Single-Channel Fluorescence Detection

In one report, the sample stream was physically focused so that it was within the illumination region of the laser probe beam. In this way, fluorescence detection of DNA was enhanced [329]. In another report, the effective observation volume was reduced by confinement within nanochannels in a fused silica chip. In this way, fluorescent detection of even single molecules was achieved [673]. 

Optical fibers (excitation and emission) have also been integrated on a PMM A chip for fluorescent detection. An intercalating near-IR dye (TOPRO-5) with an excitation wavelength of 755 nm was used to label DNA fragments. Sub-attomol detection limit of the labeled DNA fragments was achieved [674]. 

FIGURE 7.1 Schematic of the laser-induced fluorescence detection system. Excitation was provided by an argon ion laser light. It was focused with a lens (1) onto the separation channel in a microchip. The chip was held in place with a Plexiglas holder (2). Fluorescence emission (3) was collected with a microscope objective (4), focused onto a spatial filter (5), emission filter (6), and then detected with a photomultiplier tube [550]. Reprinted with permission from the American Chemical Society. 

Two-photon fluorescent (TPF) detection, which was initiated by a non-linear optical absorption process, has been performed on a quartz chip. Since the fluorescent efficiency in TPF is inversely proportional to the excitation beam area, the path length dependence problem in fluorescence is significantly reduced. This method is used for analysis of P-naphthylamine (excitation at 580 nm), which is the enzymatic product of leucine aminopeptidase (LAP) acting on the fluorogenic substrate leucine P-naphthylamide [675], 

FIGURE 7.2 Four-color optical excitation and detection system for LIF. Excitation was provided by a laser emitting at 488 nm and 514 nm. (a) Dichroic mirrors (b) emission band-pass filters. Detection was carried out at 4 wavelengths (525, 555, 580 and 605 nm) [543]. Reprinted with permission from Wiley-VCH Verlag. 

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Implementation of time domain FLIM methods is comparatively straightforward in laser scanning microscopes (LSMs). Here, pointscanning is used so that single channel lifetime detection suffices. In principle, standard fluorescence lifetime detection equipment developed for spectroscopy can be used in combination with point-scanning systems and a pulsed laser. 

Dittrich P.S., Manz A., Single-molecule fluorescence detection in microfluidic channels—The Holy Grail in mu TAS Anal. Bioanal. Chem., 382, 1771-1782 (2005). 

In addition to wavelength and time-resolved fluorescence techniques, polarization fluorescence can yield important information about an analyte." This is especially true when differentiating between chiral compounds. Combining, for example, a fluorescently tagged antibody immunoassay with polarization detection allows for very sensitive detection limits of chiral enantiomers. Laser-induced fluorescence polarization (LIFP) has been used to detect concentrations as low as 0.9 nM of an antibody-boimd cyclosporine A (an immunosuppressive drug) in human blood. A conventional single channel fluorescence detector can be easily modified to perform such measurements, simply by adding the appropriate polarization filters. 

Dittrich PS, Manz A (2005) Single-molecule fluorescence detection in mitaofluidic channels-the Holy Grail in [iTAS Anal Bioanal Chtan 382(8) 1771—1782... 

The use of sub-micrometer channels and detection by confocal fluorescence microscopy is an interesting alternative, which should allow precise control of the movement of single molecules by electrokinetic or electro-osmotic forces . ... 

In the past, molecular luminescence spectrometry was always conducted with single channel systems involving a photomultiplier tube (PMT) as the detector. The availability of multichannel detectors with internal gain has provided a new powerful tool for luminescence measurements, and several types of applications have been reported (1-15). This paper is concerned with the application of an intensified diode array dynamic molecular fluorescence and chemiluminescence measurements. In this paper the types of measurements and analytical systems for which multichannel detectors are used in our laboratory are introduced. Next the specific IDA system used is presented along with important hardware and software considerations. Third, the characteristics of the IDA detector are reviewed to give some perspective about its influence on the quality of measurements. Finally, some typical applications to chemical systems are presented to illustrate the advantages of multichannel detection. 

Photodiode Array Versus Photomultiplier Detection. The advantages of photodiode array detection, PDA, as compared to photomultiplier tube, pmt, detection for emission spectroscopy are well known (19). These advantages are especially important for the specific examples we discuss here, namely, upconverting emission spectroscopy. This is dramatically demonstrated in Figure 7 where we compare single channel pmt versus multichannel PDA detection of a small portion of the upconverted fluorescence spectrum of coumarin 520 in ethanol solvent at room temperature. 

A high-performance confocal fluorescence detection unit usable in either a single channel or multichannel mode. 

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