Principle of Fluorescence Microscopy

Principle of Fluorescence Microscopy

• A fluorescence microscope uses fluorescence to generate an image.
• The specimen would be first prepared by injecting the fluorophores, which are fluorescence indicators such as Green fluorescent protein (GFP) or Oregon Green™ 488 BAPTA-1, AM (OG488).
• After that, it would be illuminated with specific wavelengths of light which is absorbed by the fluorophores.
• When the fluorophores absorb a high-energy photon (from the light source), the chemical system would be excited electronically and vibrationally, and become unstable at high energy levels.
• This unstable chemical system would soon release the absorbed photon energy through non-radiative transition first and eventually emitted fluorescence at a longer wavelength.
• The emitted fluorescence would be separated from the illumination light source through the emission filter, and the camera would capture the image from the specimen.
• Through the fluorescence microscope, we could conduct an observation of the specific structures or activities which have been labeled for fluorescence only.

FIG. 1. The working principle of Fluorescence Microscopy. The arc lamp together with the blue excitation act as the incident light source with specific wavelengths. GFP is used as the fluorophores.	FIG. 2. Jablonski diagram including the energy level of the fluorophore. The photochemical reaction includes the absorption, non-radiative transition, and emission of fluorescence.