Linearity fluorescence

The experiment is performed with a spectrofluorometer similar to the ones used for linear fluorescence and quantum yield measurements (Sect. 2.1). The excitation, instead of a regular lamp, is done using femtosecond pulses, and the detector (usually a photomultiplier tube or an avalanche photodiode) must either have a very low dark current (usually true for UV-VIS detectors but not for the NIR), or to be gated at the laser repetition rate. Figure 11 shows a simplified schematic for the 2PF technique. 

Schmid, I., Schmid, P., and Giorgi, J V (1988) Conversion of logarithmic channel numbers into relative linear fluorescence intensity Cytometry 9, 533—538... 

Sutoo et al. developed a high sensitivity and high linearity fluorescence microphotometry system for distribution analysis of neurotransmitter and related substances in the small brain regions [42]. The method makes use of the fluorescence intensity of not more than 10,000 points in animal brain slices, which are immunohistochemically and histochemically stained. 

Cell samples are analyzed on a Becton Dickinson FACScan with a linear fluorescence channel where the fluorescence is proportional to F-actin content. Samples were excited by an argon laser at 488 nm and emission was measured at 525 nm (green fluorescence/FL1) (7). 

Several far-field light microscopy methods have recently been developed to break the diffraction limit. These methods can be largely divided into two categories (1) techniques that employ spatially patterned illumination to sharpen the point-spread function of the microscope, such as stimulated emission depletion (STED) microscopy and related methods using other reversibly saturable optically linear fluorescent transitions (RESOLFT) [1,2], and saturated structured-illumination microscopy (SSIM) [3], and (2) a technique that is based on the localization of individual fluorescent molecules, termed Stochastic Optical Reconstruction Microscopy (STORM [4], Photo-Activated Localization Microscopy (PALM) [5], or Fluorescence Photo-Activation Localization Microscopy (FPALM) [6]. In this paper, we describe the concept of STORM microscopy and recent advances in the imaging capabilities of STORM. 

Following a 10-fold dilution of the sample with 0.6 M perchloric acid, the product of this reaction absorbs maximally at 420 nm. The original assay, developed by Kissane and Robins,12 uses DABA dihydrochloride for depurination (no perchloric acid was used) and employs fluorescence measurements at 520 nm after excitation at 420 nm. More recent work shows that the assay with perchloric acid can be conducted with 10-pL DNA sample volumes and provides a linear fluorescence calibration curve over the 10-500-ng total DNA range (1-50 pg/mL).13 The DABA method has since been modified for colorimetric measurements at 420 nm, and has a detection limit of 25 pg.14 The DABA reaction is specific for DNA, but... 

This work was initiated by the problem of investigating the photophysical properties of complex protein molecules and performing the diagnostics of such molecules in water environment. At the present time, fluorescence spectroscopy (fluorimetry) is widely used to study complex organic compounds (COC) (Lakowicz, 1999). Together with spectrophotometry these methods form the basis for fast and nondestructive diagnostics of COC in the natural environment, i.e. they present the diagnostic methods in vivo and in situ. However, the conventional (linear) fluorescence spectroscopy methods can not provide complete information on fluorescent objects under study because of insufficient selectivity (fluorescence bands of most COC are broad and structureless at room temperature). 

The narrow bandwidth can ensure that the resolution limit is defined by the molecule rather than by the light source. The low divergence can facilitate the use of long path lengths to enhance weak absorption, or can be used to concentrate the power into a small volume, providing an ideal linear fluorescent volume of gas which can be efficiently imaged on to a spectrometer slit. 

Note that in liquid phase chromatography there are no detectors that are both sensitive and universal, that is, which respond linearly to solute concentration regardless of its chemical nature. In fact, the refractometer detects all solutes but it is not very sensitive its response depends evidently on the difference in refractive indices between solvent and solute whereas absorption and UV fluorescence methods respond only to aromatics, an advantage in numerous applications. Unfortunately, their coefficient of response (in ultraviolet, absorptivity is the term used) is highly variable among individual components. 

For the parallel recording of EEL spectra in STEM, linear arrays of semiconductor detectors are used. Such detectors convert the incident electrons mto photons, using additional fluorescent coatings or scintillators in the very same way as the TEM detectors described above. 

At low laser powers, the fluorescence signal is Imearly proportional to the power. Flowever, the power available from most tunable laser systems is suflFicient to cause partial saturation of the transition, with the result that the fluorescence intensity is no longer linearly proportional to the probe laser power. While more... 

