For many applications conventional widefield fluorescence microscopy is still the best choice. The CCD detected used for conventional microscopy are often more sensitive that the photomultiplier tubes used in confocal microscopes and flow cytometers. As the camera capture the whole field of view at the same time – it also allows for faster imaging in many cases. Examples where conventional microscopy may be more appropriate include the visualization of individual molecules, receptors or small organisms such as bacteria and yeast. Total Internal Reflection Fluorescence (or TIRF) is a powerful technique which combines the sensitivity of conventional fluorescence without out of focus light. User have access to a dedicated conventional fluorescence system equipped for time-lapse microscopy and a laser-based multi-color TIRF system (also used as the basis for the N-STORM system)
Applications:
>> Endocytosis and vesicle dynamics: High contrast, speed and sensitivity makes it possible to follow live processes close to the membrane with TIRF
>> Single molecule studies: The advanced EM-CCD camera allows the fluorescence of individual molecules to be detected even with relatively short exposures
>> Actin cytoskeleton and focal contact dynamics in live cells: Using fluorescent protein markers these processes can be followed using high time-lapse imaging
>> Yeast and bacteria studies: It is often difficult to fluorescence from microorganism by confocal microscopy without bleaching, whereas the combination of a 100x 1.49NA objective and a very sensitive camera allows fluorescence to be de-tected without need high laser power or very low exposures.
>> High speed imaging of calcium dynamics