Fluorescent optical sensors instrumentation

Wolfbeis O.S., Fluorescence-based optical sensors for biomedical applications, In Scheggi A.M.V., Martelluci, S., Chester, A.N., Pratesi, R. (Eds.), Biomedical Optical Instrumentation and laser-Assisted Biotechnology, Kluwer Academic Publishers, 1996, p.327-337. 

Most present-day fiber-optic sensors use linear diode arrays combined with optical gratings and measure the absorption, transmission, fluorescence, and reflection in UV, visible, and NIR regions (see Table 3.1). Light travels to the sampling probe via one fiber-optic cable and returns to the instrument via a second. Laser excitation permits long-distance transmission of excitation radiation to get a useful signal from the sample. 

A useful specialized type of analytical instrumentation is the fiber-optic sensor or optrode (253,254), in which an optical transducer monitors some chemically selective change. These are often based on the fluorescence of a reagent immobilized at the distal end of the fiber (255). 

It is important that an instrument with a good optical system is used to develop methods before they are applied to simpler instruments or fiber optic sensors. Fluorescence instrumentation was recently reviewed by Wehry (9). 

Typically, agents used to modify probes are selected so that their optical properties, i.e. fluorescence, are modified in the presence of a relevant ligand. Although within the fiber optic sensor field a distinction is sometimes made between sensors, which are said to detect events continuously, and probes, which are thought of as single use instruments, for clarity and simplicity, those distinctions will not be made here and the terms will be used interchangeably.(Vo-Dinh, Alarie et al. 2000)... 

Fig. 5 shows the instrumental arrangement of the commercially most successful optical chemical sensor between 1984 and 2000. It is used in about 70% of all critical care operations in the US to monitor pH, pC02 and p02 in the cardiopulmonary bypass operations35. It contains 3 fluorescent spots, each sensitive for one parameter, in contact with blood. Fluorescence intensity is measured at two wavelengths and the signals are then submitted to internal referencing and data processing. 

SECM instruments (77,78) will undoubtedly increase the scope and power of SECM. Further improvements in the power and scope of SECM has resulted from its coupling scanning probe or optical imaging techniques, such as AFM (57,79) or single-molecule fluorescence spectroscopy (80). The combined SECM-AFM technique offers simultaneous topographic and electrochemical imaging in connection to a probe containing a force sensor and an electrode component, respectively. 

Fig. 11. Schematic design of a fluorescence sensor. A strong light source creates radiation with low wavelengths. Optics like lenses and filters extract and focus the desired excitation light which is sent through the window into the measuring solution. Only a small fraction of the fluorescent light arrives at the window, passes this, and is collected by appropriate optics and fed to a sensitive detector (usually a photomultiplier). Variations in the light source intensity can be compensated by a comparative measurement. When optical fibers are used inside the instrument, the dichroitic mirror shown is obsolete...

Fluorescence sensors have been used since 1957 to measure cell internal NAD(P)H at 450 nm. Later on they were applied for in situ determination of the cell concentration. However, the culture fluorescence intensity is not only influenced by the cell concentration, but also by the physiological state of the cells [56] and, in addition to that, there are several other compounds that participate in the fluorescence emission besides NAD(P)H. To identify the fluorophores in the cells and cultivation medium, the excitation and the emission wave lengths are varied in a broad range [57,58]. Two instruments were applied for the 2D-fluorescence spectroscopy Model F-4500 (Hitachi) and the BioView Sensor (Delta light Optics). Each of them uses an excitation range of 250-560 nm,an emission range of 260/300-600 nm and the measuring time of 1 min [59,60]. The application of this technique for CPC production was performed by Lindemann [61]. 

A fluorescence measurement is performed by directly delivering a vacuum controlled pulse of the analyte vapour diluted with air to the distal end of the optical fibre containing the sensors (Figure 4). The optical instrument includes a fluorescence microscope and a charge coupled device (CCD) camera. The excitation light is launched into the fibre, and the... 

---