Selecting filters for your application

Labbot is equipped with a wide-spectrum light source that provides high-intensity light from the UV range to the infrared. To obtain the correct excitation light for your fluorescence experiment, the light must be sent through a bandpass filter. Labbot can accommodate up to 10 filters, giving you ample opportunities to obtain the optimal filter for each experiment. But how do you determine which filter is best for each application? This guide provides the guidelines you need to find the best filter for each experiment.

Know your sample

Before selecting the best filters, you need to know which molecules you intend to study. For each molecule of interest, it is recommended to find their absorption and emission spectra to have a complete picture of how they interact with light of different wavelengths. In most cases, spectral information is available from the supplier or from the scientific literature. Alternatively, if you  have the equipment, you can measure the spectral properties of the molecules yourself.

More light in - more light out

The absorption spectrum will tell you which wavelengths of light the molecule is most likely to absorb. To get the brightest fluorescence from the molecule, you should maximize the amount of input light in this spectral region. In a filter-based setup, this also means that a broader pass filter, which will transmit a greater total photon flux. This will give a higher fluorescence intensity, provided that the additional light is of a wavelength that the molecule can absorb.

Avoid spectral overlap

There are two main reasons you may want to limit the bandwidth of the excitation light. The first is in order to prevent any scattered excitation light from overlapping with the detected fluorescence light. Should this happen, it may distort your results and saturate the detector.

The second reason arises if you have several chromophores in your sample but only want to get a signal from one of them. In that case, you need to know the absorption spectrum of each molecule and carefully select the excitation light to intersect the absorption of your molecule of interest but not that of the other molecule(s).

Wide or narrow band?

For these reasons, the spectral width of the excitation light is a tradeoff between sensitivity and specificity. In general, it is a good idea to select the maximum bandwidth allowed by your experiment in order to maximize the light that reaches your sample. For reasons of specificity, you may sometimes need to limit this.

Finally, sometimes you may be limited by which bandwidths are available for your excitation wavelength of interest.

An example from protein fluorescence

Let us examine these principles from the perspective of intrinsic protein fluorescence. In proteins, there are three main chromophores: tryptophan, tyrosine, and phenylalanine. For most investigations of protein fluorescence, excitation light of 280 nm is used. This covers the absorption peaks of both tyrosine and tryptophan, giving a maximum total fluorescence from the protein.

However, sometimes one is interested in looking exclusively at the fluorescence from tryptophan, often because this is more responsive to the local environment. This can be done, since the absorption of tyrosine approaches zero at around 290 nm, while the absorption peak of tryptophan has a tail that extends to approximately 310 nm. By using excitation light that falls within the range of 290-310 nm, one can ensure that only tryptophan is excited. Doing so comes, however, at a massive price in terms of fluorescence intensity.

Summary

• Check the absorption and emission spectra of your chromophore
• Decide which wavelength region is your output signal
• Make sure the excitation light is of a wavelength that is shorter than that of the output signal
• Make sure that the excitation light does not intersect with any chromophores in the sample that you do not want to excite
• Within these constraints, maximize the cross-section of absorption and excitation light.