Fluorescence, laser apparatus

Fig. 8. Schematic diagram of ps fluorescence microprobe apparatus. PM Photomultiplier. D Diaphragm. XYS X-Y stage. F Filter. M Laser mirror. RS Rotating stage.

Boyer and coworkers were the first to develop instrumentation for near-infrared fluorescence immunoassays [117]. WiUiams and coworkers also developed instrumentation for detection of near-infrared fluorescence in sohd-phase immunoassays [118]. The instrument consists of a semiconductor laser coupled with a fiber-optic cable, a silicon photodiode for detection, a sample stage coupled to a motor drive, and a data acquisition device. The instrument could detect 500 pM concentrations of human immunoglobulin G (IgG) on a nitrocellulose matrix. The assay was performed in roughly two hours. The detection limits obtained on this instrument were comparable to that obtainable with ELISA. The assay developed by WiUiams suffers from excessive scatter generated from the membrane, nonspecific binding, and incompatibility with conventional microtiter plate immunoassay formats [140]. Patonay and coworkers developed a NIR fluorescence immunoassay apparatus that overcame many of these limitations. Baars and Patonay have evaluated a novel NIR dye NN382 (Fig. 14.25) for the ultrasensitive detection of peptides with capilary electrophoresis [141]. A solid-phase, NIR fluorescence immunoassay system was... 

Transient Infrared Absorption (TRISP) and laser-induced fluorescence. Because the CJ temperatures are only 2000-3000 K, most of the molecular products are in the ground electronic state. Emission spectroscopy looks selectively at only a few extraordinary molecules which are scarcely representative of most of the products. Infrared absorption, on the other hand is ideal for probing the vibrotational states of the ground state molecules, and the fast response time of TRISP makes it ideal for detonations. The technique has not been applied extensively and is difficult to implement, but our preliminary attempts have shown that we can do it with the proper laser apparatus. Broadband CARS is an alternative approach if the instrumental difficulties of TRISP cannot be overcome. 

Schematic drawing of the integrated PCR-CE microdevice (a) Laser-excited confocai fluorescence detection apparatus and an integrated PCR-CE microdevice, (b) Expanded view of the microfabricated PCR chamber, (c) Expanded cross-sectional view of the junction between the PCR and CE devices. The size of the epoxy filled gaps is exaggerated for viewing clarity. Reprinted with permission from Woolley etal. Functional Integration of PCR Amplification and Capillary Electrophoresis in a Microfabricated DMA Analysis Device. Reprinted with permission from [5]...

Figure B2.3.9. Schematic diagram of an apparatus for laser fluorescence detection of reaction products. The dye laser is syncln-onized to fire a short delay after the excimer laser pulse, which is used to generate one of the reagents photolytically.

Laboratory work involved making calibration curves which show the response of the system for various concentrations of pollutant, e.g., phenol. Typically, remote laser-induced fluorescence measurements from both the laboratory apparatus and the mobile unit are made on... 

Nelson and Zahniser used a moveable injector discharge flow apparatus in these studies, generating the HO2 from the H + O2 + M reaction. They detected both OH and OH by laser-induced fluorescence, correlating their ratio with... 

Figure 6. Tempcraiure dependence of the fluorescence lifetime of BMPC in 1 1 ethanol-mcihanol. 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 fluorescence time jirofiles by a streak camera. Since the overall insuumental 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 (An,x=2), self absorption may have reduced the lifetimes to some extent.

Figure 4.6 shows an apparatus for the fluorescence depolarization measurement. The linearly polarized excitation pulse from a mode-locked Ti-Sapphire laser illuminated a polymer brush sample through a microscope objective. The fluorescence from a specimen was collected by the same objective and input to a polarizing beam splitter to detect 7 and I by photomultipliers (PMTs). The photon signal from the PMT was fed to a time-correlated single photon counting electronics to obtain the time profiles of 7 and I simultaneously. The experimental data of the fluorescence anisotropy was fitted to a double exponential function. 

For the investigation of triplet state properties a laser flash photolysis apparatus was used. The excitation source was a Lambda Physik 1 M 50A nitrogen laser which furnished pulses of 3.5 ns half-width and 2 mJ energy. The fluorescence decay times were measured with the phase fluorimeter developed by Hauser et al. (11). 

More recently Ghiggino and co-workers(32) have applied laser scanning confocal fluorescence lifetime microscopy to the study of polyvinyl alcohol films containing rhodamine B (650 nm emission) and cresyl violet (632 nm emission). Synchronously pumped dye laser excitation and APD detection were used with optical fiber coupling. A schematic diagram of their apparatus is shown in Figure 12.5. 

In addition to the commercially available systems, several authors have described laboratory-built systems using commercially available components from companies such as Upchurch Scientific (Oak Harbor, WA). One of the first reported laboratory-built micro-bore HPLC systems was described by Simpson and Brown, which was a simple adaptation of a standard HPLC system to accept micro-bore columns built from guard columns. A complete system has been described based on dual microdialysis syringe pumps (CMA Microdialysis, Chelmsford, M A) or dual syringe pumps (Harvard Apparatus, Inc., Holliston, MA), a microinjection port, and a micro-column the latter components being obtained from Upchurch scientific (Figure 3.5). This system was coupled with a laser-induced fluorescence (LIF) detector and used to measure neuropeptides in sub-microliter samples. A further modification of this system was built to perform immunoaffinity isolations of biomedically important analytes from clinical samples. ... 

The fluorescence dynamics measurements were carried out with fluorescence up-conversion apparatus [1(b)] based on Ti Sapphire laser (820 nm, 800 mW, 76 MHz, -65 fs). The fwhm of the instrumental response was -110 fs. 

FRAP data were analysed by a non-linear least squares fit to an expression [8,23,29], defining the time dependence of the fluorescence recovery (F(t)). The apparatus as described above delivered a laser spot of uniform circular cross sectional intensity to the sample and the recovery curves obtained could be analysed with the expression ... 

The FRAP apparatus can also be used in a semi-quantitative manner to measure the surface concentration and subsequent competitive displacement of adsorbed labelled species, such as the fluorescent-labelled protein in the adsorbed layer of a/w or o/w thin films [10]. This can be achieved by focusing the low power 488 nm beam on the film and detection of the emitted fluorescence using the FRAP photon counting photomultiplier. The detected fluorescence signal is proportional to the amount of adsorbed protein at the interfaces of the thin film provided that the incident laser intensity is kept constant. Calculations have proved that the contributions from non-adsorbed protein molecules in the interlamellar region of the film are negligible [12],... 

The schematic view of the Mainz apparatus for collinear laser spectroscopy, installed at Isolde is given in fig 4. The 60 keV ion beam is set collinear with the laser beam, then accelerated (or decelerated) and finally neutralized in charge exchange cell. By Doppler tuning the atomic absorption is set resonnant with the stabilized laser frequency, and the fluorescence emitted is detected. 

The innovative thermostated separation system published by de Bokx et al. [17] represents an interesting example and comprises a capillary cross intersection for sample injection and a 100 pi fluorescence detector cell based on fiber optics. This apparatus shows basically all features that are required to perform automated fast and efficient electrophoretic separations and has been used to separate a mixture of laser dyes in 35 seconds with moderate efficiency. However, in order to keep all dead volumes at the junctions sufficiently small, the connections had to be done by tedious laser-based drilling of holes through the capillary walls. A similar approach to interconnect capillaries was described for a postcolumn derivatization reactor for CE [18], and many more inventive capillary coupling devices have been designed. 

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