Autofluorescence characteristics

AMC is still a popular dye today. According to our experience, the IC50 values determined for protease inhibitors in AMC-based protease assays may be biased and misinterpreted due to the interference of label and compound autofluorescence characteristics (Figure 2.3). In the worst case, this can lead a drug discovery program in a wrong direction with respect to prioritization of compound classes. At the excitation and emission wavelengths of 350 and 500 nm, respectively, used for AMC, many compounds also display fluorescence characteristics. 

FIGURE 2.3 Dose-response curves for protease inhibitor with autofluorescence characteristics, (a) Result of standard fluorescence intensity-based assay employing an AMC-labeled substrate at a concentration of 2 pM. Profiling data could not be obtained due to the interference of the compound s fluorescence with the readout, (b) Result obtained from assay employing a RhllO-based substrate at a concentration of 0.5 pM. The profiling data (IC50 value of 335 nM and Hill coefficient of 1.0) were obtained for the depicted data set. 

The same publication indicated that fluorescence lifetimes of compounds from the compound collection of Pfizer classified as problematic due to their autofluorescence characteristics resulted in false positive results in many FI-based assays. For most compounds, the fluorescence lifetimes were below 1 ns. Thus, FLT measurements with a reporter fluorophore displaying a lifetime significantly longer than 1 ns are suitable for application in protease assays for compound testing. However, for applications under initial velocity conditions with a substrate turnover below 20%, fluorophores with lifetimes of a few nanoseconds are still critical because the dynamic range of the assay is then too low with lifetimes below 1 ns. 

J.D. Pitts, R.D. Sloboda, K.H. Dragnev, et al. (2001). Autofluorescence characteristics of immortalized and carcinogen-transformed human bronchial epithelial cells. J. Biomed. Optics, 6, 31-40. 

Autofluorescence characteristics of minerals have been described by Henkel (1989). In this chapter, we document the visualization and identification of chalcedony crystals in electric organs, by using a Leica TCS - SP2 Laser Scanning Confocal Microscope (LSCM). This microscope has three ion lasers i.e. argon with emission band in 458 run (cyan), 476 ran (blue-green), 488 nm (green) and 514 nm (yellow) He/Ne with emission band in 543 ran (red) and He/Ne with emission band in 633 nm (blue). Since chalcedony is characteristically an autofluorescent mineral, we were allowed to obtain images of crystals. 

There may well be increasing interest in analysis of the intrinsic characteristics of cells that lead to alterations in autofluorescence. John Steinkamp, Harry Crissman, and others at the Los Alamos Laboratory have been using flow systems to study the time character-... 

Fixation Fixation is the process by which the protein of cells is denatured, or cross-linked, and preserved. Fixation in flow cytometry is used to inactivate hazardous biological material and also to preserve stained cells when there is not immediate access to a flow cytometer. Fixation is also important in preserving proteins before detergent permeabilization for intracellular staining. Formaldehyde is often the fixative of choice for flow cytometry because it preserves the forward and side scatter characteristics of cells (but does cause some increase in their autofluorescence). 

Signature Signature is a term used by marine flow cytometrists to refer to the flow cytometric characteristics of particular planktonic species in a sample of water. The signature of plankton is related, in particular, to the autofluorescent pigments that are present. Signature has also been used to describe the distin-... 

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