Microscopy, fluorescence

Fluorescence microscopy is used to estimate microbial population density and viability based on the fact that some molecules absorb light of a spe-cihc wavelength and emit light of different wavelengths (fluorescence). Compounds that fluoresce can be either present in nature (e.g., chlorophyll) or chemically synthesized (fluorophores). Fluorophores absorb blue light resulting in formation of excited but unstable electrons that rapidly 

The specific dyes used in fluorescence microscopy are directly taken up by the cells and may react indiscriminately with organic material or be incorporated and concentrated in specific subcellular organelles. Still other techniques utilize immunochemical methodology (Section 16.3.2) whereby a fluorescent dye react with specific moieties of the viable cell membrane. In these cases, the dye can be used to distinguish between viable and dead cells. These methods have been applied toward detection of viable-but-non-culturable cells because these cells cannot be cultured using standard microbiological media (Section 6.1). 

Splittstoesser (1992) described a method using fluorescent dye (acridine orange) known as direct epifluorescence filter technique (DEFT), a method also applied by Divol and Lonvaud-Funel (2005). Divol and Lonvaud-Funel (2005) used a different substrate, fluoresceine diacetate, which is hydrolyzed by viable cells to form a fluorescent product, fluoresceine. However, Atlas and Bartha (1981) observed that cell population values can differ substantially between (epifluorescence and direct plating) methods (10 vs. 10 CFU), possibly due to the presence of viable-but-non-culturable cells. In addition, Meidell (1987) reported interference 

In the more modern fluorescence microscopes the primary filter has been replaced by a system of mirrors (the Ploem illuminator in the Leitz microscope) which serves a similar function. 

The disruption of tissue culture cells and their subsequent subcellular fractionation may be more difficult than parallel processes with say rat liver. Although the cells are homogeneous they are usually small and cells grown in suspension frequently have very little cytoplasm. 

Disruption often requires the use of hypotonic media and, where possible, non-ionic detergents. For nuclear isolation a buffer containing 20 mM Tris HC1, 1 mM EDTA and 1% Tween 80 or Triton X-100 is suitable and dithiothreitol and protease inhibitors may be 

scraped from the growing surface or sedimented from a suspension culture are washed in PBS to remove serum and other unwanted medium components and then homogenised for example, using 5 strokes of a Potter-Elvehjem homogeniser or, for small volumes, by pipetting. Cell densities can be as low as 106 cells per ml. Disruption is checked using a phase contrast microscope. The nuclei are readily sedimented at 800 g for 10 min. 

When isolating cytoplasmic organelles the use of detergents is not possible and hypotonic buffers should be avoided. If this is not possible then, immediately after homogenisation, tonicity should be restored by addition of a concentrated sucrose solution. 

Some of the insights that these techniques have brought to the field of chromatin research are described in this chapter. In some cases, microscopy has served simply to confirm conclusions obtained from less direct, but powerful biochemical approaches. In other cases, however, studies with imaging techniques are described where the information could only be obtained by microscopy, because of the spatial or temporal resolution that the microscope can provide. 

Duplicating the GFP-H2B experiments with both GFP-H3 and GFP-H4 vectors, very little H3 and H4 exchange outside of S phase was observed. Fluorescence of GFP-H3 and GFP H4 in HeLa cells in G1 recovered rapidly. The extremely rapid recovery rate is similar to that of a diffuse soluble protein, indicating that at this stage of the cell cycle, GFP-tagged H3 and -H4 are not incorporated into chromatin. FRAP experiments of transfected cells in S or G2 show that there is very little recovery of GFP-H3 or -H4 fluorescence. The fluorescence imaging indicates that once the H3 and H4 proteins are incorporated into the chromatin, they are essentially immobile for the remainder of the cell cycle. Unlike histones H2A and H2B, which associate as dimers in the nucleosome histone octamer, there is very little exchange of the components of the H3/H4 tetramer. 

