Advanced fluorescence microscopy

Single-molecule detection using advanced fluorescence microscopy techniques... 

Progress in instrumentation has considerably improved the sensitivity of fluorescence detection. Advanced fluorescence microscopy techniques allow detection at single molecule level, which opens up new opportunities for the development of fluorescence-based methods or assays in material sciences, biotechnology and in the pharmaceutical industry. 

Fluorescence measurements are very important in studies on microfluidics and nanofluidics, with main applications on flow visualization and single-molecule detection. To achieve measurements with higher spatial resolution, which becomes more significant with the rapid development of nanofiuidics, research efforts should be focused on developing more advanced fluorescence microscopy setups. The particularly useful setups will be the ones that can break the classic optical diffraction limit. 

The introduction and diversification of genetically encoded fluorescent proteins (FPs) [1] and the expansion of available biological fluorophores have propelled biomedical fluorescent imaging forward into new era of development [2], Particular excitement surrounds the advances in microscopy, for example, inexpensive time-correlated single photon counting (TCSPC) cards for desktop computers that do away with the need for expensive and complex racks of equipment and compact infrared femtosecond pulse length semiconductor lasers, like the Mai Tai, mode locked titanium sapphire laser from Spectra physics, or the similar Chameleon manufactured by Coherent, Inc., that enable multiphoton excitation. 

Finally, in Chapter 11 some advanced techniques are briefly described fluorescence up-conversion, fluorescence microscopy (confocal excitation, two-photon excitation, near-field optics, fluorescence lifetime imaging), fluorescence correlation spectroscopy, and single-molecule fluorescence spectroscopy. 

There has been a continued interest in examining the properties of intact living cells using fluorescence microscopy. This field has seen considerable advances since the application of digital imaging techniques. In examining whole cells, one has to be especially aware of the location(s) of the probe. This is particularly important when bulk measurements are to be made on intact cells. 

The availability of laser scanning confocal fluorescence microscopy,68 first commercially offered in the 1980s, enabled a major advance in chemical microscopy imaging convenient summaries are available in the Handbook of Biological Confocal Microscopy.69 Confocal fluorescence microscopy works by focusing continuous-wave laser illumination to a diffraction-limited spot in a focal... 

In recent years, many other techniques have been employed to elucidate the structure of fat crystal networks including confocal laser scanning fluorescence microscopy (Heertje et al. 1987) and multiple photon microscopy (Marangoni and Hartel 1996). Another advance has been the development of three-dimensional imaging. 

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