Fluorescence Microscopy: Principles and Applications

Fluorescence microscopy

Combines light microscopy with visualization of fluorescence

Fluorescence

Involves absorbance and emission of light wavelengths by a fluorophore

Fluorophore

Fluorescent molecule responsible for absorbance and emission of light

Fluorescently label

Tag a sample with fluorescently tagged antibodies, proteins, or nucleic acid dyes

Fluorescence microscope

Includes xenon or mercury light source, filters, dichroic mirror, and shutter

Xenon or mercury light source

Provides powerful light in a wide range of wavelengths

Exciter filter

Selects the excitation wavelength for fluorescence

Dichroic mirror

Reflects excitation wavelength towards the sample

Barrier filter

Filters out contaminating light and selects for emission wavelength

Filter cube

Combines exciter filter, dichroic mirror, and barrier filter

Fluorophore absorption

Absorbs specific excitation wavelengths

Fluorophore emission

Emits light at specific emission wavelengths

Stoke's Shift

Difference between absorption and emission wavelengths

Photobleaching

Weakening or loss of fluorescence due to prolonged excitation

Anti-fade mounting medium

Reduces photobleaching by adding to the slide

Fluorescence imaging

Turn on light source, focus, select filter cube, adjust exposure time

Multiple dyes imaging

Change filter cube for each fluorophore, overlay and merge images

Fluorescently labeled antibodies

Used to image proteins, attached to fluorescent compounds

Fluorescent protein

Protein that can be expressed and fluoresces when excited

Fluorescence Speckle Microscopy

Uses fluorescently labeled macromolecular assemblies to study movement

FRAP

Fluorescence recovery after photobleaching, monitors diffusion rate