Fluorescence microscopy, presence

Fluorescent labels, by contrast, can provide tremendous sensitivity due to their property of discrete emission of light upon excitation. Proteins, nucleic acids, and other molecules can be labeled with fluorescent probes to provide highly receptive reagents for numerous in vitro assay procedures. For instance, fluorescently tagged antibodies can be used to probe cells and tissues for the presence of particular antigens, and then detected through the use of fluorescence microscopy techniques. Since each probe has its own fluorescence emission character, more... 

For the design of mitochondriotropic liposomes, we have used a method, that has been a standard procedure in liposome technology for over 30 years the lipid-mediated anchoring of artificially hydrophobized water-soluble molecules into liposomal membranes (25-28). We have hydrophobized mitochondriotropic TPP cations by conjugating them to long alkyl residues specifically, we have synthesized stearyl TPP (STPP) salts (29). Following liposome preparation in the presence of STPP, the liposomal surface became covalently modified with TPP cations, thereby rendering these liposomes mitochondriotropic as verified in vitro by fluorescence microscopy (30). 

In this chapter we explore several aspects of interferometric nonlinear microscopy. Our discussion is limited to methods that employ narrowband laser excitation i.e., interferences in the spectral domain are beyond the scope of this chapter. Phase-controlled spectral interferometry has been used extensively in broadband CARS microspectroscopy (Cui et al. 2006 Dudovich et al. 2002 Kee et al. 2006 Lim et al. 2005 Marks and Boppart 2004 Oron et al. 2003 Vacano et al. 2006), in addition to several applications in SHG (Tang et al. 2006) and two-photon excited fluorescence microscopy (Ando et al. 2002 Chuntonov et al. 2008 Dudovich et al. 2001 Tang et al. 2006). Here, we focus on interferences in the temporal and spatial domains for the purpose of generating new contrast mechanisms in the nonlinear imaging microscope. Special emphasis is given to the CARS technique, because it is sensitive to the phase response of the sample caused by the presence of spectroscopic resonances. 

Figure 12.5 Nuclear import in permeabilized cells. HeLa cells were grown on coverslips and permeabilized with digitonin as described in Wilson et al., 1999. Fluorescein-PNA-labeled plasmids (containing the SV40 enhancer, 4.2 kb) or rhodamine-labeled BSA-NLS peptide conjugates were incubated with the cells for four hours at which time they were viewed by fluorescence microscopy. With no additions, neither DNA nor protein was imported, but in the presence of nuclear and cytoplasmic extracts both substrates localized to the nuclei. While plasmids containing the SV40 enhancer were taken up by the nuclei, those lacking the sequence were excluded. The remaining panels demonstrate the need for both the import machinery (importins and Ran) and a source of adapter proteins (nuclear extract) for plasmid nuclear entry, but not for protein nuclear localization.

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