The dynamics of histone H1 in vivo has also been described by taking advantage of GFP fusions in combination with FRAP [23]. From previous biochemical 

The experimental techniques for single molecule spectroscopy described in the previous chapters differ mainly in the method employed to reduce the excitation volume of the sample (combined with different fluorescence collection methods). This was achieved in four different ways (i) the laser was focused to a tiny spot on the sample by a lens immersed in liquid helium, (ii) the excitation light was coupled into an optical fiber carrying the sample at its end, (iii) the sample was mounted behind a small aperture (pinhole with typically 5 pm diameter). All these methods reduce the excitation area to a few pm. The near-field technique (iv) allows investigations beyond the classical diffraction limit the tapered tip used had a typical diameter in the order of 50-100 mn. 

In the approach described here, microscopy is used to investigate an illuminated area of about 100 x 100 pm. The small volume to be studied is effectively defined by the optical resolution of the microscope giving as great benefit that many molecules can be investigated simultaneously in parallel. The first experiment performed to spatially localize a single fluorescing molecule in one dimension was realized by translating a focused laser spot, with a width of w5 pm, in one direction across the 

Kauppi and Verseghy-Patay 449) studied the distribution of lichen substances (especially depsides and depsidones) in lichen thalli by fluorescence microscopy. 

Mietzsch et al. 544) have developed a computer program (Wintab-olites) for identification of lichen substances based on spot tests, TLC Rf values and HPLC Rj values. The third edition of this data base contains data for over 700 lichen substances 545). 

The chemical substances to be observed are either intrinsically fluorescent, or made so by a chemical process, or attached to a fluorescent label. Samples for fluorescence microscopy are often stained with a fluorescent dye (e.g. rhodamine), with the aim of having the physical characteristics of the probe represent the specimen characteristic. The specimen is then illuminated with light at an appropriate wavelength to excite the dye, generating fluorescent light, which is collected by the microscope. The intense fluorescence of the reactive dansyl group also determines convenient use in fluorescence microscopy, which allows the lowest concentrations 

Fluorescence microscopy offers a number of advantages over other forms of microscopy (c/r. Tables 5.16 and 5.17). Its high sensitivity allows very low concentrations of specific substances to be localised. Because fiuorescence is observed as luminosity on a dark background, fluorescent constituents of the specimen can be seen even in extremely small amounts. Fluorescence microscopy can also be applied to detect particles below the resolution of a light microscope. Since fluorescence involves two wavelength bands (excitation and emission), optical specificity can substantially be increased. Fluorescence microscopy, because of its complexity, gives more difficulty than usual in interpretation of the image. 

Reviews on fluorescence techniques in polymer science have been reported [58,81,82] recent books on fluorescence imaging spectroscopy and microscopy are available [83,84] c/r. also Bibliography. 

Fluorescence microscopy is closely allied to transmission (absorption) microscopy in its range of application, but possesses particular advantages (Table 5.17). Because many substances are fluorescent, or can be made so, fluorescence microscopy is widely applicable to all kinds of material. In view of the more complex and expensive instrumentation than conventional transmitted-light microscopy, fluorescence microscopy is usually reserved for those applications in which its high sensitivity is of importance i.e. to examine substances present in low concentrations. Fluorescence microscopy is especially a valuable tool in the biological sciences. 

Fluorescence microscopy has also been employed largely for 2D surface imaging. Scanning confocal fluorescence microscopy has been applied to the investigation of subsurface morphology of foams. The general knowledge on the applications of fluorescence microscopy for polymers is rather limited. 

Ofher fluorometric assays will be of a more qualitative nature, if it comes to microscopic studies to characterize receptor expression on cells [38, 39] (Fig. 5.6) or receptor internalization upon fhe binding of a ligand [40]. 

FIGURE 6.19 Excitation and emission spectra for the fluorophore Alexa Fluor 488. Notice how the excitation spectrum (blue dashed line) does not significantly overlap the emission spectrum (red solid line). 

