Imaging multidimensional fluorescence

Multidimensional Fluorescence Imaging for Non-Invasive Tracking of Cell Responses 623... 

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... 

We developed two kinds of multidimensional fluorescence spectroscopic systems the time-gated excitation-emission matrix spectroscopic system and the time- and spectrally resolved fluorescence microscopic system. The former acquires the fluorescence intensities as a function of excitation wavelength (Ex), emission wavelength (Em), and delay time (x) after impulsive photoexcitation, while the latter acquires the fluorescence intensities as a function of Em, x, and spatial localization (%-, y-positions). In both methods, efficient acquisition of a whole data set is achieved based on line illumination by the laser beam and detection of the fluorescence image by a 2D image sensor, that is, a charge-coupled device (CCD) camera. 

Multidimensional Fluorescence Imaging for Non-lnvasive Tracking of Ceil Responses... 

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). 

Duncan, R.R., Bergmann, A., Cousin, M.A., Apps, D.K., and Shipston, M.J. et al. 2004. Multidimensional time correllated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (Aim) to Detect Fret in Cells. J. Microsc. 215 1. 

Webb SED, Needham SR, Roberts SK et al (2006) Multidimensional single-molecule imaging in live cells using total-intemal-reflection fluorescence microscopy. Opt Lett 31 2157-2159... 

It is commonly known that the proximity of the SNOM tip changes the fluorescence lifetime in the seanned point of the sample. Whether this effect makes lifetime imaging in a SNOM useless or particularly interesting is hard to say as tong as only a few results exist. However, multidimensional TCSPC may be one way to make use of the dependenee of the lifetime on the tip distance. At a t)q)ical vibration frequency of the tip of a few hundred kHz, the photons for different tip dis-tanee eould be routed into different memory blocks. The result would be several images for different tip distance. 

W. Becker, A. Bergmann, E. Haustein, Z. Petrasek, P. Schwille, C. Biskup, T. Anhut, I. Riemann, K. Koenig, Fluorescence lifetime images and correlation spectra obtained by multidimensional TCSPC, Proc. SPIE 5,700 (2005)... 

In biological imaging, confocal laser scanning microscopy (CLSM) has in the last decade significantly extended our ability to visualize highly complex samples as multidimensional datasets (space, time, colors). In parallel, the introduction of fluorescent protein variants as in vivo tags of structures of interest has opened up new ways to observe cellular processes inside the living cell or tissue (for review see Miyawaki et al., this issue). 

Claybom, M., Luget, A., and Williams, K.P. (. (1995) Raman Microscopy and Imaging of Polymers, in Multidimensional Spectroscopy of Polymers, Vibrational, NMR and Fluorescence Techniques (eds M.W. Urban and T. Provder), American Chemical Society, Washington, DC, Ch. 3. 

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