Confocal fluorescence microscopy

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. 

Combining fluorescence spectroscopy with fluorescence microscopy, confocal microscopy could be used to elucidate the pathway of siderophore-mediat iron uptake in the fungus Ustilago maydis, and visualize this pathway by providing unique fluorescent microscopic images. Using these techniques, clear images of two independent iron-uptake mechanisms have become visualized as well as their cellular compartment locahzed. 

The last two decades have seen tremendous progress in optical microscopy, especially, confocal fluorescence microscopy. Confocal laser-scanning fluorescence... 

Confocal fluorescent microscopy Confocal laser scanning microscopy Layer-by-Layer (LbL) technique Microcapsules... 

Similar spectral techniques as discussed for macroscopic tumour imaging can be employed for fluorescence microscopy. Confocal and two-photon-induced fluorescence microscopy [10.210], and imaging Fourier transform spectroscopy [10.211] are all valuable techniques for studies at the cellular level. Related to this field is the optical trapping of ceils with focused laser beams optical tweezers), which relies on gradient forces of the same kind as discussed in Sect. 9.8.5. Trapped cells and polymer strings can be manipulated in many ways to enable fundamental studies to be conducted [10.212]. 

B1.18.5.5 CONTRAST ENHANCEMENT AND PRACTICAL LIMITS TO CONFOCAL ONE-PHOTON-EXCITATION FLUORESCENCE MICROSCOPY... 

Lindek St, Cremer Chr and Stelzer E H K 1996 Confocal theta fluorescence microscopy using two-photon absorption and annular apertures Optik 02 131-4... 

Carlsson Kand Liljeborg A 1997 Confocal fluorescence microscopy using spectral and lifetime information to simultaneously record four fluorophores with high channel separation J. Microsc. 185 37-46... 

Figure C 1.5.5. Time-dependent fluorescence signals observed from liquid solutions of rhodamine 6G by confocal fluorescence microscopy. Data were obtained with 514.5 mn excitation and detected tlirough a 540-580 nm...

Osborne M A, Balasubramanian S, Furey W S and Klenerman D 1998 Optically biased diffusion of single molecules studied by confocal fluorescence microscopy J. Chem. Phys. B 102 3160-7... 

There are several other techniques Uke the fluorescent dye displacement assays, footprinting, Fourier transform infrared spectroscopy. X-ray crystallography, electron microscopy, confocal microscopy, atomic force microscopy, surface plasmon resonance etc used for hgand-DNA interactions that are not discussed here. 

Noise can be also introduced by biochemical heterogeneity of the specimen. This can be a major cause of uncertainty in biological imaging. The high (three-dimensional) spatial resolution of fluorescence microscopy results in low numbers of fluorophores in the detection volume. In a typical biological sample, the number of fluorophores in the detection volume can be as low as 2-3 fluorophores for a confocal microscope equipped with a high NA objective at a fluorescent dye concentration of 100 nM. This introduces another source of noise for imaging applications, chemical or molecular noise, related to the inherent randomness of diffusion and the interaction of molecules. 

McConnell, G. (2004). Confocal laser scanning fluorescence microscopy with a visible continuum source. Opt. Express 12, 2844—50. 

As mentioned above, spectral imaging microscopy is a form of multidimensional fluorescent microscopy where a fluorescent emission spectrum is acquired at each coordinate location in the sample. This mode of imaging has been implemented for wide field, confocal, and two-photon laser scanning microscopy, and several excellent... 

On the intergrowth structure of zeolite crystals as revealed by wide field and confocal fluorescence microscopy of the template removal processes... 

Keywords zeolites fluorescence confocal fluorescence microscopy template removal intergrowth... 

Bowman R.D., Kneas K.A., Demas J.N., Periasamy A., Conventional, Confocal And Two-Photon Fluorescence Microscopy Investigations of Polymer-Supported Oxygen Sensors, J. Microscopy 2003 211 112-120. 

Plant cytochemistry/histochemistry continues to evolve as fluorescence microscopy (16-19), confocal fluorescence microscopy (20,21), and microspectrophotometry (22) expand our quantitative knowledge of the distributions of chemical constituents in plant cells and tissues. With regard to microspectrophotometry, this is possible for single cells, as the Arcturus Corporation (Mountain View, C A) has developed an instrument capable of isolating single cells. 

Shuming N, Chiu DT. Probing molecules with confocal fluorescence microscopy. Science 1994 266 1018. 

Hepler PK, Gunning BES. Confocal fluorescence microscopy of plant cells. Protoplasma 1998 201 121-157. 

Quader H. Formation and disintegration of cistemae of the endoplasmic reticulum visuahsed in hve cells by conventional fluorescence and confocal laser scanning microscopy evidence for the involvement of Ca2+ and the cytoskeleton. Protoplasma 1990 155 166-175. 

Eukaryotic cells utilize an efficient transport system that delivers macromolecules fast and secure to their destination. In the case of the small GTP binding proteins of the Ras family the modified C-terminus seems to be sufficient for addressing the polypeptide to its target membrane (in the case of Ras itself the plasma membrane). Lipopeptides with the C-terminal structure of N-Ras (either a pen-tamer with a C-terminal carboxymethylation and farnesylation or a heptapeptide with a palmitoyl thioester in addition) and a N-terminal 7-nitrobenz-2-oxa-l,3-diazolyl (NBD) fluorophore were microin-jected into NIH3T3 fibroblast cells and the distribution of the fluorophore was monitored by confocal laser fluorescence microscopy. Enrichment of the protein in the plasma membrane was efficient only for peptides with two hydrophobic modification sites, while the farnesylated but not palmitoylated peptide was distributed in the cytosol.1121... 

Various solutions have been proposed for the reduction or elimination of autofluorescence. One way is to chemically suppress the autofluorescence signal with some reagents such as sodium borohydride, glycine or toluidine blue. However, in many cases, these approaches are either infeasible or ineffective, and none of them fully eliminates the problem. The second way is to use spectral unmixing algorithms subtracting the background fluorescence. This is only possible if you have at your disposal complicated, expensive confocal optics with sophisticated automated software (http //www.cri-inc.com/applications/fluorescence-microscopy.asp). 

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