Confocal Microscopy
Céline Hoffmann
Address: 84 Val Fleuri - 1526 Luxembourg
Phone. (+352) 26 970 253
Fax. (+352) 26 970 390
celine.hoffmann@crp-sante.lu
TIRF SYSTEM
The TIRF microscope: Total Internal Reflection Fluorescence microscope
Illumination source: Laser Argon 100mV: 458/488/514nmn
Objective: 100x, oil, 1.46NA, DIC
The Total Internal Reflection Fluorescence (TIRF) microscope is a new powerful technique based on evanescent wave illumination. This technique generates high contrast images with a strong reduction of out of focus intracellular background. Due to the limited penetration of the evanescent wave; the TIRF is especially dedicated to the observation of near membrane structure as vesicles trafficking, receptors behavior and cortical actin for examples.
Laser excitation is set with an incident angle inducing a reflection at the interface between the glass and the cell. Depending on the angle the laser can be totally reflected creating in the same time an evanescent wave that spread into the cell. The wave will excite only few fluorochromes close to the surface.
Example: Dynamics of actin-fluorescent filaments polymerized in vitro on a NEM-Myosin coated slide. After few minutes an Actin Depolymerizing Factor is added inducing filament depolymerization
