Time-resolved imaging

Marriott, G., Clegg, R. M., Arndt-Jovin, D. J. and Jovin, T. M. (1991). Time resolved imaging microscopy. Phosphorescence and delayed fluorescence imaging. Biophys. J. 60, 1374—87. 

Conventional TCSPC equipment has been successfully employed in LSM for fluorescence spectroscopy on discrete microscopic volumes [18, 19] and for fluorescence lifetime imaging at a low acquisition speed [1], The use of conventional TCSPC equipment for imaging results in very long acquisition times, several to many minutes per (time-resolved) image. Importantly, operating the TCSPC detection system at too high detection rates, above 5% of the excitation frequency, results in distortion of the recorded decay curve [20],... 

Steinkamp [91] for the Luminex microspheres. Recently, Mayr et al. stained magnetic microspheres with luminescent ruthenium(II) metal ligand complexes [92] which were encoded by time resolved imaging in the microsecond domain. 

S. Charbonneau, L. B. Allard, J. F. Young, G. Dyck, and B. J. Kyle, Two-dimensional time resolved imaging with 100-ps resolution using a resistive anode photomultiplier tube, Rev. Sci. Instrum. 63(11), 5315-5319 (1992). 

The operation and application of streak cameras in fluorescence lifetime spectroscopy has been reviewed previously (see, e.g., Refs. 91 and 92). Streak cameras are useful in 2-D time-resolved imaging applications such as microscopy or multiwavelength array fluorometry. The operating principle is based on converting an optical pulse into a photoelectron pulse and spatially dispersing the electron image on a phosphor by means of a synchronized deflection voltage across two plates. 

Fujino, T, Fujima, T, and Tahara, T. 2005. Picosecond Time-Resolved Imaging by Nonscaiming Fluorescence Kerr Gate Microscope. Appl. Phys. Lett. 87 131105. 

Table 1 Luminescence properties of oxygen-sensitive probes applicable to time-resolved imaging...

Fig. 7 Set-up for time-resolved imaging of 96-microwell plate formats, a Fast gateable CCD-camera, b Optical emission filter, c Light-guiding adapter, consisting of 96 optical fibers, d 96-microwell plate (black with transparent bottom), e Optical excitation filter, f Pulseable LED array with 96 light emitting diodes...

The combination of different fluorescent metal indicators with inert luminescent reference beads consisting of poly(acrylonitrile) containing Ru(dpp)3 leads to a sensor array in a microwell plate format, suited for ratiomet-ric time-resolved imaging [95]. The data can be acquired with the help of the f-DLR method (for details see Sect. 2.3). A cross-reactive sensor array was arranged for the determination of mixtures of calcium(II), copper(II), nickel(II), cadmium(II), and zinc(II) ions by nine different commercially available fluorescent indicators (Table 3). For a successful application, it is mandatory that all luminophores can be excited at the same wavelength range between 400 and 500 nm, and that the excitation and emission spectra of all indicators overlap with those of the reference dye encapsulated in the nanobeads. 

Fig. 17 Decay profiles of [Eu(Tc)] and the complexes with d- and L-malate (left). Time-resolved imaging of enantiomer malates with [Eu(Tc)] (50 xM), [L-malate] + [D-malate] = 500 xM, time gate 140-220 xs (right) [108]...

It should be noted that this scheme does not require the presence of enzymes (or even multi-enzyme systems). Furthermore, chiral discrimination can also be accomplished by fluorescence imaging with help of the [Eu(Tc)] probe. As shown in Fig. 17, the enantiomeric excess (ee) of l- or D-malates can be screened by means of time-resolved imaging with the help of microwell plates [108]. 

The [Eu(Tc)] assay for H202 can also be used for the determination of enzyme activities. This comprises all enzymes that produce (oxidases) or consume (catalase, peroxidases) H2O2. For example, the activity of glucose oxidase can be directly imaged after the addition of [Eu(Tc)] as indicator and glucose as substrate. Steady-state and time-resolved imaging schemes can be applied for the read-out of a microwell plate-based assay. The best results were obtained by the RLI method (Fig. 19) [115]. 

Fig.26 Pseudo-color time-resolved image of polystyrene nanobeads containing Pt porphyrin and conjugated to streptavidin immobilized to a biotinylated microarray surface (black teflon coated 96-well glass slide, spot diameter 1 mm, Erie Scientific) in different concentrations (25, 15, 10, 5, Ong streptavidin per well) [167]...

New europium probes have been presented that can indicate changes in hydrogen peroxide concentration and are applicable for time-resolved imaging. As H2O2 is a product of the activity of almost all oxidases, quantitative assays for different substrates, e.g glucose, or the determination of the activity of the respective oxidases can be carried out. On the other hand, peroxidases and catalases consume H2O2, which can also be monitored via the europium tetracycline probe and can therefore be used for the imaging of ELIS As performed in microwell plates. 

Metal complexes like lanthanide chelates (mainly europium or terbium), ruthenium phenanthrolines or bipyridyls, and platinum porphyrins can be used as fluorescent labels for biomolecules. Their long decay times are perfectly suited for a detection by time-resolved imaging, and the labeled target molecules can be used for the determination of intracellular recognition processes or for the screening of DNA and protein arrays. Ratiometric lifetime-based imaging methods in combination with sophisticated data acquisition and evaluation tools can substantially contribute to the development... 

Figure 1.6 (a) In a time-resolved imaging configuration, the interferogram is obtained by observing the... 

M 4] [P 3] Using the improved EKI mixer device, mixing times of about 2.5 s are obtained [25]. This was deduced from time-resolved images showing the point when a randomly distribution of a fluorescence tracer is achieved (see also Flow perturbation upon alternating (AC) current operation above for the lower performance of the first-generation device). 

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