Microscopes are organized and grouped based on what method of magnification they use. Optical microscopes, for example, use light and a series of mirrors to produce a magnified image of a sample. The two main categories of optical microscopes are simple and compound.

The difference between simple and compound microscopes is that simple microscopes only use one mirror, while compound microscopes use multiple. The most common optical microscopes use visible light as the light source. However, microscopy utilizes other light spectrums as well.

Fluorescence refers to the emission of light due to the absorption of light. When a material with GFP or other fluorescent dye is exposed to electromagnetic radiation, it absorbs that radiation and re-emits some of it. The time difference, less than a millisecond, between when the organic or inorganic object absorbs the radiation and emits it is short due to the photons’ inherent absorption and emission time.

The object will stop emitting fluorescent light once it stops being hit by radiation. When the materials emit light after the radiation source has gone, it is called phosphorescence. The fluorescent objects’ re-emitted light from the fluorochromes has lower energy and thus a lower wavelength signature, making it exploitable in different analytical techniques.

An example of fluorescence can be seen when one uses a blacklight, and previously unseen colors suddenly are illuminated. Other common examples of autofluorescence include minerals and various bioluminescent animals and plants like algae, fish, and insects.

A fluorescence microscope is a type of light microscope that exploits fluorescence and phosphorescence to identify and observe specific microscopic objects. The area of the material to be observed is dyed with fluorophore tags that will illuminate only the intended parts of the sample.

The specimen is struck with electromagnetic radiation of a specific excitation wavelength that is subsequently absorbed by the fluorophores, which then fluoresce radiation with longer wavelengths than the incident light. This difference in wavelengths can then be used to filter out the light from the light source and only analyze light from the fluorescent sample by using a spectral emission filter.

Detailed imaging of live cells, antibodies, and more can be easily achieved using the fluorescence microscope with proper microscopy techniques. The main components that make up these imaging systems include:

• Light source: Common types are xenon arc lamps and mercury vapor lamps
• Excitation filter: Filters out undesirable specific wavelengths from a light source.
• Dichroic mirror: Filter out specific spectra of light or colors. Also called interference filters or accurate color filters.
• Emission filter: Filters out non-fluorescent light spectra to analyze the desired fluorescence.

Different filter cubes and beam splitters are chosen depending on the specific fluorescent protein used to label the specimen. Single fluorophores are imaged at one time, meaning that a narrow wavelength or one “color” that is emitted from the sample is observed at one time.

High-resolution multi-colored images can be composed by sewing multiple one-color images together. Understanding how to properly perform your fluorescence imaging is critical to fluorescence microscopy since running a sample too many times can cause photobleaching, causing the fluorescent molecules to stop responding.