Fluorescence semiconductor lasers

The introduction and diversification of genetically encoded fluorescent proteins (FPs) [1] and the expansion of available biological fluorophores have propelled biomedical fluorescent imaging forward into new era of development [2], Particular excitement surrounds the advances in microscopy, for example, inexpensive time-correlated single photon counting (TCSPC) cards for desktop computers that do away with the need for expensive and complex racks of equipment and compact infrared femtosecond pulse length semiconductor lasers, like the Mai Tai, mode locked titanium sapphire laser from Spectra physics, or the similar Chameleon manufactured by Coherent, Inc., that enable multiphoton excitation. 

The application of semiconductor lasers to a broad range of areas in spectrometry has recently been reviewed by Imasaka. 67, 68) Topics covered include photoacoustic, absorption, and thermal lens, as well as steady-state and time-resolved fluorescence. Patonay et al. have reviewed the application of diode lasers to analytical chemistry.(69) The performance of several commercially available laser diodes for fluorimetry has recently been compared. 70 ... 

The methods range from simple, inexpensive absorption spectroscopy to sophisticated tunable-laser-excited fluorescence and ionization spectroscopies. AAS has been used routinely for uranium and thorium determinations (see for example Pollard et al., 1986). The technique is based on the measurement of absorption of light by the sample. The incident light is normally the emission spectrum of the element of interest, generated in a hollow-cathode lamp. For isotopes with a shorter half life than and Th, this requires construction of a hollow-cathode lamp with significant quantities of radioactive material. Measurement of technetium has been demonstrated in this way by Pollard et al. (1986). Lawrenz and Niemax (1989) have demonstrated that tunable lasers can be used to replace hollow-cathode lamps. This avoids the safety problems involved in the construction and use of active hollow-cathode lamps. Tunable semiconductor lasers were used as these are low-cost devices. They do not, however, provide complete coverage of the spectral range useful for AAS and the method has, so far, only been demonstrated for a few elements, none of which were radionuclides. 

Several of the polymethine dyes absorb in the red, far-red, and near-infrared and fluoresce efficienlly as well in this spectroscopic region. Functional derivatives of these may provide excellent fluorescent labels for semiconductor laser excitation. Several research groups are currently actively involved in the synthesis and development of large polyunsaturated dye molecules that are excited and show luminescence in the red and near-infrared. This promises to be one of the most exciting areas of luminescence spectroscopy for the foreseeable future. 

Novotny (18) has pointed out an alternate trend in the effort to utilize the potential power of the laser for HPLC detection, namely the "tuning" of the chemistry of the system to match the available laser wavelengths (19). The use of naphthalenedlaldehyde as discussed above is a good example. An even more striking example is the work of Sauda, Imasaka and Ishibashi (20), who used indocyanine green as a fluorescent tag for protein labeling. The dye/proteln complex absorbs at about 765 nm and emits at 820 nm. This couples well with the output of a pulsed semiconductor laser diode (780 nm,... 

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... 

Larson, A. R, Ahlberg, H., and Folestad, S., Semiconductor laser-induced fluorescence detection in picoliter volume flow cells, Appl. Opt., 32, 794,1993. 

Higashijima, T., Fuchigami, T., Imasaka, T., and Ishibashi, N., Determination of amino-acids by capillary zone electrophoresis based on semiconductor-laser fluorescence detection, AnoZ. Ghent., 64, 711, 1992. 

Lemmer, U., and Mappes, T. (2011) Plastic lab-on-afluorescence excitation with integrated organic semiconductor lasers. Opt. Express, 19,8179-8186. 

Lasers represent a special type of light source [16], [21], [60], [61]. They are used in trace analysis by fluorescence measurement or laser-induced fluorescence (LIF) (- Laser Analytical Spectroscopy) [62] - [64], in high-resolution spectroscopy, and in polarimetry for the detection of very small amounts of materials. Lasers can be of the gas. solid, or dye type [21]. In dye lasers, solutions of dyes are pumped optially by another laser or a flash lamp and then show induced emi.s-sion in some regions of their fluorescence bands. By tuning the resonator the decoupled dye laser line can be varied to a limited extent, so that what may be termed sequential laser spectrometers can be constructed [65]. In modern semiconductor lasers, pressure and temperature can also be used to detune the emission wavelength by 20-30nm [66], [67]. 

A direct use of the fluorescence approach is the photoreduction of noble [75] (for example Au and Ag+) and or rare-earth (for example Sm and Eu +) [76, 77] metals. For instance, Sm + in glass can be space-selectively photoreduced with an IR femtosecond laser. It is found that after photoreduction from Sm + to Sm +, a pronounced fluorescence band appears at 650-775 nm, which has been used as readout signal of 3D memory [77]. Another outstanding characteristic is that photoreduced Sm +, stable at room temperature, can be converted back to Sm by photo-oxidation with a CW laser, such as an argon-ion laser or a semiconductor laser. This enables rewritable optical memory with two stable statuses. 

In this chapter, we review the instrumentation presently available for studying near-IR fluorescence. This includes modern semiconductor devices such as diode lasers and photodiode detectors and also more conventional devices such as discharge lamps and photomultipliers which are traditionally more usually associated with the study of UV/visible fluorescence. Throughout the chapter emphasis will be placed on the novel red/near-IR aspects of instrumentation and we will assume that the reader has a knowledge of the basics of steady-state and time-resolved techniques to the level consistent with Volume 1 of this series. 

Electrothermal atomizers are also suitable for AFS as, when an inert gas atmosphere is used, quenching will be minimized. In the nuclear, electronic, semiconductor and biomedical industries where detection limits have to be pushed as low as 1 part in lO (or 0.1 pg g- in the original sample), electrothermal atomization with a laser as excitation source (LIF-ETA) may be used. Figure 6.5 shows schematically a common way of observing the fluorescence in LIF-ETA. The fluorescence signal can be efficiently collected by the combination of a plane mirror, with a hole at its centre to allow excitation by the laser, positioned at 45° with respect to the longitudinal axis of the tube and a lens chosen to focus the central part of the tube into the entrance slit of the fluorescence monochromator. 

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