Fluorescent molecules have many applications in the biosciences. They can be incorporated into macromolecular probes for measuring the selective binding of the probes to cellular targets. Fluorescence-based bioassays are routinely used to detect, spatially localize and quantitate diverse analytes in biological and clinical samples.
The major advantages of fluorescence detection for bioassay purposes are specificity, spatial resolution, and sensitivity. The sensitivity of fluorescence detection is potentially very high provided that background noise is adequately suppressed and collection of specimen-emitted fluorescence is efficient. Under optimal conditions, it is feasible to detect single fluorescent molecules using fluorescence microscopy (Mathies and Stryer, 1986, cited in Chapter 13, Methods in Cell Biology 29: Part A, Eds. Y -L. Wang & D. L. Taylor, 1989).
Background noise is the single most important limitation in the quantitative measurement of fluorescence. Noise from any of the following sources may be substantial: sample fluorescence excited by stray light; background fluorescence from materials in the field of view but not in the focal plane; and contamination of emitted fluorescence with stray excitation light. These problems can be managed by using confocal scanning microscopy with a high sensitivity photodetection device (reviewed in Handbook of Biological Confocal Microscopy (Ed. J. B. Pawley) Plenum Press, NY, 1990; and Electronic Light Microscopy, Chapters 10 and 12, (Ed. D. Shotton), John Wiley, 1992).
In confocal imaging, a point light source is focused on or within the specimen, thus restricting the field of view to a well-defined focussed spot. The spot is imaged onto a point detector, e.g., a photomultiplier tube (PMT) or a charged couple device (CCD). A CCD array is a preferred detection means for confocal imaging because of its signal integration capability, high detection efficiency and potentially high spatial resolution. See, e.g., Aikens, Agard & Sedat, Chapter 16, In: Methods in Cell Biology 29: Part A, Eds. Y -L. Wang & D. L. Taylor, 1989; G J Brakenhoff and K Visscher (1990), Trans R Microsc Soc 1: 247-250; Scanning 13(Suppl 1): 65-66; and U.S. Pat. No. 5,233,197.
Because of the restricted field of view of the specimen in confocal imaging, scanning in x and y directions (for 2-D image) or x, y and z directions (for 3-D image) is required obtain a complete image. Commonly used techniques for scanning a fluorescent sample include mechanical stage scanning, beam scanning or combined stage and beam scanning. In stage scanning, the illumination and light collection optics are held fixed while the sample is moved laterally through the confocal point of the point light source, where the light intensity is greatest, and the point detector, where the collection efficiency is greatest. Stage scanning provides a wide field of view but a limited scan rate. In beam scanning, the specimen is held stationary while the illuminating light beam is caused to move laterally in the focal plane by an optical system containing a two-dimensional galvanometer-driven scanning apparatus. The galvanometric scanners can be quite fast by comparison with translation stages, typically between 0.1 and greater than 2 Hz for art image of 512.sup.2 or 512.times.768 pixels (Shotton, ibid), but the image field of view is limited to that of the objective used. For this reason a field flattening lens, or a post-scanner objective may be required. In combined stage and beam scanning, galvanometric scanning of the beam in one dimension is combined with translation of the sample in a second dimension using a linear mechanical stage.
In confocal imaging, out-of-focus information is rejected by spatially filtering the emitted fluorescence at the primary image plane before the light reaches the detector. Typically, the filter consists of a single small on-axis detector aperture. In order to achieve true confocal operation, the confocal detector aperture is ideally very small. This limits the light that reaches the photodetector and can result in a poor signal to noise level with weakly fluorescing specimens. Thus spatial filtering prior to image detection acts to diminish the fluorescence signal intensifying effects of high numerical aperture (NA) confocal imaging lenses. What is needed is a spatial noise discriminator that combines good background noise suppression with high resolution imaging and high detection efficiency without sacrificing the advantages gained by using high numerical aperture lenses.
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