Fluorescence spectral imaging

Fluorescence spectral imaging microscopy (FSPIM)—a method that uses a spectroscopic approach to quantify changes in the acceptor intensity at donor excitation wavelengths [24, 27, 51, 52] (see Chapter 8) ... 

We have shown an application of fluorescence spectral imaging for the detailed study of thylakoid membranes in three types of organisms with oxygenic photosynthesis. 

We have not addressed the problem of analyzing the spectral image and transforming it into the chemical image. We only note that many of the algorithms we have used in the past (10-12,20,30) for convoluting fluorescence spectral images can be readily and efficaciously applied to this problem. 

Recently, new instruments have been developed, in combination of confocal microscope with atomic force microscope (AFM), enabling topographic and fluorescence spectral imaging simultaneously. The unique combination of these two microscopes not only offers the best resolution at x-axis, j/-axis (confocal), and z-axis (AFM), but also provides the ability to study the topographic distribution of certain chemical components, or proteins on the biological surface (see next section for more details). 

Hanley, Q. S., Arndt-Jovin, D. J. and Jovin, T. M. (2002). Spectrally resolved fluorescence lifetime imaging microscopy. Appl. Spectrosc. 56,155-66. 

Hanley, Q. S. and Ramkumar, V. (2005). An internal standardization procedure for spectrally resolved fluorescence lifetime imaging. Appl. Spectrosc. 59, 261-6. 

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... 

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... 

Multi-spectral imaging and linear unmixing add a whole new dimension to laser scanning fluorescence microscopy. Biotechniques 31, 1272-8. 

Spectral imaging fluorescence microscopy. Genes Cells 7, 881-7. 

Ecker, R. C., de Martin, R., Steiner, G. E. and Schmid, J. A. (2004). Application of spectral imaging microscopy in cytomics and fluorescence resonance energy transfer (FRET) analysis. Cytometry A 59, 172-81. 

M-5 (Quin-2) was the first practical fluorescent indicator for cytosolic calcium with a simple 6-methoxyquinoline as its fluorophore. Ca2+-binding increases the fluorescence intensity about six-fold (without spectral displacement, in contrast to Fura 2 see Section 10.3.3). The fluorescence lifetime of Quin-2 is highly sensitive to calcium concentration Quin-2 can thus be used as a probe in the technique of fluorescence lifetime imaging. 

Tinnefeld, R, Herten, D.P., and Sauer, M. 2001. Photophysical dynamics of single molecules studied by spectrally-resolved fluorescence hfetime imaging microscopy (SFLIM). J. Phys. Chem. A 105 7989. 

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