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Fluorescence basics: how to choose the right fluorophore and filter set

Maria Milla

We have joined forces with Harvard's Wyss Institute.

Collaboration to improve CRISPR-based multiplexed gene editing.

Most of the applications of the FluidFM systems can be easily followed through fluorescence microscopy: from spotting a fluorescent solution in a glass surface to the injection of specific complexes into single adherent cells. 

Fluorescence and fluorophores

Fluorescence refers to the physical property of an object absorbing light at one specific wavelength and then reemitting it at another one. Then, when a molecule absorbs light (or it is excited) of one wavelength and emits in another one, that molecule is called fluorophore. 

Each fluorophore has a characteristic excitation and emission spectra. Maximum values are the peaks of excitation and emission, respectively. However, most fluorophores absorb and emit in a range of wavelengths; for imaging, it is useful to think not just about their peaks, but also about the full spectrum of absorption (or excitation) and emission:

Figure 1. Excitation (blue) and emission (orange) spectra for Lucifer Yellow; peaks for excitaion and emission are 428 and 544 nm, respectively. 

The properties in the excitation and emission spectra can also determine the visualization under the fluorescence microscopy: the closer the spectra are, the more difficult it will be to see the emitted light from the labeled object as a separate from the one used for excitation. 

Fluorescence filters

In order to collect the light form the specific fluorophore, a set of filters is introduced in the microscope: exciter filter, dichroic mirror and emission filter (or barrier filter). In our system, the three filters are located into a cube (figure 2). 

Figure 2. (a) Scheme of a fluorescence filter set (cube). (b) Available filters in the FluidFM BIO Series. Images adapted from Olympus website

The exciter filter has generally a defined band of wavelengths that it allows through. After going through this filter, it reflects onto the sample. Fluorophores in the sample will then become excited and, thus, emit light at a different wavelength. The dichroic mirror and, then, the emission filter will filter this emitted light. 

In the FluidFM BIO Series, the filter sets available are the following ones:

FilterExcitation FilterEmission FilterCorresponding color in the visible spectrum
U-FUNA360-370 nm420-460 nmBlue
U-F39002480 nm535 nmGreen
U-F39004540 nm605 nmRed

What should be considered when selecting a fluorophore and a filter set?

In order to obtain a better resolution of our samples and adapt the fluorescence intensity to our needs, it is important to take into account the following concepts when selecting the fluorescence presets and fluorophores for the experiment:

1. Selectivity of the fluorescent label. The fluorophore must be specific for a target: a molecule, a biological activity or a cellular location, without nonspecific background signal. 

2. Photostability. The fluorophore must be able to maintain its properties after repeated exposures to illumination light. 

3. Excitation and emission properties of the fluorophore. The filter set should be compatible with the range of wavelengths of the fluorophore (and vice versa). 

4. Environmental stability. Some fluorophores can be sensitive to air, light or temperature. 

Here, there is a list of the most common fluorophores used in FluidFM experiments, together with the filter sets used for its observation:

FluorophoreExcitation wavelength Emission wavelengthFluorescence filter
Lucifer Yellow428 nm544 nmU-F39002 (green)
DAPI359 nm461 nmU-FUNA (blue)
Hoechst352 nm455 nmU-FUNA (blue)
Propidium Iodide300 nm610 nmU-F39004 (red)
Alexa Fluor 555 dextran555 nm585 nmU-F39004 (red)
Alexa Fluor 568580 nm600 nmU-F39004 (red)
mCherry587 nm610 nmU-F39004 (red)
GFP480 nm500 nm

U-F39002 (green)