Auto fluorescence

Figure 5. Fluorescence of collagen IV conjugated with Oregon Green 488 (Molecular Probes, Eugene, OR, USA.) spontaneously adsorbed to unmodified polyethylene foils (A) or polyethylene modified with 1014 0+ ions/cm at the energy of 30 keV (B). Collagen was diluted in phosphate-buffered saline to the concentration of 0.02 mg/ml (10 pg/cm ) and incubated with the foils for 24 h at room temperature. For auto fluorescence control.

BD. PPT1-/- mice present neuronal loss in the hippocampus and cerebellum, as well as an accumulation of auto-fluorescent storage material within these areas [647969]. 

Smith, M. M., and O Brian, T. P., 1979, Distribution of auto fluorescence and esterase and peroxidase activities in the epidermis of wheat roots, Austr. J. Plant Physiol. 6 201-219. 

Fig. 7.6. A and B. Confocal laser scanning microscographs of schistosome adults showing auto-fluorescence 488 nm was used for excitation and 520 nm for emission. A. Mid-body of a male schistosome with the focus on the lumen of the bipartite gut (g). In bombarded worms, a pale auto-fluorescence sometimes occurs under in vitro culture conditions. This auto-fluorescence occurs over a wide spectrum of wavelengths.

Due to the auto-fluorescence of mature females, other reporter genes should be envisaged for transformation experiments in this gender such as luciferase whose applicability for schistosomes had already been demonstrated... 

By taking advantage of the deep penetration depth offered by CARS microscopy with near-infrared laser pulses, CARS tissue imaging has been demonstrated to complement other label-free optical techniques, such as auto-fluorescence that is limited to a few chemical species and optical tomography, second harmonic generation (SHG), and third harmonic generation (THG)... 

PBS is also commonly used as a wash buffer for IHC. PBS s advantages are reduced auto fluorescence in immunofluorescent assays, and it is relatively inexpensive compared to Tris-based buffers. However, in some cases PBS can cause higher levels of nonspecific staining, and it has been observed to reduce the specific binding abilities of certain monoclonal antibodies (Anti-CD30, for example). 

TR-FRET measurements present interesting alternatives to the FRET quench format to minimize compound interference. Time-gated measurements allow the exclusion of compound auto-fluorescence from the detection. The technology is very popular due to its sensitivity, ease of use, and simplicity of assay development. The high costs for the reagents are the major disadvantage of... 

Due to the use of a confocal volume, FCS is particularly suited for miniaturization in HTS and relatively insensitive to auto fluorescent test compounds. Moreover, in compound testing, the small path length of the confocal volume greatly limits any filter effects on fluorescence intensity. As in FP, the requirement for large differences in mass in the assay design is a limitation to the applicability of FCS. However, it can be overcome by methods like Fluorescence Intensity Distribution Analysis (FIDA) or two-colour cross correlation derived from the original FCS concept. 

In addition to the general criteria discussed above, each assay technology can also suffer from technology-specific interference of chemical compounds. Auto-fluorescent compounds can interfere with any fluorescence-based readout. In assays that use the biotin-streptavidin interaction, biotin analogs are potential false positives. 

Fluorescence measurements on proteins require both an appropriate fluorescence technique and the presence of a suitable fluorophore. The techniques used for the application of fluorescence to proteins are described later in this article. In this section, we briefly consider three classes of fluorophores that are used widely to study proteins native fluorophores including fluorescent amino acids, extrinsic fluorescent labels, and auto-fluorescent proteins. Each has advantages for probing proteins and has distinct drawbacks No perfect fluorophore exists for studying proteins. 

An important factor for in vivo studies is the auto fluorescent cellular background. To overcome this problem, pyrene-labeled binary probes have been used to take advantage of the long fluorescence lifetime (>40ns) of the pyrene excimer, compared with that of the cellular extracts (7 ns), that allows selective detection of the excimer using time-resolved spectroscopy (35). 

GFP Auto fluorescent. No homologue endogenous activity in most systems. Stable at biological pH. Moderate sensitivity. Need post-transductional modification. Auto fluorescent for some compounds (aromatic amino acids). Potentially cytotoxic for some cellular types. 

Optical assay methods, including the scintillation readout for radioactive assays, suffer from interference through absorbance by colored test compounds, which give rise to a so-called inner filter effect or color quenching . Additional sources of artefacts in fluorescence assays are auto-fluorescence of compounds, plates, and reagents, as well as scattering of excitation Hght. 

Bone, Y. (1989) Paleotemperature analysis, Eromanga Basin, South Australia—fluid inclusion microthermometry auto-fluorescence microscopy and cathodolumines-... 

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