Lamp intensity fluctuations

A Spex Fluorolog 212 spectrophotometer was used for recording the emission and excitation spectra of the polyimide films and the model compounds. The slit width used for the films was 2 mm and for the model compounds was 1 mm. Excitation and emission spectra were subsequently normalized with respect to the lamp intensity fluctuations by dividing each spectrum by that obtained with a Rhodamine-B standard solution. Absorption spectra were obtained with... 

This provides a means for correcting the emission of both dyes for the aforementioned effects of dilution, lamp intensity fluctuation, etc. In a medical device, such corrections could be made with the aid of a simple processor. We corrected the raw signals for TSPP and HPTS using the reference signal from SR-B. The raw and corrected binding curves of both HPTS and TSPP reporter dyes are given in Figure 5.20. 

Variations in lamp intensity and electronic output between the measurements of the reference and the sample result in instrument drift. The lamp intensity is a function of the age of the lamp, temperature fluctuation, and wavelength of the measurement. These changes can lead to errors in the value of the measurements, especially over an extended period of time. The resulting error in the measurement may be positive or negative. The stability test checks the ability of the instrument to maintain a steady state over time so that the effect of the drift on the accuracy of the measurements is insignificant. 

Thacker [24] reported the design of a miniature flow fluorimeter for liquid chromatography. The body of the fluorimeter was machined from a block of aluminium and contained a low-pressure mercury lamp, an excitation filter, a quartz flow cell, an emission filter, a photomultiplier tube and a photoconducter in order to compensate for fluctuations in lamp intensity. Fluorescence was examined at a direction perpendicular to that of the excitation light. The cell was small enough for it to be attached directly to the end of the column with a minimum dead volume. 

In this laboratory we use an SLM-Aminco 8100, equipped with Glan-Thompson polarizers. The electronics have been updated by the ISS Phoenix system. Measurements of FRET efficiency are performed under photon counting conditions, with the polarizers crossed at the magic angle (54.7°) to remove polarization artifacts. Fluctuation of lamp intensity is corrected using a concentrated rhodamine B solution as a quantum counter. 

Another factor which can cause spectral distortions is the fluctuation of the output energy of the radiant source. The OMA 2 system incorporates a capability to correct spectral data for variations in source output energy (17). The source compensation mode continuously monitors the output energy of the lamp and automatically corrects incoming spectral data for source intensity fluctuations prior to storing that data. 

Decay of Transient Absorption. The optical absorption decayed significantly over a few milliseconds only at the longer wavelength side of the maximum absorption—i.e., in the region of 300 m/. (The behavior of the absorption on appreciably longer time scales was obscured by the combination of low optical density and fluctuations in lamp intensity.)... 

We have performed fluorescence depolarization measurements on both compressed and uncompressed gels, containing similar concentrations of chlorosomes. To obtain good results the absorption in the excitation band was kept low, typically 0.05. We excited at 460 nm and detected at 750 nm. Several thousands of counts were needed to minimize statistical errors. To keep the measuring time low, bandwidths of 16 nm were used. The determination of 1 intensity took 30 seconds. To eliminate the effect of fluctuating lamp intensities, 20 series of measurements were poformed on both a compressed and an uncompressed gel. One series consists of the determination of 4 intensities for both the uncompressed and the compressed gel. From one series the parameters , , , and the constant C were determined. These led to the following average parameters and standard errors ... 

Kienle and Stearns described 23 modifications of the G. E. Spectrophotometer that enable it to be used for applications other than those for which it was designed. Richardson has also described techniques and accessories for facilitating the use of this instrument. We have taken G. E. reflectance spectra at widely varying lamp voltages as a crude measure of relative fluorescence of the specimen. While the true reflectance spectrum of the sample is unaffected by lamp intensity (provided that enough light reaches the phototube), the fluorescence must fluctuate with the source of excitation. 

In some applications, one requires corrected spectra. Corrections in excitation spectra have to be made to account for the wavelength depemdemce of the lamp source and the efficiency of the excitation monochromator throughput, as well as for temporal fluctuations in lamp intensity (9). These corrections are commonly made within the fluores-cence spectrometer by use of a quantum counter. A quantum counter is a substance such as rhodfimine B which has a fluorescence quantum efficiency independent of the excitation wavelength. A small fraction of the incident light is directed onto the quantum counter, whose emission intensity serves as a reference for the emission intensity of the sample itself. The ratio of the two intensities is independent of distortions arising from the excitation source. 

A) Fluctuations in the intensity of a 514-nm argon ion laser line (left) and a mercury arc lamp (right), measured every 20 s for a 3 h time period. 

A fluorescence emission spectrum is generally measured by setting the excitation monochromator, Mi, to the chosen wavelength and scanning the second monochromator, M2, with constant slit width. The fluorescent screen monitor, F-P2, now serves to correct for variations in the intensity of the exciting light caused by fluctuations in lamp output. The emission spectrum so recorded has to be corrected for the spectral sensitivity of the apparatus to give the true emission spectrum. 

Mercury/Xenon arc lamp If the arc is not focused sharply on the back aperture, the specimen plane will be unevenly illuminated. In addition, fluctuations in the illumination intensity of the lamp will also result in variable intensity over the whole field of view. In order to avoid this, it is better to consider a fluorescence microscope wherein the light coming from the mercury/xenon lamp goes through a quartz optical fiber for light scrambling, resulting in an even illumination and less fluctuations (Fig. 4). 

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