Imaging spectral

Confocal microscopy already allows the mapping of slices of a sample by spectral imaging at different depths. By combining traditional spectroscopy with microscopic and macroscopic imaging capabilities, complex heterogeneous samples can be analysed. In summary, the technique allows both the physical characteristics e.g. particle size and distribution, and chemical characteristics, e.g. the structural properties, of a sample to be investigated simultaneously. 

The spectral ranges for these instruments vary from close to Onm up to ISOOOnm. Bulk images or specihc pixel-based images can be obtained by zooming in on a certain region of the sample. The spectroscopy can be used to detect individual absorption or emission features due to specihc components. 

This type of spectral imaging has most of its applications in space exploration - for studying solar radiation, comets and planet atmospheres. 

Based on infrared array detector technology, NIR chemical imaging techniques are the most popular in the market. Spatial, chemical, structural and functional information can 

Raman microscopy can be applied to many areas of scientific analysis, such as surface and materials science, forensic research and industry. The technique is noninvasive and 

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Snively, C.M. and Lauterbach, J. (2002) Sampling accessories for the high-throughput analysis of combinatorial libraries using spectral imaging. Spectroscopy, 17, 26. 

The fourth chapter by James McGuinty et al. describes the more advanced forms of time-domain FLIM. While not immediately available on commercial instruments this chapter should give the reader an idea what the current state-of-the-art is in terms of FLIM instrumentation, and perhaps what to expect on future commercial instruments. Real-time FLIM, combined FLIM-spectral imaging, hyperspectral FLIM-imaging, combined lifetime-anisotropy imaging and some of their applications are covered here. 

Chapter 8 written by Steve Vogel et al. also deals with sensitized emission based FRET methodology, but now using a spectral imaging detector device. Because a spectral detector and spectral unmixing software nowadays are standard options on the major commercial confocal microscopes, here a complete description is given how to quantify FRET from unmixed spectral components. 

Hanley, Q. S., Verveer, P. J. and Jovin, T. M. (1999). Spectral imaging in a programmable array microscope by hadamard transform fluorescence spectroscopy. Appl. Spectrosc. 53, 1-10. 

Zimmermann, T., Rietdorf, J., Girod, A., Georget, V. and Pepperkok, R. (2002). Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2-YFP FRET pair. FEBS Lett. 531, 245-9. 

Spectral imaging and its use in the measurement of Forster resonance energy transfer in living cells... 

As mentioned above, spectral imaging microscopy is a form of multidimensional fluorescent microscopy where a fluorescent emission spectrum is acquired at each coordinate location in the sample. This mode of imaging has been implemented for wide field, confocal, and two-photon laser scanning microscopy, and several excellent... 

Standard linear unmixing of a spectral image of a sample composed of two fluorophores yields a measure of the concentration of each fluorophore present for each pixel. If FRET is occurring, linear unmixing will produce an apparent donor concentration ( apparent) that underestimates the true donor concentration (d) by a factor of 1 -ED ... 

Linear unmixing is applied to each spectral image to produce four measurements at each pixel, Apparent an[Pg.384]

Strategies for measuring FRET using spectral imaging... 

One criticism of this approach, however, is that in addition to requiring the specialized hardware for obtaining spectral images, additional instrumentation is often required to measure the FRET efficiency. Furthermore, the limitations specific to the FRET method used in conjunction with spectral imaging will also apply to this hybrid approach. 

Linear unmixing of the spectral image acquired at will also yield two observables ... 

Measuring FRET from spectral images sRET... 

Many of the strategies for measuring FRET from spectral images that were mentioned above have been implemented to study FRET. We will now cover sRET [12], a specific implementation that uses the last approach where FRET is measured from a pair of spectral images collected at different excitation wavelengths. Recently, the sRET approach has been extended to explicitly consider paired and unpaired fluorophores, the impact of incomplete labeling (or for fluorescent proteins fractional maturation), and the... 

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