Confocal Imaging

Three types of widefield confocal scanners are common for imaging microarrays. Scanning beam systems obtain a wide field of view using a telecentric f-theta laser scan lens combined with a slow-moving scanning stage;[1,2] scanning objective systems rapidly move a small objective lens across the width of the microarray while slowly moving the microarray in the perpendicular direction on a moving stage; and scanning stage systems use rapid stage motion to move the specimen under a stationary-focused laser beam. Confocal scanning laser microscopes can also be used, but their small field of view requires automated stage motion combined with image tiling to produce an image of the entire microarray. In addition to the problems of tiling separate subimages together, especially when the focus position may have changed from one subimage to the next, the boundaries of the subimages are exposed twice to the laser beam (and the corners four times), so that some fluorophore bleaching may occur at the edges of the images, resulting in poor quantification of probe spot intensity. The same problems arise in microarray readers based on CCD microscopes. Here the subareas are illuminated with a white light source, and nonconfocal images are collected using a CCD camera. A computer-controlled stage is used to move from one subarea to the next, and tiling is used to stitch the images together. In addition to multiple exposures of the fluorophores near the edges of the subareas, these systems have the additional disadvantages of collecting out-of-focus fluorescence from the glass substrate or an aqueous buffer solution (as they are not confocal).

Figure 1 shows a simple confocal microscope. Light from a point source (often a pinhole illuminated by a focused laser beam) passes through a beam splitter, expands to fill a microscope objective, and is focused to a tiny volume (at the focal point) inside the specimen (shown using solid lines). Light reflected (or emitted) from that point in the specimen is collected by the objective lens (a microscope objective) and is partially reflected to the right to pass through a pinhole to reach the detector. At the same time, light is reflected from parts of the specimen above the focal point (shown with dashed lines). This light is also collected by the objective lens and is partially reflected to the right, converging toward a focus behind the pinhole. Most of this light runs into the metal surrounding the pinhole and is not detected. Similarly, light reflected from parts of the specimen below the focal point (shown as dotted lines) converges toward a focus in front of the pinhole and then expands to hit the metal area surrounding the pinhole. Again, this light is blocked from reaching the detector. Thus the pinhole blocks light from above or below the focal point, so the detector output is proportional to the amount of light reflected back from only the parts of the specimen at the focal point. Images of the source pinhole and the detector pinhole formed by the objective lens are at its focal point (the source pinhole, detector pinhole, and the focal point are ''confocal'' with each other).

An image is collected by moving the specimen under the fixed laser beam in a raster scan (a scanning-stage microscope), by moving the beam using mirror scanners (a scanning-beam system), by moving the objective lens (a scanning-head system), or by moving the beam in one direction while moving the specimen in the perpendicular direction. Confocal images consist of sharp and empty areas; nonconfocal images consist of sharp and blurry areas. In a confocal microscope, light from out-of-focus parts of the specimen is rejected by the confocal pinhole, and it is this absence of out-of-focus information that allows three-dimensional images to be formed using a series of confocal slices.

Fig. 1 Confocal laser microscope.

For many applications, an infinity-corrected confocal microscope is more useful than the simple microscope shown above. In an infinity-corrected mxicroscope (shown in Fig. 2), a parallel beam from a laser or other light source is focused by an infinity-corrected microscope objective onto a specimen at the focal plane. Light reflected from the specimen is collected by the microscope objective, and partially reflected by the beam splitter toward a detector lens that focuses the beam to pass through the detector pinhole to reach the detector. Just as before, light reflected (or emitted) from above or below the focal plane is rejected by the pinhole, and the microscope is confocal.

Fig. 3 Widefield confocal scanning laser MACROscope1

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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