Multicolor fluorescence

Buchwalow IB, Minin EA, Bocker W (2005) A multicolor fluorescence immunostaining technique for simultaneous antigen targeting. Acta Histochemica 107 143 148 Harlow E, Lane D (1999) Using Antibodies A Laboratory Manual. Cold Spring Harbor Labora... 

Whereas multicolor immunoenzyme staining is applicable only for separately located antigens (see Chap. 7), multicolor fluorescence immmunostaining makes it possible to colocalize antigens not only in the same cell but also in the same cellular compartment. Simultaneous immunolocalization of antigens using fluorescent antibodies can be fulfilled both by the direct (see Sect. 4.1) and indirect (see Sect. 4.2) methods. With the direct method, primary antibodies are labeled with fluorescent dyes, while with the indirect method, primary antibodies are applied as unlabeled antibodies and the visualization is performed with secondary antibodies that are labeled with fluorescent dyes. 

Bentolila LA, Weiss S (2006) Single-step multicolor fluorescence in situ hybridization using semiconductor quantum dot-DNA conjugates. Cell Biochem Biophys 45 59-70... 

Stich MIJ, Schaeferling M, Wolfbeis OS (2009) Multicolor fluorescent and permeation-selective microbeads enable simultaneous sensing of pH, oxygen, and temperature. Adv Mater 21 2216-2220... 

Eastmond, D.A., Rupa. D.S. Hasegawa, L.S. (1994) Detection of hyperdiploidy and chromosome breakage in interphase human lymphocytes following exposure to the benzene metabolite hydroquinone using multicolor fluorescence in situ hybridization with DNA probes. Mutat. Res., 322, 9-20... 

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. 

The practical limitation on library oversampling depends on screening instrumentation, for which flow cytometry must be considered the gold standard. Commercially available flow cytometers analyze multicolor fluorescence signals and sort desired cells within user-defined gates at a rate of 50,000 cells per second [44], A salient comparison would be to microplate-based robotic screening. Considering each cell in a combinatorial library to be functionally equivalent to a microplate well, a flow cytometer can screen the equivalent of several million 1536-well microplates per day. 

A NOVEL MULTICOLOR FLUORESCENT PROTEIN FROM THE SOFT CORAL SCLERONEPHTHYA GRACILLIMA KUEKENTHAL... 

A Novel Multicolor Fluorescent Protein from the Soft Coral S. gracillima Kuekenthal 313... 

Karlinsey, J. M. and Landers, J. P., Multicolor fluorescence detection on an electrophoretic microdevice using an acoustooptic tunable ftUev, Analytical Chemistry 78, 5590-5596, 2006. 

Fig. 6 (a) Molecular structure of 15 and (b) Photos of the polymorphs A-D under 365 nm UV irradiation at room temperature. Multicolor fluorescence of its polymorphs through tuning (c) the molecular packing structure (B and C) or (d) the molecular conformation (A and B). Adapted with permission from ref. 36. 2014, Wiley-VCH. 

High-content screen (HCS) is a phenotypic screening that monitors multiple cellular parameters simultaneously. HCS employs fluorescence-based reagents (antibodies, dyes that bind or localize to a given cell component, sensors) to generate a multicolor fluorescence readout that is usually recorded using automated optical image acquisition devices. 

The three APS-dye conjugates were mixed at desired ratios and added to a synthesis mixture of TEOS nanoparhcles via a modified Stober method. The resulting nanoparhcles exhibited high, multicolored fluorescence intensity under single-wavelength excitation and excellent photostability [55]. 

Kitaguchi, A. Yamaguchi, N. Nasu, M. Simultaneous enumeration of viable Enterobacteriaceae and Pseudomonas spp. within three hours by multicolor fluorescence in situ hybridization with vital staining. J. Microbiol. Methods 2006, 65, 623-627. 

M. Kikawada, A. Ono, W. Inami, Y. Kawata, Enhanced multicolor fluorescence in bioimaging using deep-ultraviolet surface plasmon resonance. Appl. Phys. Lett. 104, 223703 (2014)... 

Saleh, S. M. Mueller, R. Mader, H. S. Duerkop, A. Wolfbeis, O. S. Novel multicolor fluorescently labeled silica nanoparticles for interface fluorescence resonance energy transfer to and from labeled avidin. Anal. Bioanal. Chem. 2010,398,1615-1623. 

Transfection techniques in vitro and ex vivo (organotypic cultures) offer an array of possibilities to investigate the consequence) s) of the introduction of foreign nucleic acids (DNA but also RNA) and or other biologically active molecules into neurons, and to combine observations with immunocytochemistry. In particular, a wide number of fluorescent reporter proteins (FRPs) can be employed for multicolor fluorescence imaging. Here we present a series of protocols for in vitro and ex vivo transfection of DNA or RNA sequences into cerebellar neuron cultures and organotypic slices based on the use of plasmid vectors and multicolor laser scanning confocal microscopy. These protocols allow analysis of live transfected cells, and, after fixation, correlative neurochemical studies. 

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