by Bjoern Ch. Ludwar January, 25th 2006

Alexa-Dyes are a family of fluorescent dyes from Molecular Probes. Most of them can be electrophoretically injected into cells, that is, being electrically driven out of an electrode and into a cell. This tutorial takes you from the dye to taking a photo of a filled cell to document its morphology. While it uses the nervous system of Aplysia californica, but the basic procedure should work with most other animal systems.

Background

As your physics teacher tried to explain, light of different color differs in its wavelength. Light of short wavelength (blue) has much energy, light of long wavelength (red) little energy (Fig. 1).

Figure 1: The electromagnetic spectrum. ©Abrisa Glass

What's special about a fluorescent dyes is that they absorb light (energy) of a specific wavelength and in return emit light of a different, longer wavelength (Fig. 2). The first wavelength is called the excitation wavelength, the second is the emission wavelength (the difference is the Stokes-shift).

Figure 2: A fluorescent dye is excited by light of short wavelength and emits light of longer wavelength.

After filling a cell look at it with a fluorescence microscope. A fluorescence microscope typically has a broad spectrum light source like a mercury arc lamp that is filtered through an excitation filter (only short wavelength passes). The dye filled cells absorb this light and emitt light of longer wavelength. The emitted light passes through an emission filter (only long wavelength passes) to the eye-piece. Light that is not emitted, but reflected (and thereby has a short wavelength) is blocked by the emission filter. The result is a dark picture with the filled cells lighting up brightly (Fig. 3).

Figure 3: Basic principle of a fluorescence microscope.

Well, that's the principle. In reality the microscope looks a bit different a) because of mechanical constraints and b) because the Stokes-shift is never long enough and filters are never sharp enough. Light of all wavelengths is filtered through the excitation filter and falls on a dichromatic mirror. A dichromatic mirror reflects light of one wavelength (short), while transmitting that of a different wavelength (long) and thereby acts as a second filter. The mirror reflects the exciting light on the object. The filled cells emit light of longer wavelength that passes through the mirror and is filtered by the emission filter before it reaches the eyepiece (Fig. 4). The excitation filter, Dichromatic Mirror, and Emission filter are normally combined in a filter block (Fig. 5). A fluorescence microscope typically has several of these blocks and you need to select the one appropriate for the dye used.

Figure 4: More realistic fluorescence microscope