Improvements in conventional fluorescence microscopy

A conventional fluorescence microscope differs from a standard microscope by the light source (mercury or xenon lamp), which produces UV-visible light. The excitation wavelength is selected by an interference filter or a monochromator. Observation of the fluorescence is made by eye, photographic film or CCD (charge- 

For samples thicker than the depth of field, the images are blurred by out-of-focus fluorescence. Corrections using a computer are possible, but other techniques are generally preferred such as confocal microscopy and two-photon excitation microscopy. It is possible to overcome the optical diffraction limit in near-field scanning optical microscopy (NSOM). 

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By setting properly all the aforementioned parameters, a proper imaging of the sample will be obtained. At those conditions, lateral or xjy resolution (minimum spacing that can just be resolved) is about 1.4 times better than in widefield fluorescence microscopy (180-200 run are typical values), while the improvement in viewing axis or z resolution at optimal conditions is about 3.0 times poorer than the lateral resolution (500-800 nm). This represents a marked improvement compared with conventional microscopy, which arises from the rejection of fluorescence light from out-of-focus regions of the specimen. 

Fluorescence Microscopy. Improvements in fluorescence observation have resulted in images that are twice as bright as conventional fluorescence images. Fluorescence microscopy is used for staining and... 

At the moment, LEDs are constantly being improved in terms of efficiency, output power, and available wavelength. If developments continue, it could very well be that, in time, LEDs are the hght source of choice, not only for wide-field FLIM, but also for conventional steady-state wide-field fluorescence microscopy. 

Presently karyotypes of human metaphase chromosomes are used to detect genetic defects like deletions or translocations, where the chromosomes are treated by the trypsin-Giemsa protocol, to produce a typical banding pattern and imaged by optical microscopy. Because of the diffraction limit in optical microscopy, even the smallest visible band contains around 1 million base pairs. Improved resolution has been demonstrated using fluorescence NSOM on the treated chromosomes compared to conventional light microscopy. 

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