Spectrofluorometers

The sample cells for molecular fluorescence are similar to those for optical molecular absorption. Remote sensing with fiber-optic probes (see Figure 10.30) also can be adapted for use with either a fluorometer or spectrofluorometer. An analyte that is fluorescent can be monitored directly. For analytes that are not fluorescent, a suitable fluorescent probe molecule can be incorporated into the tip of the fiber-optic probe. The analyte s reaction with the probe molecule leads to an increase or decrease in fluorescence. 

Fluorometry and Phosphorimetry. Modem spectrofluorometers can record both fluorescence and excitation spectra. Excitation is furnished by a broad-band xenon arc lamp foUowed by a grating monochromator. The selected excitation frequency, is focused on the sample the emission is coUected at usuaUy 90° from the probe beam and passed through a second monochromator to a photomultiplier detector. Scan control of both monochromators yields either the fluorescence spectmm, ie, emission intensity as a function of wavelength X for a fixed X, or the excitation spectmm, ie, emission intensity at a fixed X as a function of X. Fluorescence and phosphorescence can be distinguished from the temporal decay of the emission. 

A variety of commercial instruments are available for PL measurements. These include spectrofluorometers intended primarily for use with liquids in a standard configuration, and simple filter-based systems for monitoring PL at a single wavelength. For use with opaque samples and surfaces, a few complete commercial systems are available or may be appropriately modified with special attachments, but due to the wide range of possible configuration requirements it is common to assemble a custom system from commercial optical components. 

Rabbit peritoneal neutrophils were harvested and their release of p-glucuronidase was measured at 37°C, as described previously (13). For indo-1, neutrophils were washed twice in a calcium-free buffer, then loaded with 15 indo-1 acetoxymethyl ester (24) for 40 min at 37 C at a density of 5 x 10 cells/ml. The cells were then washed twice more in calcium-free buffer, resuspended to a density of 1 X 10 cells/mL, and kept on ice. Prior to fluorometry, cells were diluted 4x with the appropriate buffer at 37 C. For CTC, neutrophils were incubated with 20 pH CTC at 37°C in the spectrofluorometer cuvette. All measurements were carried out using an SLH-Aminco SPF 500C fluorospectrometer interfaced with an IBM PC microcomputer. 

As mentioned previously (11), the wavelength position and stability of spectral lines from xenon or mercury excitation sources of spectrofluorometers may be variable with time-, and such sources are difficult to use with certainty for the calibration of monochromators. ... 

Another important linear parameter is the excitation anisotropy function, which is used to determine the spectral positions of the optical transitions and the relative orientation of the transition dipole moments. These measurements can be provided in most commercially available spectrofluorometers and require the use of viscous solvents and low concentrations (cM 1 pM) to avoid depolarization of the fluorescence due to molecular reorientations and reabsorption. The anisotropy value for a given excitation wavelength 1 can be calculated as... 

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. 

Glanzmann, T., Ballini, J. P., van den Bergh, H. and Wagnieres, G. (1999). Time-resolved spectrofluorometer for clinical tissue characterization during endoscopy. Rev. Sci. Instrum. 70, 4067-77. 

The fluorescence studies were performed on 90% acetone solutions with an Aminco-Bowman spectrofluorometer (Model J4-8203G). 

Fluorescence Measurement Fluorescence spectra were measured on a Spex Fluorolog 212 spectrofluorometer equipped with a 450 W xenon arc lamp and a Spex DM1B data acquisition station. Spectra were recorded in the front-face illumination mode using 343 nm as the excitation wavelength. Single scans were performed using a slit width of 1.0 mm. PDA fluorescence emission spectra were recorded from 360 to 600 nm, with the monomer and excimer fluorescence measured at 376.5 and 485 nm, respectively. Monomer and excimer peak heights were used in the calculation of the ratio of excimer to monomer emission intensities (Ie/Im). Excitation spectra were recorded from 300 nm to 360 nm and monitored at 376.5 and 500 nm for the monomer and excimer excitation, respectively. 

Fluorescence Instrumentation and Measurements. Fluorescence spectra of the FS samples were obtained on a steady state spectrofluorometer of modular construction with a 1000 W xenon arc lamp and tandem quarter meter excitation monochromator and quarter meter analysis monochromator. The diffraction gratings In the excitation monochromators have blaze angles that allow maximum light transmission at a wavelength of 240 nm. Uncorrected spectra were taken under front-face Illumination with exciting light at 260 nm. Monomer fluorescence was measured at 280 nm and exclmer fluorescence was measured at 330 nm, where there Is no overlap of exclmer and monomer bands. 

Fluorescence spectra of the novolac samples were measured on a Spex Fluorolog 212 spectrofluorometer with a 450 W xenon arc lamp and a Spex DM1B data station. Spectra were taken with front-face Illumination using a 343 or 348 nm excitation wavelength for solutions or films, respectively, which are near the maximum transmission region of this spectrometer. Spectra were corrected using a rhodamlne B reference. Monomer fluorescence was measured at 374 or 378 nm and exclmer fluorescence was measured at 470 nm. Monomer and exclmer peak heights were used In calculations of Ie/Im. The 1 monomer peak of pyrene was used to reduce overlap with the exclmer emission. 

When an analyte is fluorescent, direct fluorometric detection is possible by means of a spectrofluorometer operating at appropriate excitation and observation wavelengths. This is the case for aromatic hydrocarbons (e.g. in crude oils), proteins (e.g. in blood serum, in cow milk), some drugs (e.g. morphine), chlorophylls, etc. Numerous fields of applications have been reported analysis of air and water pollutants, oils, foods, drugs monitoring of industrial processes monitoring of species of clinical relevance criminology etc. 

Emission and excitation spectra are recorded using a spectrofluorometer (see Chapter 6). The light source is a lamp emitting a constant photon flow, i.e. a constant amount of photons per unit time, whatever their energy. Let us denote by N0 the constant amount of incident photons entering, during a given time, a unit... 

When the emission monochromator of the spectrofluorometer is set at a certain wavelength AF with a bandpass AAF, the reading is proportional to the number of photons emitted in the wavelength range from AF to AF + AAF, or in the corresponding wavenumber range from to 1/AF to 1/(AF + AAF). The number of detected photons satisfies the relationship ... 

Spectrofluorometers equipped with a photodiode instead of a quantum counter provide excitation spectra that should be further corrected because, in addition to the reasons explained above, the wavelength response of the photodiode is not strictly flat over the whole wavelength range available. 

C. D. Tran and R. J. Furlan, Spectrofluorometer Based on Acoustic-Optic Tunable Filters for rapid scanning and multicomponent sample Analyses, Anal. Chem. 65, 1675-1681 (1993). 

Excitation and emission spectra for the flavoprotein preparation was obtained in 100 mM acetic acid using an Aminco Bowman Spectrofluorometer. 

In this detector, the solute is excited with UV radiation and emits radiation at a longer wavelength. The limits of this detection by a spectrofluorometer depend upon the physico-chemical properties of the compound (degree of aromacity), the solvent, and the pH. The eluents (mobile phase) should neither fluoresce nor should they absorb at the excitation and emission wavelengths used. The pH is important because some compounds show fluorescence only in particular ionic forms. There are only a few... 

A discussion on steady state fluorescent monitoring necessitates a distinction between spectroscopic and photometric measurements. The former involves a grating-based spectrofluorometer where full spectrum excitation and emission multivariate spectra are acquired. In contrast a filter photometer involves optical elements (e.g., optical Alters) to isolate excitation and emission bands thereby resulting in a univariate output emission response. 

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