Time- and Spectrally-Resolved Fluorescence Imaging

Figure 32.2 Experimental set-up for time- and spectrally-resolved fluorescence imaging based on the line illumination.

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). 

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. 

LSM 510 META software to process time-lapse fluorescence images, and spectrally resolved images of Lambda Stack using the Linear Unmixing Function of LSM510 META. 

Basic principles and applications of time-resolved fluorescence spectroscopy have been outlined in a very illustrative way by Valeur [16]. Although punctiform spectroscopy is still the best way to get a detailed knowledge of all the important parameters that characterize fluorescence emission (exact spectral properties, decay time behavior, polarization), imaging is always preferred whenever the localization of the distribution of any biomolecule of interest is required or a great number of samples have to be analyzed [22]. 

To make multispectral time-resolved fluorescence measurements into a diagnostic tool, the technique must be combined with imaging. Direct imaging techniques by position-sensitive TCSPC are optically easy. They can be used with wide-field illumination and are easily integrated into endoscopes. However, it is difficult to obtain simultaneous spectral and temporal resolution. Several detectors must be used or several images of different wavelength be projected on one detector. True multispectral resolution cannot be obtained this way. 

Figure 7.5 Ultrafast spectrally resolved dynamics of two CdS tSei x nanobelts. The fluorescence image is shown in the upper left corner. Note that the broad, time-integrated photoluminescence spectrum shown on the left consists of two distinct features centered at 628 and 636 nm with vastly different dynamics that is clearly discernible in the 2D map.

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