Fluorescence Microscopy
Making the specimen the light source.
Every mode covered so far shines light through the specimen. Fluorescence inverts that: the specimen is made to emit its own light, and the illuminating light is filtered out entirely. The result is a bright signal on a black background, and contrast far beyond anything transmitted light achieves.
Excitation and emission
A fluorophore absorbs a photon and is raised to an excited state. Some energy is lost as heat, then a photon of lower energy and therefore longer wavelength is emitted. That gap between absorbed and emitted wavelength is the Stokes shift, and it is what makes the technique possible: because the two wavelengths differ, a filter can block the excitation light while passing the emission.
The filter cube
Three components, usually in one removable block:
- Excitation filter — passes only the wavelengths that excite the fluorophore.
- Dichroic mirror — reflects excitation light down to the specimen but transmits the longer emitted wavelengths upward.
- Emission filter — blocks stray excitation light and passes the emission.
Most fluorescence microscopes are epifluorescent: excitation light arrives through the objective, which then also collects the emission. One lens does both jobs.
Common fluorophores
| Fluorophore | Excitation | Emission | Labels |
|---|---|---|---|
| DAPI | ~358 nm | ~461 nm blue | DNA, nuclei |
| FITC | ~495 nm | ~519 nm green | Antibody conjugates |
| GFP | ~488 nm | ~509 nm green | Genetically expressed |
| Texas Red | ~596 nm | ~615 nm red | Counterstain |
Photobleaching
Fluorophores degrade under illumination and the signal fades irreversibly. Minimise exposure, reduce excitation intensity, and use antifade mounting media. Plan the experiment so the important image is captured first — you do not get a second chance at the same field.