Fluorescence beads

Sheetz, M.P. Spudich, J.A. (1983). Movement of myosin-coated fluorescent beads on actin cables in vitro. Nature 303, 31-35. 

Although several types of fluorescent beads were proposed as a microscopic fluorescence standard 30 years ago,2 beads have not been used as a proteinembedding matrix for routine IHC on FFPE tissue. We recently tested primary coated beads ( Dynabeads, Dynal, New York) that are coated with a goat anti-mouse antibody on the surface of the beads. In the first experiment, a monoclonal antibody to cytokeratin 7 (DAKO, 50pL/34.5pg) was bound to the beads by incubating with the beads (150 pL at a concentration of 109 beads/1 pL) at 4°C in a cold room with an automatic shaker for overnight. Incubation was followed by three phosphate-buffered saline (PBS) washes,... 

C. Application 1 Femtosecond Time-Resolved Fluorescence erom a Fluorescent Bead... 

In the first example, we describe time-resolved fluorescence measurements of a fluorescent bead (Fujino and Tahara 2004). Figure 3.6a shows the CCD image of a commercial fluorescent bead that has a diameter of -4.85 pm (Mag Sphere). This bead was laser trapped near the focus point by the excitation pulse. In fact, when the irradiation... 

FIGURE 3.6 The CCD image of a fluorescent bead under (a) laser trapping, and (b) without laser trapping, (c) The femtosecond time-resolved fluorescence at 520 nm observed with lOOx objective lens. (Form, Fujino, T. and Tahara, T.,Appl. Phys. B 79 145-151, 2004.)... 

An impressive demonstration that myosin heads do move along the actin filaments was provided by Sheetz and Spudich, who found that myosin-coated fluorescent beads 0.7 pm in diameter will move along actin filaments from cells of the alga Nitella in an ATP-dependent fashion at velocities similar to those required in muscle.149 The myosin heads literally glide along the thick cables of parallel actin filaments present in these algae. 

A positive test is communicated by red beads for the AliR test and green or fluorescent beads in the case of the fluoresceine test. 

Meldal and co-workers26,27 first described the fluorescence-quenching approach to screen the OBOC combinatorial libraries for protease substrates. A fluorescence-quench combinatorial library prepared from the porous PEGA bead resin (see above) is first inspected under a fluorescent microscope and the fluorescent beads are removed and discarded. After all of the false-positive beads have been removed, the peptide beads are transferred into an Eppendorf tube and washed 5 x with water, followed by a 5 x wash with the appropriate protease buffer. Protease is then added... 

The fluorescent beads are manually isolated, washed with 8 M guanidine-HCl and water, and then submitted for sequence analysis. We expect that some of the peptides on the positive beads will be cleaved at the proteolytic site. As a result, the PTH-amino acids detected during each cycle of Edman degradation will have been generated from both of these peptides. This complicates the sequence analysis somewhat. However, it also allows us to determine the proteolytic cleavage site of the peptide, in addition to its uncleaved sequence. 

Fig. 3.11. Intensity signals from fluorescent beads (of five different intensities) acquired with linear amplification (top) and logarithmic amplification (bottom). Log amplification permits all five intensities to be on scale (that is, within the 1024 channel range). Additionally, the spread (the CY) and the peak height of the distributions for each bead are visually similar with a log but not with a linear amplifier. From Givan (2001).

One way around this problem is to compare cell fluorescence not with unstained cells but with the fluorescence of an external standard. This can be done by the use of fluorescent beads. In brief (this is an insiders joke you would be amazed at how much has been written about the use of beads in flow cytometry), there are commercially available polystyrene beads ( microspheres ) that have standardized... 

Flow switching in a PDMS chip can be achieved by deflection from two side channels (see Figure 3.34) [359]. When there is zero hydrostatic pressure, the two streams of fluorescent beads flow equally to two channels. When the hydrostatic pressure from the bottom channel is greater (i.e., 2 mm liquid level differ-... 

Egner BJ, Rana S, Smith H, Bouloc N, Frey J, Brocklesby WS, Bradley M, Tagging in combinatorial chemistry the use of colored and fluorescent beads, Chem. Commun., pp. 735-736, 1997. 

The importance of proper instrument and experimental standards to evaluate the machine performance and experimental variables should not be underestimated. We recommend to check for even illumination, PSF, system/laser stability and correct deviations, when necessary, using proper procedures such as fluorescence slides and triple or double-labeled fluorescence beads containing defined amounts of fluorophores. 

Point spread function (PSF) If a tiny population of 100 nm fluorescent beads sandwiched between a coverslip and a microscope slide are examined at high resolution (i.e. at 100x objective magnification, 1.4 NA. and in a correctly matched refractive index of oil), it can actually show a tiny set of rings in the horizontal (XY) view (also called an airy disk (see Fig. below). This airy disk cannot be avoided due to diffraction and the wave nature of light. If a specimen is optically sectioned and projected in a vertical (XZ) view (see Fig. xx), a set of concentric rings will flare from the center. When a three-dimensional image of this specimen is collected, a complete point spread function is said to be recorded for each bead. The (PSF)... 

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