We describe a simple method of obtaining optical sectioning in a conventional wide-field microscope by projecting a single-spatial-frequency grid pattern onto the object. Images taken at three spatial positions of the grid are processed in real time to produce optically sectioned images that are substantially similar to those obtained with confocal microscopes.

A preparation of isolated small intestine of the rat or golden hamster (Mesocricetus auratus) is described which permits convenient measurement ofrespiration and glycolysis during periods of active transference of substances across the wall. The difficulty of adequate oxygenation is overcome by everting a piece of intestine, tying it at both ends and filling it with sufficient fluid to distend the wall. The eversion exposes the highly active mucosa to the well-oxygenated suspending medium, while the distension increases the surface area of the sac and reduces the thickness of the sac wall. The oxygenation of the inner layer of the serosal surface is facilitated by an oxygen bubble which is introduced into the sac along with the inner fluid.The relatively small volume of fluid contained in the sac (serosal side) allows a rapid rise in concentration of transferred substances. A number of adjacent segments of intestine from the same animal may be studied simultaneously.The method was tested with glucose and methionine, both substances being known to be transferred against a concentration gradient (Fisher & Parsons, 1949b;Wiseman, 1953). Aerobically both were transferred against a concentration gradient in this preparation, but no active transference took place anaerobically. EXPERIMENTAL Preparation of tissue. The animal (rat or hamster) was killed by a blow on the head, the abdomen opened by a midline incision, and the entire small intestine washed out with a solution of 0.9% (w/v) NaCl containing 03 % (w/v) glucose. The whole of the small intestine was then removed by cutting across the upper end of the duodenum and the lower end of the ileum and manually stripping the mesentery from the intestine. In the case of the hamster all the fat and mesentery were removed by this procedure, but in the rat a small amount of fat remained on the * Exchange Fellow of the American Cancer Society.

We demonstrate wavefront sensorless aberration correction in a two-photon excited fluorescence microscope. Using analysis of the image-formation process, we have developed an optimized correction scheme permitting image-quality improvement with minimal additional exposure of the sample. We show that, as a result, our correction process induces little photobleaching and significantly improves the quality of images of biological samples. In particular, increased visibility of small structures is demonstrated. Finally, we illustrate the use of this technique on various fresh and fixed biological tissues.

The main advantage of confocal microscopes over their conventional counterparts is their ability to optically ''section'' thick specimens; the thin image slices thus obtained can be used to reconstruct three-dimensional images, a capability which is particularly useful in biological applications. However, it is well known that the resolution and optical sectioning ability can be severely degraded by system or specimen-induced aberrations. The use of high aperture lenses further exacerbates the problem. Moreover, aberrations can considerably reduce the number of photons that reach the detector, leading to lower contrast. It is rather unfortunate, therefore, that in practical microscopy, aberration-free confocal imaging is rarely achieved. Adaptive optics systems, which have been used widely to correct aberrations in astronomy, offer a solution here but also present new challenges. The optical system and the source of aberrations in a confocal microscope are considerably different and require a novel approach to wavefront sensing. This method, based upon direct measurement of Zernike aberration modes, also exhibits an axial selectivity similar to that of a confocal microscope. We demonstrate an adaptive confocal fluorescence microscope incorporating this modal sensor together with a deformable membrane mirror for aberration correction. Aberration corrected images of biological specimens show considerable improvement in contrast and apparent restoration of axial resolution.

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