The fluorescence signal is linearly proportional to the fraction/of molecules excited. The absorption rate and the stimulated emission rate 1 2 are proportional to the laser power. In the limit of low laser power,/is proportional to the laser power, while this is no longer true at high powers 1 2 <42 j). Care must thus be taken in a laser fluorescence experiment to be sure that one is operating in the linear regime, or that proper account of saturation effects is taken, since transitions with different strengdis reach saturation at different laser powers. 

Two-photon excited fluorescence detection at the single-molecule level has been demonstrated for cliromophores in cryogenic solids [60], room-temperature surfaces [61], membranes [62] and liquids [63, 64 and 65]. Altliough multiphoton excited fluorescence has been embraced witli great entluisiasm as a teclmique for botli ordinary confocal microscopy and single-molecule detection, it is not a panacea in particular, photochemical degradation in multiphoton excitation may be more severe tlian witli ordinary linear excitation, probably due to absorjDtion of more tlian tire desired number of photons from tire intense laser pulse (e.g. triplet excited state absorjDtion) [61],... 

Let us consider tire case of a donor-acceptor pair where tire acceptor, after capturing excitation from tire donor, can emit a photon of fluorescence. If tire excitation light is linearly polarized, tire acceptor emission generally has a different polarization. Common quantitative expressions of tliis effect are tire anisotropy of fluorescence, r, or tire degree of polarization,... 

Standardizing the Method Equations 10.32 and 10.33 show that the intensity of fluorescent or phosphorescent emission is proportional to the concentration of the photoluminescent species, provided that the absorbance of radiation from the excitation source (A = ebC) is less than approximately 0.01. Quantitative methods are usually standardized using a set of external standards. Calibration curves are linear over as much as four to six orders of magnitude for fluorescence and two to four orders of magnitude for phosphorescence. Calibration curves become nonlinear for high concentrations of the photoluminescent species at which the intensity of emission is given by equation 10.31. Nonlinearity also may be observed at low concentrations due to the presence of fluorescent or phosphorescent contaminants. As discussed earlier, the quantum efficiency for emission is sensitive to temperature and sample matrix, both of which must be controlled if external standards are to be used. In addition, emission intensity depends on the molar absorptivity of the photoluminescent species, which is sensitive to the sample matrix. 

Selectivity The selectivity of molecular fluorescence and phosphorescence is superior to that of absorption spectrophotometry for two reasons first, not every compound that absorbs radiation is fluorescent or phosphorescent, and, second, selectivity between an analyte and an interferant is possible if there is a difference in either their excitation or emission spectra. In molecular luminescence the total emission intensity is a linear sum of that from each fluorescent or phosphorescent species. The analysis of a sample containing n components, therefore, can be accomplished by measuring the total emission intensity at n wavelengths. 

Subsequent studies (63,64) suggested that the nature of the chemical activation process was a one-electron oxidation of the fluorescer by (27) followed by decomposition of the dioxetanedione radical anion to a carbon dioxide radical anion. Back electron transfer to the radical cation of the fluorescer produced the excited state which emitted the luminescence characteristic of the fluorescent state of the emitter. The chemical activation mechanism was patterned after the CIEEL mechanism proposed for dioxetanones and dioxetanes discussed earher (65). Additional support for the CIEEL mechanism, was furnished by demonstration (66) that a linear correlation existed between the singlet excitation energy of the fluorescer and the chemiluminescence intensity which had been shown earher with dimethyl dioxetanone (67). 

L-pyrenyldiazomethane to form stable, highly fluorescent L-pyrenyhnethyl monoesters (87). These esters have been analy2ed in human blood by ce combined with lif detection. To mimini e solute adsorption to the capillary wall, they were coated with polyacrjiamide, and hydroxypropyl methylceUulose and dimethylfoTTnamide were used as buffer additives to achieve reflable separations. Separation was performed in tris-citrate buffer, pH 6.4, under reversed polarity conditions. The assay was linear for semm MMA concentrations in the range of 0.1—200 p.mol/L. 

Mercury generally is found in low and trace concentrations. So there is need to determine Hg in ranges corresponding to various types of water samples. Detection levels of Hg can be improved by the use of vapour generation technique. This technique allows to sepai ate the analyte from the sample matrix and so to overcome the matrix interference. The fluorescence technique, with its high sensitivity and linearity, in combination with vapour generation, provides for a possibility to detect Hg in parts per trillion per liter regions. 

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