Instrumentation instrument-response profile

Figure 6. Fluorescence decay profiles of trans-7,8-dihydroxy-7,8-dihydro-BP and 8,9,10,11-tetrahydro-BA measured at 23 °C with and without native DNA. Taken from refs. 14 and 15. The upper left-hand corner contains an instrument response profile. Emission and excitation wavelengths, lifetimes, and values of x2 obtained from deconvolution of the lifetime data are also given.

The fluorescence lifetime is obtained from this curve by an iterative curve-fitting program that deconvolutes the lifetime from the instrument-response profile (IRF). This describes the response of the instrument to scattered excitation pulses. A typical... 

There is significant debate about the relative merits of frequency and time domain. In principle, they are related via the Fourier transformation and have been experimentally verified to be equivalent [9], For some applications, frequency domain instrumentation is easier to implement since ultrashort light pulses are not required, nor is deconvolution of the instrument response function, however, signal to noise ratio has recently been shown to be theoretically higher for time domain. The key advantage of time domain is that multiple decay components can, at least in principle, be extracted with ease from the decay profile by fitting with a multiexponential function, using relatively simple mathematical methods. 

All resolution methods mathematically decompose a global instrumental response of mixtures into the contributions linked to each of the pure components in the system [1-10]. This global response is organized into a matrix D containing raw measurements about all of the components present in the data set. Resolution methods allow for the decomposition of the initial mixture data matrix D into the product of two data matrices C and ST, each of them containing the pure response profiles of the n mixture or process components associated with the row and the column directions of the initial data matrix, respectively (see Figure 11.2). In matrix notation, the expression for all resolution methods is ... 

The field of curve resolution was bom in response to the need for a tool to analyze multivariate experimental data from multicomponent dynamic systems. The common goal of all curve-resolution methods is to mathematically decompose the global instrumental response into the pure-component profiles of each of the components in the system. The use of these methods has become a valuable aid for resolving complex systems, especially when obtaining selective signals for individual species is not experimentally possible, too complex, or too time consuming. 

Multivariate curve resolution-alternating least squares (MCR-ALS) uses an alternative approach to iteratively find the matrices of concentration profiles and instrumental responses. In this method, neither the C nor the ST matrix have priority over each other, and both are optimized at each iterative cycle [7, 21, 42], The general operating procedure of MCR-ALS includes the following steps ... 

Figure 9. The time profile for the decay of fluorescence from a solution of anthracene in degassed hexane. The data were obtained with a time-correlated single-photon-counting instrument such as that shown in Figure 8. The upper panel shows the raw data with a superimposed linear fit and the instrument-response function. The extracted lifetime was 5.14 ns. The lower panel shows the residuals. (Courtesy of Dr. F. N. Castellano).

Curve 5 is the laser pulse, detected through ND filters without the bloeking filters. The laser pulse was recorded to get a time reference as to where the fluores-cenee is to be expeeted. Please note that the recorded laser pulse is not the effective profile of the instrument response function (IRF). The two-photon effeet is proportional to the square of the power, therefore the two-photon IRF is different from the one-photon IRF. 

Figures 2 and 3 show how the appearance of the residuals and autocorrelation function for a pulsed excitation experiment typically depend on the appropriateness of the fitting function. In panel A of both figures, L shows an actual excitation pulse profile (more properly, the instrument response function) that was generated by an argon ion laser that pumped a dye laser circulating rhodamine 6G, the tuned output of which was frequency-doubled to 295 nm (nanometer = 10" m = 10 A) by passage through a p-barium borate crystal (cf.

Figure 6. Tempcraiurc dependence of the fluorescence lifetime of BMPC in 1 1 ethanol-methanol. Measurements were carried out at the LENS laboratory of Florence by a picosecond apparatus using as an excitation source (at 380 nm) a dye laser pumped by a frequency-doubled cw Nd-YAG laser and recording the Huorescence time profiles by a streak camera. Since the overall instrumental response time was 75-80 ps, decays with t>200 ps, observed at T<130 K, were analyzed without deconvolution. At 177, 178 and 193 K. the lifetimes were roughly estimated as i=(FWHM -77 ) , where FWHM was the width at half maximum of the decay. Because of the rather high sample absorbances (A .°°2), self absorption may have reduced the lifetimes to some extent.

Figure 5-8. Upper left-hand side frame Refractive index profile at 514.5 nm of a three-layered Si02-Ti02 planar waveguide. Left-hand side column Calculated squared electric-field patterns of the five TE modes. Right-hand side column BriUouin experimental spectra (open circles), calculated spectra (dotted line), and convolution of the calculated spectra with the instrumental response (solid line). The longitudinal sound velocity used in the fit is vi = 5.9 km/s,for m= 0,1, 2, and 3, and Pl = 5.75 km/sjbr m = 4 (Chiasera, 2003b).

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