What is the Inner Filter Effect and How Does it Impact Fluorescence Measurements?

The origins of the inner filter effect

The inner filter effect is a loss of observed fluorescence intensity caused by absorption of light by the sample. The effect is closely linked to the path that the light takes through the sample, before it reaches the detector, and for this reason we need to talk a bit about the geometry of the measurement setup.

When we shine light on the sample to excite the fluorescent molecules, the light beam will typically not cover the entire sample. The part of the sample that is exposed to the excitation light is called the excitation volume.

The emitted light is sent in all directions, and only a fraction of it is collected by the detector. The subset of the excitation volume that is observable by the detector is called the detection volume.

Primary inner filter effect vs Secondary inner filter effect

As the excitation light travels through the sample, some of it is absorbed, decreasing the intensity the further into the sample it gets. This means that with more absorbance, the detection volume is exposed to less light, leading to a lower degree of excitation and thus less fluorescence. Loss of intensity due to absorption of the excitation light is called the primary inner filter effect. The primary inner filter effect is unavoidable, as a certain amount of light needs to be absorbed for fluorescence to occur. The presence of additional chromophores in the sample will add to this effect.

The emitted light can also get absorbed on its way to the detector, resulting in the secondary inner filter effect. The presence of a secondary inner filter effect will vary greatly depending on the sample composition.

When is the inner filter effect a problem?

The inner filter effect is always present to some extent, since the absorbance of the sample is never zero. The question is, will it be a problem for your measurements?

A simple, static loss of intensity will not distort the results in most applications. However, problems can arise when the loss of intensity due to the inner filter effect varies between measurements. This can happen if the absorbance is affected by ongoing reactions, or if new chromophores are added to the sample. To illustrate this, let us follow the fluorescence intensity as we increase the concentration of a fluorescent molecule. In the beginning, we see a linear increase of fluorescence, as might be expected. However, as the concentration increases, so does the absorbance, leading to a less than linear increase in fluorescence intensity.

The inner filter effect is distinct from quenching

The inner filter effect is sometimes confused with quenching. It is not surprising, as both phenomena result in a decrease in fluorescence intensity, and both become more prominent at high concentrations. However, quenching refers to non-radiative effects caused by thermal motion, molecular collisions, and chemical modifications. Quenching effects are consistent throughout the sample, while the inner filter effect is determined by the geometry of the light paths. Conversely, the inner filter effect is not affected by temperature, while quenching is. Understanding wether you are dealing with loss of intensity from quenching or from the inner filter effect is important if you want to mitigate it effectively.

How can you handle the inner filter effect?

The most common approach to avoid the inner filter effect is to work with concentrations low enough for the effect to be negligible. What this means exactly will vary, depending on the measurement setup, but working in an absorbance of 0.1 or less will keep intensity losses to a few percent. The best practice is to take full spectrum absorbance measurements of any samples that will be used for fluorescence, to make sure that you are within an acceptable range.

There are, of course, many applications where a significant inner filter effect is unavoidable. In these cases, one can compensate for the effect using numerical methods. The perhaps most straightforward version involves calculating the loss of intensity using Beer-Lamberts law and correcting the signal. This requires continuously measuring the absorbance of the sample, both at the wavelength of excitation and emission, as well as a thorough understanding of the geometry of the instrument light path.

With this in mind, stay tuned for our upcoming application note on how to deal with the inner filter effect using Labbot.

Summary

• The inner filter effect is a loss of observed fluorescence intensity caused by absorption of light by the sample.
• It is important to have a full understanding of the inner filter effect to accurately interpret fluorescence experiment results.
• The effect is influenced by the geometry of the measurement setup, including the excitation volume and detection volume.
• There are two types of inner filter effects: primary (due to absorption of excitation light) and secondary (due to absorption of emitted light).
• The inner filter effect can be a problem when the loss of intensity varies between measurements, especially if the absorbance changes or new chromophores are added to the sample.
• The inner filter effect should not be confused with quenching, which refers to non-radiative effects caused by molecular collisions and chemical modifications.
• To mitigate the inner filter effect, it is best to work with low concentrations where the effect is negligible and measure the absorbance of samples.
• In cases where the inner filter effect is significant, compensation can be done using numerical methods, such as using Beer-Lambert's law and understanding the instrument's geometry.

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