Fluorescence Microscopy: How It Works and What It Shows

The short version

A fluorescence microscope lights the specimen with light of one color (usually ultraviolet, blue or green) and then filters out that light completely, so only the light the specimen emits in response reaches your eye or camera. That emitted light is a longer-wavelength color. Because everything that doesn’t fluoresce stays dark, the glowing structures stand out against a black background with very high contrast and specificity. You see exactly what you labeled, or what fluoresces naturally, and little else.

What fluorescence is

Certain molecules, called fluorophores or fluorochromes, can absorb a photon of light and jump to a higher-energy excited state. A moment later, typically within nanoseconds, they drop back down and release a new photon. Some energy is lost in the process, so the emitted photon has less energy and therefore a longer wavelength than the one absorbed. In practice:

• UV light in → blue light out
• Blue light in → green light out
• Green light in → red or orange light out

The gap between the absorption peak and the emission peak is called the Stokes shift. It’s what makes fluorescence microscopy possible: because the two colors differ, filters can separate them. Fluorescence stops almost immediately when the excitation light is switched off. A longer-lasting glow is called phosphorescence, which is a different process.

Inside a fluorescence microscope

Most modern fluorescence microscopes use epi-illumination: the excitation light comes down through the objective, and the emitted light travels back up through the same objective. This is the same geometry as a reflected light microscope.

The light path looks like this:

1. Light source. Traditionally a mercury or xenon arc lamp. Today it’s increasingly high-power LEDs, and lasers in confocal systems.
2. Excitation filter. Passes only the band of wavelengths that excites your fluorophore, such as blue for fluorescein or GFP.
3. Dichroic mirror (beam splitter). Set at 45°, it reflects the short-wavelength excitation light down into the objective but lets longer wavelengths pass straight through.
4. Objective and specimen. The objective focuses the excitation light onto the specimen, and the fluorophores emit light in all directions. The objective collects part of it.
5. Back through the dichroic. The longer-wavelength emitted light passes through the dichroic mirror toward the eyepieces or camera.
6. Emission (barrier) filter. Blocks any remaining excitation light and passes only the emission band.

The excitation filter, dichroic mirror and emission filter are usually mounted together in a filter cube. A microscope often has several cubes, each matched to a different fluorophore, so you can switch colors and then combine the images.

Fluorophores: where the glow comes from

• Autofluorescence. Some materials fluoresce naturally. Chlorophyll glows deep red under blue or UV excitation, which makes algae and leaf cells spectacular. Many minerals, some plant cell walls, pollen and some animal tissues also autofluoresce.
• Fluorescent dyes. Synthetic stains bind to particular structures. DAPI and Hoechst bind DNA and fluoresce blue. Acridine orange stains nucleic acids. Fluorescein and rhodamine are long-established general labels. Many modern dye families are designed for brightness and resistance to fading.
• Fluorescent proteins. Green fluorescent protein (GFP), first found in a jellyfish, and its relatives in many colors can be genetically encoded so that cells produce their own label. This lets researchers watch specific proteins in living cells.
• Immunofluorescence. Antibodies carrying a fluorescent dye bind to a specific target molecule, which is then lit up wherever it occurs in the cell or tissue.

Each fluorophore has its own excitation and emission spectrum, so the filter cube has to match it.

What fluorescence microscopy shows

Fluorescence microscopy doesn’t show the whole specimen the way bright field does. It shows where a particular molecule or structure is. That makes it suited to:

• Locating specific structures: nuclei, mitochondria, the cytoskeleton, cell membranes
• Multiple labels at once: different colors for different targets in the same cell
• Living processes: cell division, protein movement and changes in calcium or pH, using specialized indicators
• Microbiology and clinical labs: certain fluorescent stains are used, for example, to look for the bacteria that cause tuberculosis in prepared samples
• Materials and geology: fluorescent minerals, cracks penetrated by fluorescent dye, and contamination checks

Because it has so much contrast, fluorescence can reveal even sparse or tiny labeled objects. Its specificity is the main reason it has become so central in cell biology.

Limitations

• Photobleaching. Fluorophores gradually fade under continued excitation. Minimize exposure, use anti-fade mounting media for fixed slides and choose photostable dyes.
• Phototoxicity. Intense light, especially at shorter wavelengths, can damage or kill living cells.
• Out-of-focus blur. In a standard widefield fluorescence microscope, fluorescence from above and below the focal plane adds a haze, especially in thick specimens.
• Unwanted autofluorescence. Natural fluorescence that’s beautiful in a leaf can be background noise when you’re trying to see a specific label.
• Only what fluoresces. Unlabeled structures are invisible. Many researchers overlay a fluorescence image on a brightfield, phase or DIC image to show context.
• Cost and complexity. Filter cubes, suitable light sources and sensitive cameras add up, and dyes and labeling take expertise.

Widefield vs confocal

Widefield epi-fluorescence illuminates the whole field at once. It’s fast, relatively simple and the most common type, but it includes out-of-focus light.

Confocal microscopy scans a focused laser spot across the specimen and places a pinhole in front of the detector, which rejects most out-of-focus light. The result is sharp “optical sections,” which can be stacked into 3D images, at the cost of much more expensive equipment and slower imaging. Further techniques, including multiphoton, light-sheet and super-resolution methods, push depth and resolution further in research settings.

Fluorescence for amateurs

Fluorescence used to be out of reach for hobbyists, but blue high-power LEDs and inexpensive filters have changed that. A common amateur approach:

• Excite with blue light, using a blue LED with a blue excitation filter, either epi-style through the objective or from below at an angle.
• View through a yellow or orange barrier filter that blocks the blue.
• Start with autofluorescence. Leaf cross-sections, mosses and algae show chlorophyll glowing red. Some pollen and plant tissues glow in other colors.

Commercial LED fluorescence attachments and complete fluorescence microscopes are also available in a wide range of prices, and used lab equipment occasionally appears secondhand; our used microscope checklist can help you assess it.

Safety: never look directly at a UV or high-power LED or arc source, and never look into the eyepieces with excitation light on unless a barrier filter is in place. UV light can harm the eyes and skin, and intense blue light can also damage the eyes. Use UV-blocking eye protection when working with UV sources, follow the manufacturer’s instructions, and treat any fluorescent stains and chemicals with the precautions on their safety data sheets.

For how fluorescence fits alongside darkfield, phase contrast and other methods, see our overview of contrast in microscopy.