Lecture 6 -- Fluorescence Microscopy

Lecture 6 -- Fluorescence Microscopy

Emission

release of energy from a system after excitation

Fluorescence

• process of absorbing high-energy light and then emitting lower-energy light

• a specific type of emission

Photoluminescence

• absorption and re-emission of light

• Fluorescence — Fast emission and stops immediately

• Phosphorescence — slow emission and lasts

Chemiluminescence

• chemical reaction

• energy from chemical bonds like glow stick

• not fluorescence

Bioluminescence

• enzymatic reaction

• biological light like fireflies

Jablonski Diagram

• shows ground state energy level correlated with Energy

Fluorophores

• fluorescent molecules used to label parts of specimens

• different fluorophores excited at different wavelengths

• lets you distinguish between different structures

Photobleaching

• process in which fluorophore loses its ability to fluoresce due to prolonged exposure to light

• permanent irreversible photochemical damage

• good for looking at cell death

Widefield Fluorescence Microscopy

• uses broad field of light

• excitation filter for target wavelength

• not as detailed

• detects emission of photon

• quick and cheap

Confocal Fluorescence Microscopy

• pinhole aperture for resolution

• reduces background out of focus light

• longer, expensive, destructive photobleaching

• pseudo 3D

• used to visualize structures