Epi-fluorescence configuration

Recently, manufacturers have designed objectives with even higher numerical apertures that would be ideal for TIR experiments with high refractive index samples (see Section 3.6). However, these objectives require special immersion fluid and coverslip materials, which may have implications for other aspects of the experiments such as surface immobilization chemistry and cost. In essence the TIR configuration is identical to the epi-fluorescence configuration discussed earlier, with the exception of the excitation beam convergence and positioning with respect to the optical axis of the objective. 

TIRF is easy to set up on a conventional upright or inverted microscope with a laser light source or, in a special configuration, with a conventional arc source. TIRF is completely compatible with standard epi-fluorescence, bright-field, dark-field, or phase contrast illumination so that these methods of illumination can be switched back and forth readily. Some practical optical arrangements for observing TIRF through a microscope are described in Section 7.4. 

A schematic of the imaging configuration used for the measurement of temperature via singledye fluorescence is presented in Fig. 2. Light from an illumination source, like an Nd YAG laser, argon-ion laser, or a mercury-arc lamp, is directed through an epi-fluorescent Alter cube and through a microscope objective lens to excite the fluorescent dye molecules in the microfluidic... 

The laser beam can be directed along one of several optical paths available in either the upright or inverted microscope configurations. For fluorescence applications, it is often convenient to make use of the standard epi-illumination... 

A second fluorescent Chl-catabolite, Ca-FCC-2, was isolated from another in vitro system, based on enzymatic activity obtained from ripe (red) sweet pepper (Capsicum annuurri) and its structure was analyzed (67). The new fluorescent catabolite could be shown by mass spectrometry to be an isomer of 10 Further NMR-spectroscopic analysis revealed Ca-FCC-2 to have the same constitution and to differ from pFCC (10) only in the absolute configuration at C(l). Ca-FCC-2 was thus assigned as the epimeric primary I -epz-pFCC (epi-10) (67). 

The red chlorophyll catabolite RCC (11) is bound strongly to PaO and inhibits it. In an in vitro assay, the soluble reductase from oilseed rape converted 11 to the primary fluorescent chlorophyll catabolite pFCC (10, 31,32-didchydro-1,4,5,10,17,18,20-(22//)-octahydro-132-(mcthoxy-carbonyl)-4,5-dioxo-4,5-seco-phytoporphyrin) (62, 83). The reductase, which was named red chlorophyll catabolite reductase (RCC-reductase) (68, 80, 83), introduced the chiral center C(l) via a stereo-selective reduction step. However, early studies with oilseed rape and sweet pepper indicated a remarkable stereo-dichotomy of the respective reductases (see above) (67, 68, 69). Screening of a variety of plant species for their type of primary FCC revealed the broad existence of two classes of the RCC-reductases , whose stereo-selectivity was species specific (84). At present, the (absolute or relative) configuration at C(l) in the two pFCCs (10 and epi-10) is not yet established (2). Indeed, the existence of the two epimeric pFCCs (10 and epi-10) (see Scheme 6) indicated the absolute configuration at the newly generated chiral center to have no apparent functional relevance (67, 68, 69). 

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