Epifluorescence Methods

In the wide-field epifluorescence method, a CCD detects fluorescence excited by a laser and scattered back from the sample (Fig. 12.47a). If the fluorescing molecules are well separated in the specimen, then it is possible to obtain a map of the distribution of fluorescent molecules in the illuminated area. For example. Fig. 12.47b shows how epifluorescence microscopy can be used... 

In addition to these time-honoured methods, newer techniques involving bio-luminescense, fluorescent dyes (epifluorescence) and physical methods such as impedance, calorimetry and flow cytometry have been developed. A feature being sought in these methods is rapidity see section 5.6. 

The technique of fluorescence spectral measurements has become very sensitive over the past decade. In order to obtain more information on the surface monolayers, a new method based on fluorescence was developed. It consisted of placing the monolayer trough on the stage of an epifluorescence microscope, with doped low concentration of fluorescent lipid probe. Later, ordered solid-liquid coexistence at the water-air interface and on solid substrates were reported. The theory of domain shapes has been extensively described by this method. 

All the imaging modes of light microscopy are amenable to handsectioning methods but none more so than epifluorescence. This is shown clearly by the work of Fulcher and later by that of Yiu [26]. A range of products is examined in these papers, from cereal seeds to cheese and yet the resolution obtained approaches that of sections from embedded product. 

Two pools of organic matter were prepared from exponentially growing LFe and HFe P. antarctica (i) dissolved organic matter (DOM) obtained by gentle filtration (<0.3 atm) on 0.1 pm polycarbonate Nucle-pore filters, (ii) total organic matter (TOM), unfiltered, including DOM and POM obtained after phytoplankton lysis by sonication (10 min of sonica-tion at 22 W with pulse of 0.2 s/s) and ffeeze/thaw ( 80°C deep freezer/60°C water bath cycles, repeated three times) methods. The broken cells were observed by epifluorescence microscopy, evidencing the efficiency of this method. 

Fig. 6.10 Distributions of bacterial abundance at the sea surface during various seasons. Two different methods - epifluorescence microscopy (upper panels) and flow cytometry (lower panels) - were used to enumerate bacteria. Redrawn from Ducklow etal. (2001) with permission from Elsevier Science.

Answers to the questions should point to a label type and a method of application for the antibodies. For high-resolution samples, generally the needed label is fluorescence. For lower-resolution samples, both fluorescence and enzymes can be used. However, before deciding on a label type, determine whether the correct microscope is available. A wide field fluorescence (epifluorescence) microscope or a confocal microscope is required for fluorescent type of label. Also, check the fluorescence microscope to determine whether it has the correct filter sets (Chapter 13, Microscopy and Images). 

Direct counting can be improved by the use of fluorescent dyes, such as acridine, especially if combined with the recovery of cells by membrane filtration. Direct epifluorescent filter techniques (DEFT) are used in the milk and dairy industries to estimate both bacteria and fungi. They can produce results in less than 25 minutes which correlate closely with traditional methods. Further developments have automated the counting procedure by the use of image analysers thus removing the problem of operator fatigue. 

Recent decades have witnessed spectacular developments in in-situ diffraction and spectroscopic methods in electrochemistry. The synchrotron-based X-ray diffraction technique unraveled the structure of the electrode surface and the structure of adsorbed layers with unprecedented precision. In-situ IR spectroscopy became a powerfiil tool to study the orientation and conformation of adsorbed ions and molecules, to identify products and intermediates of electrode processes, and to investigate the kinetics of fast electrode reactions. UV-visible reflectance spectroscopy and epifluorescence measurements have provided a mass of new molecular-level information about thin organic films at electrode surfaces. Finally, new non-hnear spectroscopies such as second harmonics generation, sum frequency generation, and surface-enhanced Raman spectroscopy introduced unique surface specificity to electrochemical studies. 

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