Physical Properties that Define Fluorescence

The visible spectrum of light

When we talk about light in microscopy, it is usually noted as wavelength, even though photons (the packets of energy that make light) can act as both particles and waves. Visible light, or light that we can see with our eyes, is usually in the range of 400–700 nm and encompasses all colors in the rainbow, with blues starting at around 400 nm and reds finishing at around 700 nm.

Figure 1. The electromagnetic spectrum, with visible wavelengths and their corresponding colors highlighted.

Range of detection in fluorescence microscopy

The range for fluorescence imaging extends a bit beyond where our eyes can see. Using this extended range is no problem because the CCD cameras that collect light emitted from our sample in a typical fluorescence microscopy setup have a wider range than our eyes do. In practical terms, imaging wavelengths for cell biology are usually in the range of 300–800 nm. Figure 2. The range and efficiency of light detection for a CCD camera compared to that of the human eye.

What is fluorescence?

But what is special about the term fluorescence? Fluorescence refers to the physical property of an object absorbing light at one wavelength and then reemitting it at another wavelength. If a molecule absorbs the light of one wavelength and emits it in another (i.e., fluoresces), we call that molecule a fluorophore. Usually the wavelength the molecule emits will be lower energy than what it absorbed, so in simpler terms we could say that something absorbs blue light and emits green, or absorbs green light and emits red. Further study:Watch "Introduction to Fluorescence" Figure 3. The inverse relationship between energy and wavelength in the visible spectrum.

Excitation and emission spectra

Most fluorophores don’t just absorb light at one discrete wavelength and emit light at another discrete wavelength: they usually absorb and emit a range of wavelengths. So when we think about using fluorophores in imaging, it’s useful to also think about the entire spectrum of their absorption and emission, while at the same time keeping in mind the maximum excitation and emission wavelengths. The maximum values are the peaks of the excitation and emission spectra.

Further study: Watch "Anatomy of Fluorescence Spectra"

Figure 5. Excitation and emission spectra of a nuclear dye (DAPI). Shows both the fraction of light absorbed by the dye over a range of wavelengths (excitation, shown in purple) as well as the light emitted from the dye over a range of wavelengths (emission, shown in blue).

The all-important Stokes shift

The difference between the excitation and emission maxima for a given fluorophore is called the Stokes shift. A fluorophore with a large Stokes shift will be much easier to use in your imaging then a fluorophore with a small Stokes shift. When there is only a small difference in wavelength between excitation and emission, it will be very difficult for you to see the emitted light from your labeled object as separate from the light used for excitation, and there will be more problems with background fluorescence.

Figure 6. A fluorophore with good separation between the excitation and emission maxima typically results in more reliable detection than a fluorophore with little separation. Compare the fluorophore with a large Stokes shift (purple and blue maximum peaks) to that of a fluorophore with a small Stokes shift (orange and red peaks).