Dual-color analysis

Phycoerythrin Phycoerythrin is a fluorochrome derived from red sea algae. It is particularly useful in flow cytometric applications requiring dual-color analysis because, like fluorescein, it absorbs 488 nm light from an argon laser. However, it has a longer Stokes shift than fluorescein, and therefore the fluorescences of the two fluorochromes can be distinguished. 

In the most recent microarray experiments, multicolor fluorescence labeling is used for simultaneous analysis of two or more samples in a single assay. For this, total RNA or mRNA are labelled with fluorescent nucleotides by a reverse transcription reaction. Cyanines Cy3 and Cy5 are used for dual color analysis. 

Microarray slide scanner. You may use any scanner that is compatible with 75 X 25 X 1-mm slides and capable of dual-color analysis. The scanner must be capable of measuring Cy5 and Cy3 fluorescent labels. 

Figure 6.15 Applying dual-color ratiometric gene expression labeling approach to protein expression analysis. (From Haab, B.B. etal., Genome Biol., 2, 0004.1-0004.13, 2001. With permission.)...

Quadrant For dual-parameter analysis, a two-dimensional plot of particles according to the correlated fluorescence intensities of two colors is, traditionally, divided into four quadrants. The division is based on the background fluorescence of the unstained control sample. The quadrants, from this division, will contain (1) cells that have stained with the first fluorochrome only (2) cells that have stained with both fluorochromes (3) cells that remain unstained and (4) cells that have stained with the second fluorochrome only. In other words, the quadrants are defined so that they delineate the two types of single positive cells (1 and 4), double-negative cells (3), and double-positive cells (2). 

Figure 4. A detection method for chromogenic visualization of dual-color FISH signals with anti-hapten antibody. After FISH-staining with two different fluorochromes such as FITC and Texas Red (a and inset photo a HER2 FISH analysis in breast carcinoma), archival tissue can be immunohistochemically detected using the corresponding anti-fluorochrome antibodies and two different chromogens (b and inset photo b HER2 CISH analysis in the same lesion as in the upper photo).

Heinze, M.G., Kolterman, A., and Schille, P. (2000) Simultaneous two-photon exitation of distinct labels for dual-color fluorescence crosscorrelation analysis, Proc. Natl. Acad. Sci. USA 97, 10377-110382. 

Lai P, Salazar PA, Hudis CA, et al. HER-2 testing in breast cancer using immunohistochemical analysis and fluorescence in situ hybridization A single-institution experience of 2,279 cases and comparison of dual-color and single-color scoring. Am J Clin Pathol. 2004 121 631-636. 

Tryphonas H, Lacroix F, FEayward S, Izaguirre C, Parenteau M, Fournier J. Cell surface marker evaluation of infant Macaca monkey leukocytes in peripheral whole blood using simultaneous dual-color immunophenotypic analysis. J Med Primatol 1996 25 89-105. 

P. Schwille, F.J. Meyer-Almes, R. Rigler, Dual-color fluorescence crosscorrelation spectroscopy for multicomponent diffusional analysis in solution, Biophys. J. 72, 1878-1886 (1997)... 

Although dual color systems prevail at the moment in microarray analysis, the scope... 

Rigler, R. Mets, U. Widengren, J. et al. Fluorescence Correlation Spectroscopy with high Count Rate and low Background - Analysis of Translational Diffusion. Eur. Biophys. J. Biophys. Lett. 1993, 22, 169-175. Bacia, K. Majoul, I. V. Schwille, P. Probing the endocytic pathway in live cells using dual-color fluorescence cross-correlation analysis. Biophys. J. 2002, , 1184-1193. 

Fig. 3.4 Scheme of fluorescence correlation spectroscopy (FCS). In single (A) and dual (B) color FCS, the fluorescence in the observation volume of one (X) or two (X, Y) distinct classes of fluorophores, respectively, is monitored over time. The correlation functions are then used for the temporal analysis of the signals to determine the characteristic residence times of the labeled probes and their diffusion or binding constants. The correlation plots depict ideal cases. G(0) is the correlation amplitude, which is inversely proportional to the average number of fluorescent labels simultaneously present in the observation volume (N). 

Thermooptical detection has been combined with CE and used for protein and peptide Edman degradation sequencing detection, native protein detection, as well as the analysis of derivatized amino acid mixtures. " Frequency-doubled argon-ion lasers have been employed to supply an UV (257 nm) pump beam for the analysis of dansylated amino acids as well as the analysis of etopside and etopside phosphate in human blood plasma. In addition, two-color thermooptical absorbance detectors have been constructed, where each laser serves a dual role of pump and probe this system can be used to detect analytes that absorb in differing regions of the electromagnetic spectrum. 

FIGURE 9.17 Analysis of two copolymers using RI/UV 254 nm dual detection, the response factors for the detectors have been determined using homopolymers of butadiene and polystyrene. (See insert for color representation of the figure.)... 

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