Methodologies fluorescent imaging

In this section, based on the methodology presented in the previous section, we describe multidimensional fluorescence imaging and its application to tracking cell responses. We developed the time- and spectrally-resolved fluorescence imaging system based on line illumination, which is capable of rapid acquisition of fluorescence intensities as a function of Em, x, and xy-positions. We applied it to the analysis of an induced plant defense response, that is, the accumulation of antimicrobial compounds or phytoalexins, in oat (Avena sativa). 

In addition, the methodology was applied to fluorescence imaging based on the autofluorescence signals of native molecules in cells or tissues. The imaging system is capable of the rapid acquisition of fluorescence intensities as a function of Em, t, and xy-positions, which is achieved by line illumination of the excitation laser beam. We applied this system to the analysis of a plant defense response, accumulation of phytoalexin in oat leaves, induced by elicitor treatment. In oat leaves treated with an elicitor, we successfully observed weakly fluorescent components, one of which possibly originated from avenanthramide A as a phytoalexin, in addition to the strong fluorescence from chlorophyll molecules. 

Figure 10.29 Fluorescent image of five 200 nm wide, 55 nm high template channels with polyaniline grown chemically. (Reprinted with permission from Nano Letters, A "Grow-in-Place" Architecture and Methodology for Electrochemical Sythesis of Conducting Polymer Nanoribbon Device Arrays by C.-Y. Peng et al., 5, 3. Copyright (2005) American Chemical Society)...

The following example presents the methodology to determinate chlorpyrifos based on hyperspectral fluorescence imaging technology. 

The first lecture given by Tomas Hokfelt, who pioneered anatomical studies based on amine fluorescence, was entitled, Neuroanatomy for the Neurochemist while the second lecture given by Louis Sokoloff, who invented the methodology of functional brain imaging originally based on metabolism of radionuclide labeled-2-deoxyglucose, was entitled Neurochemistry for the Neuroanatomist . The history and further information... 

Starting with the first IPCR study, gel electrophoresis retains its potential as a fast and easy method for end-point determination of DNA amplificate for IPCR assays [10, 24, 25, 29, 31, 35, 36, 38, 39, 64], Readout is performed by intercalation fluorescence markers (e.g., ethidium bromide) and photometric/densitometric quantification of band signal intensities. The direct addition of a double-strand specific intercalation marker to the PCR amplificate and subsequent measurement of fluorescence in microwells proved to be of insufficient sensitivity for the quantification of IPCR amplificate [37]. Alternative approaches, such as radioactive labeling during PCR and subsequent imaging [33], were carried out but are not well suited for routine clinical application because of additional methodological requirements. An advantage of gel electrophoresis is the possibility of simultaneous amplificate detection for multiplex IPCR [41] and the ability to detect nonspecific amplification products. 

As methodologies in FISH and mFISH on the fluorescent microscope evolve, so must the software and hardware used to unravel the information contained in the specimen. A proper combination of filters, dyes, imaging hardware, and software is desirous for obtaining the resolution and contrast necessary for accurate image capture and analysis. 

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