Fluorescent converters

Klimant I., Huber C., Liebsch G., Neurauter G., Stangelmayer A., Wolfbeis O.S., Dual Lifetime Referencing (DLR) - a New Scheme for Converting Fluorescence Intensity... 

Klimant I, Huber C, Liebsch G, Neurauter G, Stangelmayer A, Wolfbeis OS (2001) Dual lifetime referencing (DLR) - a new scheme for converting fluorescence intensity into a frequency-domain or time-domain information. In Valeur B, Brochon JC (eds) Springer Ser Fluoresc 1 257-274... 

Absolute values for transporter abundance (Bmax) have been obtained with this assay by using fluorescent calibration beads to convert fluorescence signals to MESF (Wiley et al., 1994), or by calibrating the assay with a standard cell population in which Bma was determined by radioligand binding (Gati et al., 1997). Figure 13-4 illustrates an extension of the SAENTA-fiuorescein assay in which the correlation of es transporter content with the expression of immunophenotypic markers was tested in a clinical sample. 

FIGURE 3.4 Performance of the fluorescence up-conversion microscope, (a) Evaluation of the time-resolution with the 100 x objective lens , up-converted fluorescence -F-, the first derivative. By the fitting analysis, the time-resolution of the microscope was evaluated as 520 fs. (b) Evaluation of the transverse (XY) spatial resolution with the 100 x objective lens. A CCD image of the excitation pulses (inset) and the beam profile along the lateral (X) direction. By the fitting analysis, the transverse resolution was evaluated as 0.34 pm. (c d) Evaluation of the axial (Z) spatial resolution with the 100 x objective lens , up-converted fluorescence -I-, the first derivative. By fitting analysis on the first derivative coefficient, the axial resolution was evaluated as 1.1 pm with the 50 pm pinhole (c) and 5.3 pm without pinhole (d). (Rhodamine B, 2 x 10" mol dm in methanol, 600 nm.) (Erom Eujino, T. and Tahara, T., Appl Phys. B 79 145-151, 2004. Used with permission.)... 

Two-photon excitation can be used for the fluorescence up-conversion microscope, and high axial resolution was achieved without a pinhole in this case. Figure 3.5 shows the up-converted fluorescence from a coumarin 522B solution at a fluorescence wavelength of 520 nm observed in the same manner of Figure 3.4d without pinhole. In this measurement, a fundamental laser pulse at 800 nm was used for excitation. The axial resolution with two-photon excitation was evaluated to be 0.97 pm (FWHM) by fitting for the first derivative of the obtained data. This result indicates... 

The flnorescence dynamics imaging was carried ont by monitoring time-resolved fluorescence at 530 nm, where the rise of the tetracene fluorescence is observed, at different positions of the microcrystal. The np-converted fluorescence data were recorded every 1 pm distance. To shorten the measnrement time at each point, the up-converted signal was sampled at only fonr delay time points (- 5, 5,15, 30 ps), and then the energy transfer time, x, was evalnated by htting with the following single-exponential fnnction ... 

Fig. 28 a Single-photon fluorescence readout of data recorded by single-photon writing scale bar 100 ixm) b intensity profile (the direction is shown hy the arrows) of (a) c two-photon fluorescence readout of data recorded by single-photon writing (scale bar 100 xm) d intensity profile (the direction is shown by the arrows) of (c) e quadratic dependence of up-converted fluorescence of fluorene 17 on the input intensity. The smallest readout pattern achieved in this system was 3.5 xm... 

Fig. 30 Two-photon fluorescence image of storage medium before (a) and after (b) two-photon recording, c DIG readout of the storage data after two-photon recording, and d input intensity dependent up-converted fluorescence of fluorene 17...

The paper is organized in the following way. First, a brie introduction to fluorescence upconversion and other non-linear processes is given. This is followed by a description of an apparatus for making up converted fluorescence measurements, and experimental examples for this instrument. The following text describes the advantages of optical multichannel detections for... 

Optical detectors in the form of a photomultiplier tube or CCD to convert fluorescence intensity into electronic pulses that can be manipulated by computers. 

The recent work at the Polymer Branch at US Air Force Wright Laboratory and that at Photonics Research Laboratory at SUNY, Buffalo have produced organic structures that exhibit strong two- and three-photon absorption and exhibit strong up-converted fluorescence. Furthermore, we have shown that with the same pump source for multiphoton excitation, one can excite a number of different fluorophores to generate multicolors to conduct multiphoton multicolor microscopy for the study of microstructures of multilayer coatings, their interfaces and bulk as a function of depth. 

The evaluation of TPA activity of the materials can be made by various techniques such as up-converted fluorescence emission, nonlinear transmission, transient absorption, Z-scan, and four-wave mixing [122]. Among them, the up-converted fluorescence emission method is a simpler technique and the setup is shown in Fig. 49.10. In this method, the TPA cross-section value, 0-2 can be estimated according to the following equation [123] ... 

FIGURE 49.10. Schematics of experimental arrangement for measuring <72 values by the up-converted fluorescence emission method. 

The first step in interpreting the image data is image processing. Image processing is required to both remove any non-uniformities present in the imaging system, and to convert fluorescence or absorbance levels to concentration. 

The time-resolved emission experiment was performed with the fluorescence up-conversion technique. (For details see Ref. 11). Excitation parameters were Xexc = 865 nm (Rb. sphaeroides), tp 200 fs, less than 10 % of the RC absorb a photon. Up-conversion process collinear type II phase matching in a 1mm BBO crystal up-converted fluorescence emission 910-930 nm width of the instrumental response function cr 400 fs. 

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