Fluorescence scanning stage

Fig. 1. Diagram of the optical path of a fluorescence-imaging system (ACAS 570 Interactive Laser Cytometer). This system consists of a tunable 5W Argon ion laser, an acousto-optic modulator (AOM) to control the duration and intensity of excitation, an inverted microscope with an X-Y scanning stage, a Z-axis stepper motor, and two photomultiplier (PMT) tubes. A computer performs command, control, and analytical routines.

The term microfluorometer or microspectrofluorometer is used for systems that excite a small, usually diffraction-limited volume of a sample under a microscope and record the fluorescence, either with wavelength resolution or without. The borderline with FLIM techniques is not clearly defined. A TCSPC FLIM system can be used to record the fluorescence of a single point, and a microspec-trofluorometer combined with a scanning stage can be used as a FLIM system. Some typical principles of microfluorometry are shown in Fig. 5.97. 

Fig. 6. Schematic diagram of the steps involved in the cookie cutter method of cell selection. (A) Cells are grown on plastic Petri dishes covered with a darkened nylon film. (B) Based on fluorescence intensity image scans using a stage-scanning laser microscope, rare event cells are identified, and octagonal welds are made around those cells to fuse the film to the dish. (C) The film is then peeled away from the dish. (D) The cookies containing the desired cells remain on the dish so that the cells may be analyzed further or subsequently cloned.

A fluorometer is more sensitive than a spectrophotometer and enables a very low concentration of substrate to be used. This can be convenient in the first stage of pharmaceutical studies. Unfortunately the dependence of fluorescence intensity on temperature cannot be avoided. A blank scan at the end of the reaction enables a temperature-independent signal [45] useful for VTK processing to be obtained. The results were consistent (AS = -109 1J/K mol, AIR = 71 lkJ/mol, R2 = 0.99999) with those obtained spectrophotometrically under both CTK and VTK conditions. 

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