Fluorescence confocal

B1.18.5.7 THE FUTURE RESOLUTION BEYOND THE DIFFRACTION LIMIT IN CONFOCAL FLUORESCENCE... 

Schrof W, Klingler J, Heckmann W and Horn D 1998 Confocal fluorescence and Raman microscopy in industrial research Colloid Polym. Sc/. 276 577-88... 

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... 

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...

Fries J R, Brand L, Eggeling C, Kdllner M and Seidel CAM 1998 Quantitative identification of different single molecules by selective time-resolved confocal fluorescence spectroscopy J. Phys. Chem. A 102 6602-13... 

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... 

Kim, H.-B., Hayashi, M., Nakatani, K., Kitamura, N., Sasaki, K., Hotta, J.-I., and Masuhara, H., In situ measurements of ion exchange processes in single polymer particles laser trapping microspectroscopy and confocal fluorescence microspectroscopy, Anal. Chem., 68, 409, 1996. 

Monolithic columns, formed from the co-polymerization of divinylbenzene and vinylbenzyl chloride or styrene, were observed to be resistant to bubble formation.11 Application of pressure in electrochromatography, discussed below, also reduces bubble formation. A massively parallel detector capable of scanning up to 1000 capillaries using planar confocal fluorescence has been used for DNA sequencing.1213 Recovery of fluorescence following pho-tobleaching has been used to measure DNA mobility in agarose gel.14... 

In this chapter, it was shown that filterFRET is an easy, intuitive and quantitative alternative to record sensitized emission and FRET efficiency. The major advantages of filterFRET over donor-based FRET detection methods (FLIM) are that it can be carried out with standard wide-held or confocal fluorescence microscopes that are available in most laboratories, and that it yields additional data on the acceptor population. FilterFRET is also fast, requiring just two confocal scans (if need be on a line-by-line basis) which minimizes the risk of artifacts due to, for example, organelle movement in living cells, and acquisition can be optimized for each channel independently. However, quantitative... 

Sanders, R., Draaijer, A., Gerritsen, H. C., Houpt, P. M. and Levine, Y. K. (1995). Quantitative pH imaging in cells using confocal fluorescence lifetime imaging microscopy. Anal. Biochem. 227, 302-8. 

On the intergrowth structure of zeolite crystals as revealed by wide field and confocal fluorescence microscopy of the template removal processes... 

Keywords zeolites fluorescence confocal fluorescence microscopy template removal intergrowth... 

Figure 2. a) Wide field fluorescence microphotographs of a single CrAPO-5 crystal taken during heating b,c) confocal fluorescence images (top and side respectively) of the CrAPO-5 crystal at 660 K. Detection channel of 575-635 nm are represented in gray scale. 

In order to illustrate the general applicability of the methodology we have extended our approach to other large zeolite crystals, such as SAPO-34, SAPO-5 and ZSM-5. Our study on the rhombic SAPO-34 crystals reveals a four-pointed star fluorescence pattern at 445 K, which is transformed into a square-shaped feature at 550 K. This is illustrated in Figure 4a. Confocal fluorescence slices, summarized in Figures 4b-d, recorded at different temperatures show the cubical pattern, which proceed from the exterior of the crystal inwards. Both observations are consistent with a model which involves six components of equal tetragonal pyramids as illustrated in Figure 3b. 

Figure 5. a) Fluorescence microphotographs of ZSM-5 crystals taken during template removal and b) confocal fluorescence images taken at 700 K (561-nm laser, detection at 575-635 nm, intensity presented as a gray scale). 

Fig. 12 CNTs act as a vector for drug delivery into living cells. After incubation of HeLa cells with AlexaFluor594-labeled SWNTs for 12 h at 37 °C, living cells were observed under confocal fluorescence microscope for a CNT uptake study, (a) Images show dual confocal detection of AlexaFluor594-SWNT (red) internalized into cells with the membrane stained by AlexaFluor488 (green), (b) Series of images of different z-focal scanning planes down through cells. (Adapted from [61])...

Fig. 17. Confocal fluorescence imaging of [Zn(ATSM)] in IGROV cells (100 pM, where Q1 — Q2 = Me, M = Zn(II), R1 = R3 = H and R2 = R4 = Me, /ex = 488 nm, DMEM with 1% DMSO). Brightfield image shows formation of needle-like crystalline material on the cell plate (N.B. Small crystallites may be endocytosed by the cells rather than passively diffuse through the cell membrane).

Fig. 19. Typical bright-field and confocal fluorescence images of the glucose-ATSM Zn(II) derivative in cancer cells (/.ex = 360 nm, Aem = 530 nm). Diagrams adapted from Ref. (102).

Fig. 21. Molecular structures of new aromatic [M(ATSM)] analogs (a) M — Zn(II) and (b) M = Cu(II), (c) cytotoxicity tests in MCF-7 cells for the Zn(II) complex (group 2) and Cu(II) complex (group 3) and comparison with control and with cis-platin over a range of concentrations, (d) cell uptake profile monitored over 90 min, (e) confocal fluorescence imaging of Zn(II) complex in MCF-7 cells, at 100 pM cone, in DMEM, 1% DMSO (112,113).

Plant cytochemistry/histochemistry continues to evolve as fluorescence microscopy (16-19), confocal fluorescence microscopy (20,21), and microspectrophotometry (22) expand our quantitative knowledge of the distributions of chemical constituents in plant cells and tissues. With regard to microspectrophotometry, this is possible for single cells, as the Arcturus Corporation (Mountain View, C A) has developed an instrument capable of isolating single cells. 

Shuming N, Chiu DT. Probing molecules with confocal fluorescence microscopy. Science 1994 266 1018. 

Hepler PK, Gunning BES. Confocal fluorescence microscopy of plant cells. Protoplasma 1998 201 121-157. 

Figure 14. Confocal fluorescence images of single molecules of (a) the model compound 69 and (c) the multichromophoric dendri-mer 70 in a polyvinylbutyral (PVB) film. The fluorescence intensity transients (fluorescence intensity versus time) exhibit typical on-off behavior for 69 (b) and jumps between different emissive levels for 70 (d). Pictures taken from ref. [48],...

Fig. 3 (a) Normalized excitation and emission spectra of 5-(and-6)-carboxy-fluorescein, succini-midyl ester, rhodamine 6G (R6G), and 6-carboxy-X-rhodamine dyes in pH 7.4 phosphate buffer, (b) Confocal fluorescence image of a mixture of five types of microsphere-DDSN complexes under 488-nm Argon-ion laser excitation. Reproduced with permission from Ref. [12]... 

Confocal fluorescence microscopy has been extensively used in cell biology. Single living cells can indeed be studied by this technique visualization of organelles, distribution of electrical potential, pH imaging, Ca2+ imaging, etc. (Lemasters, 1996). Interesting applications in chemistry have also been reported in the fields of colloids, liquid crystals and polymer blends. 

Confocal fluorescence microscopy can be combined with time-domain and frequency-domain techniques to produce lifetime imaging (see Section 11.2.2.3). 

As shown in Section 11.2.1.1, more details can be obtained by confocal fluorescence microscopy than by conventional fluorescence microscopy. In principle, the extension of conventional FLIM to confocal FLIM using either time- or frequency-domain methods is possible. However, the time-domain method based on singlephoton timing requires expensive lasers with high repetition rates to acquire an image in a reasonable time, because each pixel requires many photon events to generate a decay curve. In contrast, the frequency-domain method using an inexpensive CW laser coupled with an acoustooptic modulator is well suited to confocal FLIM. 

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