Fluorescent detection, instrument photomultiplier tube

Fluorescent detection technology applicable to biochips is evolving rapidly, resulting in detection instruments that are more powerful, user-friendly, and less expensive. Most systems employ photomultiplier tube (PMT) technology in conjunction with multiple colors, lasers, and a variety of filters. It is essentially a fluorescent microscope that... 

Laser devices are the most sophisticated image-acquisition tools. They are particularly useful for gels labeled with fluorescent dyes because the lasers can be matched to the excitation wavelengths of the fluorophores. Detection is generally with photomultiplier tubes. Some instruments incorporate storage phosphor screens for detection of radiolabeled and chemiluminescent compounds (not discussed in this chapter). Resolution depends on the scanning speed of the illumination module and can be as low as 10 pm. 

CIO and BrO abundances are detected simultaneously and continuously as the airstream passes through the instrument. They are not detected directly but are chemically converted to Cl and Br atoms by reaction with reagent nitric oxide gas that is added to the airstream inside the instrument. The Cl and Br atoms are then detected directly with resonance fluorescence in the 2D5/2 -> 2P3/2 transitions in the vacuum ultraviolet region of the spectrum. In resonance fluorescence, the emissions from the light sources are resonantly scattered off of the Cl and Br atoms in the airstream and are detected by a photomultiplier tube set at right angles to both the light source and the flow tube. The chemical conversion reactions... 

A detector, for example, a UV absorbance detector, through which the solution flows, is placed near or at one end of the capillary. A focused beam is passed through the capillary and may be collected by an optical fiber coupled to a photomultiplier tube. The short pathlengths (10 to 100 /rm) involved make sensitive detection a challenge. But the small peak volumes, often less than 1 nL, lead to very low detection limits, even with moderately sensitive detectors (i.e., the solute is concentrated in a very small volume). The use of laser sources, especially for fluorescence detection, has pushed detection limits to zeptomoles (10"- mol) A capillary electrophoresis instrument is shown in Figure 21.19. 

The back-scattered fluorescence is collected back up the same fiber, reflected from the mirrored face of the perforated mirror (PM), and imaged by another lens onto the entrance slit of a monochromator (SLM-Aminco, Inc.) with a spectral band-pass of 8 nm. Fluorescence is detected with another photomultiplier tube (D), identical to the reference channel unit. The cross-correlation (heterodyne) frequency (16,18) is produced with a second PTS 500 frequency synthesizer amplified by a 5W power amplifier (PA2 Amplifier Research, model 5W1000). The output from this amplifier is directed simultaneously to the second dynodes of the both reference and detection photomultiplier tubes using a simple power splitter (PS Adam Russell, model H-9). The cross-correlation frequency employed for this instrument is 25 Hz. 

The widely used continuous instrumental method for determination of SO2 is based upon gas-phase fluorescence. Pulsed ultraviolet (UV) light (214 nm) is used to irradiate the air sample, which flows continuously through an optical cell. The SO2 reemits fluorescent radiation at 340 nm that is detected by a photomultiplier tube (PM), with the signal obtained being converted to concentration. This method is almost specific with a detection limit of 1 pgm in commercially available analyzers. 

A new instrument for automatic colorimetric and fluorimetric titrations is described. Titrant delivery is automatically stopped at the end point by the use of a microammeter with a built-in relay which stops a motor-driven buret equipped with digital read-out or a gravity driven buret equipped with solenoid control. Monochromatic light is used to excite fluorescence, which is received at 90° by a photomultiplier tube with the microammeter in its anode circuit. Light transmitted through the titration vessel is detected by a barrier-layer photocell, the output of which is fed to the microammeter. 

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