Fluorescence microscopy materials

Since our backbone 2 aPNA incorporates six Lys residues in its peptide sequence and is cationic at a physiological pH, we were optimistic that this aPNA would be taken up into cells without the need for any external carrier system. To answer the simple question of whether b2 aPNAs are intemahzed, a standard fluorescence microscopy experiment was performed to see if whole cells that were incubated with a fluorescent-labeled aPNA would internahze labeled material [70]. Chinese Hamster Ovary (CHO) cells in culture were incubated with BODIPY-la-beled TCCCT(b2) at 37 °C for various periods of time. Following incubation, the cells were rinsed in phosphate-buffered sahne (PBS), fixed with 4% formaldehyde at ambient temperature for 20 min, then washed with PBS and stored in a refrigerator until examined by fluorescence microscopy. 

Progress in instrumentation has considerably improved the sensitivity of fluorescence detection. Advanced fluorescence microscopy techniques allow detection at single molecule level, which opens up new opportunities for the development of fluorescence-based methods or assays in material sciences, biotechnology and in the pharmaceutical industry. 

Figure 1.18 shows fluorescence microscopy images of a bipolar three-dye antenna material with POPOP in the middle, followed by Py+ and then by Ox+. The different color regions that can be observed in this simple experiment are impressive. The red color of the luminescence (1) disappears, when the crystal is observed trough a polarizer parallel to the crystal axis while the blue emission disappears when turning the polarizer by 90°. This material is very stable and is easy to handle. 

Figure 4. Pea stem material was sequentially extracted with hot 70% ethanol, 0.1 M EDTA and 4% KOH-0.1% NaBH4 to leave xyloglucan-cellulose cell wall ghosts . Binding of fluorescent fucose-binding lectin from Ulex europeus as visualized by fluorescence microscopy shows xyloglucan distributed over the whole wall surface. Photograph courtesy of Dr. T. Hayashi.

The other important technique for the study of films at the air/water interface which has recently been introduced is fluorescent microscopy. This technique was introduced by von Tscharner and McConnell [90] and Mohwald [91, 92]. It depends on the fact that certain amphiphilic fluorescent dyes become incorporated into islands of the surface active material under study. Furthermore, where two phases of the surface active material coexist, the dye can often be chosen so that it segregates preferentially into one phase. A shallow Teflon trough is employed with a water immersion objective incorporated into the bottom. The depth of water is adjusted so that the objective focuses on the water surface. The layer of material at the air/water interface is illuminated by a xenon lamp. The fluorescent light so generated passes via the objective and suitable filters to an image-intensified video camera and the image is displayed on a television screen. In some versions of this technique the fluoresence is viewed from above. Most of the pioneering work in this field was devoted to the study of phospholipids, a topic to which we will return. Recently this technique has been applied to the study of pen-tadecanoic acid and this work will be considered here as it relates directly to other papers discussed in this section. 

Confocal laser scanning fluorescence microscopy was used to study the exposure of the avidin-specific binding sites in the Av-GEB platform by the immobilization of a small and flexible biotinylated fluorescein molecule as a fluorescence marker. Fluorescence microscopy thus confirms that Av-GEB platform exposes active binding sites for biotin, acting as affinity matrix (Fig. 21.2B). After use, the electrode surface can be renewed by a simple polishing procedure for further uses, highlighting a clear advantage of this new material with respect to surface-modified approaches such as classical biosensors and other common... 

Figure 1.49 shows an example of using confocal fluorescent microscopy to reveal microscopic features in a specimen. The specimen is low density polyethylene (LDPE) containing fluores-cently labeled silica particles. The particle size and distribution in the polymer matrix can be clearly revealed by 3D confocal microscopy. Thus, confocal microscopy provides us a new dimension in light microscopy for materials characterization, even though its applications in materials science are not as broad as in biology. 

This review describes some of the recent developments in materials which exhibit enhanced two-photon absorption that can initiate photopolymerization or up-converted emission. Various optical methods including femtosecond time-resolved pump-probe experiments to characterize the two-photon properties are discussed. Finally, the applications of two-photon processes to optical power limiting, up-converted lasing, 3-D data storage, 3-D micro-fabrication, two-photon fluorescence microscopy and bio-imaging, and two-photon photodynamic therapy are presented. 

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