoresces when illuminated by incident light.

Option 2: Probe with fluorescent tag

Figure 4-2 shows an example of a probe that is tagged with a chemical that exhibits fluorescence.

There may be two experimental approaches for this.

probe agged with fluorescent chemical

Figure 4-2. A) Probe is tagged with another chemical that exhibits fluorescence. B) Probe is detected with a second chemical probe which is tagged with a fluorescent chemical.

In this case of what is shown in figure 4-2 panel A (left), the probe is an antibody that detects the actual target molecule in the sample. This type of antibody that directly detects the actual or primary target in the sample is called a primary antibody. In this case, the primary antibody is tagged with a chemical that can fluoresce when excited appropriately.

For the case shown in panel B of figure 4-2, the primary antibody detects the target in the sample, but does not have a fluorescent tag. Instead, another antibody serves as a second probe that detects and binds to this primary antibody probe. This antibody is a secondary antibody.

The secondary antibody probe has a fluorescent tag and exhibits fluorescence when excited with a light wave of appropriate wavelength.

For a detailed understanding of how primary and secondary antibodies work and are used in experiments, please refer to this article.

Part A of Figure 4-2 shows how the primary antibody is used to bind to the target protein, after which, fluorescence occurs.

Part B of figure 4-2 is similar but employs a secondary antibody with a fluorescent tag.

Option 3: Probe with enzyme tag

The third option is where a probe is tagged with an enzyme that can act on a substrate to cleave it to a product that exhibits fluorescence.

These types of substrates are called fluorogenic substrates.

This method involves the extra step of adding the fluorogenic substrate to the experimental surface.

Detailed look at an antibody tagged with an enzyme that cleaves a fluorogenic substrate

Figure 4-3. Detailed look at an antibody tagged with an enzyme that cleaves a fluorogenic substrate.

Figure 4-3 shows the steps involved when using an antibody tagged with an enzyme that cleaves a fluorogenic substrate.

In the first stage, the primary antibody with a conjugated enzyme is added. The antibody is specific only for the target protein, which is among many proteins fixed to a membrane.

After the antibodies are given enough time to bind to the target protein, there is a wash step where any remaining unbound antibodies are washed away (second stage).

In the third stage, fluorogenic substrates are added. At this point, these substrates have not yet been acted upon by the enzyme and therefore no fluorescence should occur.

However, once the enzyme acts on the substrate (fourth stage of figure 4-3), changes occur to the substrate. The substrate is cleaved which leads to a new, fluorescent product (fifth stage of figure 4-3).

In either case, the final step uses specialized equipment such as a fluorescence microscope for hitting the experimental surface with a short wavelength radiation. Most of the time, the short-wave radiation will be a UV wave.

All that is left is observing your target glow somewhere in the visible spectrum.

The higher the intensity of the fluorescent glow, the higher the concentration is of your target in the sample.

Intensity of fluorescence emitted from the detected target biomolecule or organelle is used as the readout for measuring its concentration in the experimental sample.

Biochemicals and reagents in fluorometric detection assays

In fluorometric assays common chemicals required are:

  • fluorescent dyes that act as the probe to detect the cellular target, or
  • primary antibodies conjugated (attached) to a fluorophore, or
  • secondary antibodies conjugated with a fluorophore. These antibodies are used to detect a non-fluorescent primary antibody that detects the actual cellular target – like a specific protein.
  • a primary antibody conjugated to an enzyme. This enzyme in turn cleaves a substrate that is added at a later step, to produce a fluorescent product that is detected.

In this section we will look at the chemicals and components that aid in fluorogenic detection.

Fluorescent proteins

Fluorescent proteins GFP, YFP, RFP and a few others, that occur naturally, are extensively used in fluorogenic assays.

This is how scientists use these proteins in research experiments. Using the advances in modern recombinant DNA technology, fluorescent proteins, such as RFP (red fluorescent protein), are cloned in the cells or tissues that are aimed to be studied. This enables visualization and analysis of these fluorescent protein-expressing cells under a microscope that can trigger fluorescence and detect fluorescent images.

This technique is called fluorescence microscopy.

Nowadays mutants of naturally occurring fluorescent proteins that are optimized for better results are more commonly used in these experiments.