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The ability to image lateral heterogeneity in Langmuir monolayers dates back to Zocher and Stiebel s 1930 study with divergent light illumination [166]. More recently the focus shifted toward the use of fluorescence microscopy of mono-layers containing a small amount of fluorescent dye [167]. Even in single-corn-... 

There has been extensive activity in the study of lipid monolayers as discussed above in Section IV-4E. Coexisting fluid phases have been observed via fluorescence microscopy of mixtures of phospholipid and cholesterol where a critical point occurs near 30 mol% cholesterol [257]. 

B1.18.5.5 CONTRAST ENHANCEMENT AND PRACTICAL LIMITS TO CONFOCAL ONE-PHOTON-EXCITATION FLUORESCENCE MICROSCOPY... 

Lindek St, Cremer Chr and Stelzer E H K 1996 Confocal theta fluorescence microscopy using two-photon absorption and annular apertures Optik 02 131-4... 

Hell S W and Kroug M 1995 Ground-state-depletion fluorescence microscopy a concept for breaking the diffraction resolution limit Appl. Phys. B 60 495-7... 

Carlsson Kand Liljeborg A 1997 Confocal fluorescence microscopy using spectral and lifetime information to simultaneously record four fluorophores with high channel separation J. Microsc. 185 37-46... 

A wide variety of measurements can now be made on single molecules, including electrical (e.g. scanning tunnelling microscopy), magnetic (e.g. spin resonance), force (e.g. atomic force microscopy), optical (e.g. near-field and far-field fluorescence microscopies) and hybrid teclmiques. This contribution addresses only Arose teclmiques tliat are at least partially optical. Single-particle electrical and force measurements are discussed in tire sections on scanning probe microscopies (B1.19) and surface forces apparatus (B1.20). 

Figure C 1.5.5. Time-dependent fluorescence signals observed from liquid solutions of rhodamine 6G by confocal fluorescence microscopy. Data were obtained with 514.5 mn excitation and detected tlirough a 540-580 nm...

Sekatskii S K and Ketokhov V S 1996 Single fluorescence centres on the tips of crystal needles first observation and prospects for application in scanning one-atom fluorescence microscopy Appl. Phys. B 63 525-30... 

Klar T A and Hell S W 1999 Subdiffraction resolution in far-field fluorescence microscopy Opt. Lett. 24 954-6... 

Guttler F, Irngartinger T, Plakhotnik T, Renn A and Wild U P 1994 Fluorescence microscopy of single molecules Chem. Phys. Lett. 217 393-7... 

Jasny J, Sepiol J, Irngartinger T, Traber M, Renn A and Wild U P 1996 Fluorescence microscopy in superfluid helium single molecule imaging Rev. Sc/. Instrum. 67 1425-30... 

Strickler J FI and Webb W W 1990 Two-photon excitation in laser scanning fluorescence microscopy Proc. SPIE 13948107-18... 

Osborne M A, Balasubramanian S, Furey W S and Klenerman D 1998 Optically biased diffusion of single molecules studied by confocal fluorescence microscopy J. Chem. Phys. B 102 3160-7... 

Tokunaga M, Kitamura K, Saito K, Iwane A H and Yanagida T 1997 Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy Biochem. Biophys. Res. Commun. 235 47-53... 

Lloyd, C.W. (1987). The plant cytoskeleton The impact of fluorescence microscopy. Ann. Rev. Plant Physiol. 38, 119-139. 

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. 

Fluorescence microscopy measurements were performed with a Zeiss Axioplan microscope (Zeiss Co. Germany) equipped with a mercury lamp and a 40X objective. Images were acquired by CCD camera CH250 (Photometrix Co., Germany) cooled at -40°C by a liquid cooling unit CH260 (Photometrix Co., Germany). 

It had been found that if bacteria are stained with acridine orange and examined under fluorescent microscopy, viable, as dishnct from dead, cells fluoresce with an orange-led hue. This basic observation has been adapted to an ingenious method of determining bacterial content and may be completed within 1 hour. 

W. W. (1990) Two-photon laser scanning fluorescence microscopy. Science, 248, 73-76. 

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