Fluorescence Microscopy

Fluorescence microscopy is a useful tool in the world of bioimaging. I am currently in the BIO-477H course Bioimaging, and one of our experiments was to work with cells tagged with fluorescent molecules called fluorophores. Fluorophores produce fluorescent light and fantastic, colorful images, but caution must be taken when imaging with fluorescence. A phenomena known as photobleaching occurs when fluorophores are exposed to fluorescent light of high intensity or for a prolonged time period. This occurs because the fluorophore absorbs the photon of the receiving light, goes to an excited state, and releases a photon when it returns to the ground state. The emitted photon is what we perceive as "fluorescence". When fluorophores are exposed to high intensity fluorescent light, the fluorophore is modified covalently and remains in an excited state, losing the ability to return to ground state and release a photon. Thus, the fluorophore no longer fluoresces. Strategies such as using neutral density filters, and using a shutter to help take the photos reduces the fluorophore's exposure to fluorescent light and result in better images.