Here is an example of how RFP, along with fluorescence microscopy might get very useful in an experiment.

Imagine you have a bacterial strain that causes pneumonia. You want to study how fast this pathogenic strain multiplies within human lung cells, in comparison to a benign non-pathogenic bacterium.

For this, you transform the clinical isolate with RFP and the non-pathogenic strain with GFP and infect a lung cell line culture with equal numbers of both strains.

After a few hours of incubation, you observe the lung cells under a fluorescence microscope. You can easily quantify the number of both the pathogenic as well as the non-clinical bacterium inside each lung cell because they fluoresce red and green respectively.

Fluorophores and fluorescent stains

Fluorophores, also called fluorochromes, are compounds that exhibit fluorescence. They are conjugated to the probe, for example, an antibody, that detects the actual cellular target.

A fluorescence microscope triggers and detects the fluorophore’s fluorescence. Indirectly the tagged antibody, and the bound target can in turn be detected.

Instead of a fluorophore, a fluorescent stain may also be tagged with the detection probe. In this case, the principle of detection is the same as that of a tagged fluorophore.

In some cases, the fluorescent stain may itself be the probe that detects the cellular target.

A classic example of this is DAPI (4′,6-diamidino-2-phenylindole). DAPI binds to A-T regions in DNA.

DAPI is used as a direct probe for the cell’s nucleus (DNA rich region) and can be detected using its fluorescent properties because DAPI absorbs maximally in the UV spectrum (358nm) and fluoresces in the visible blue wavelength of 461nm.

Consider DAPI as the fluorescent probe in figure 4-1. Of course, in that case, the target for detection would be nucleic acids, and not a protein.

Antibodies for immunofluorescence assays

Immunofluorescence is the process of detecting a target using both its immunogenic properties, that is, using an antibody, as well as by using fluorescence in the same experiment.

This is depicted in figure 4-2.

If the primary antibody itself is conjugated to a fluorophore and no secondary antibody is required, the assay is called primary immunofluorescence (fig 4-2A).

More commonly, after the primary antibody detects the actual target protein or cell organelle in the sample, after a wash step that eliminates unbound primary antibody, the secondary antibody is added and detects the primary antibody already bound to the target.

This secondary antibody is conjugated to a fluorophore and is detected using the latter’s fluorescence.

The secondary antibody-conjugated fluorophore aids in the visual detection of the underlying complex comprising of three components:

  • the target protein
  • the primary antibody
  • the secondary antibody.

This type of detection is known as secondary immunofluorescence.

Common techniques where immunofluorescence is useful are fluorescence microscopy, Western blots and ELISAs.

Fluorogenic substrates

Fluorogenic substrates are compounds that are not fluorescent by themselves, but when acted upon by a specific enzyme.

When acted upon by an enzyme, fluorogenic substrates are cleaved to a product compound that is fluorescent. Figure 5 is a closeup showing the relationship between enzyme and fluorogenic substrate.

how fluorescent substrates work with enzymes

Figure 5. Substrates (orange) are added and are acted upon by the enzyme (pink). Action leads to changes in the substrate. Cleavage of the substrate (by the enzyme) yields a new fluorescent product (green).

Find Fluorogenic Substrates

GoldBio has a wide variety of fluorogenic substrates for many enzyme types. You can browse the selection here.

Table 1. Classification of fluorogenic substrates based on the enzymes and the emission wavelength of the fluorescent product.

Fluorogenic Substrate


Emission wavelength of
fluorescent product

Excitation wavelength of
fluorescent product



375 nm

316 nm

4-Methylumbelliferyl beta-
D-glucuronide (MUG)


445 nm

365 nm

4-Methylumbelliferyl-α- L-iduronic
acid cyclohexylammonium salt



4-Methylumbelliferyl Palmitate

lysosomal acid lypase

449 nm

360 nm

4-Methylumbelliferyl Phosphate

Alkaline phosphatase

449 nm

360 nm

(H-Ala-AMCTFA ) L-Alanine
7-amido-4-methylcoumarin, Trifluoroacetate Salt


440 nm

365 nm

L-Glutamic Acid α-


450 nm

365 nm

L-Leucine 7-amido-
4-methylcoumarin HCl


440 nm

380 nm