No abstract

We show the point-spread function of a confocal microscope with an increased detection aperture. The increase in aperture is accomplished by coherent collection of the light from the specimen with two opposing objective lenses, i.e., type-B 4Pi confocal microscopy. We demonstrate experimentally its feasibility for detecting scattered or fluorescently emitted light. The 4Pi confocal point-spread functions are shown for constructive and destructive interference of the collected wave fronts and are compared with the point-spread functions of comparable confocal microscopes.The major advantage of the confocal microscope is its axial discrimination that permits optical sectioning of the sample.' Axial discrimination is equivalent to axial resolution, which is quantified by the extent of the intensity point-spread function (PSF) along the optical axis. Therefore a reduction of the extent of the PSF is important for the optical-sectioning properties of the microscope. We can obtain this reduction either by decreasing the wavelengths used for illumination and for detection or by increasing the numerical aperture.4Pi confocal microscopy was proposed as a means to increase the aperture angle and therefore improve the axial resolution of a confocal microscope. 2 -4 Since in a confocal arrangement the PSF is given by the product of the illumination and the detection PSF's, three types of 4Pi confocal microscope have been described. In a type-A 4Pi confocal microscope the illumination aperture is enlarged, whereas in a type-B 4Pi confocal [4Pi(B) confocal] microscope the detection aperture is increased. A type-C 4Pi confocal microscope combines both types A and B, leading to further resolution enhancement along the optical axis. The potential of axial resolution with a type-A 4Pi confocal microscope was shown by measurement of the z response of a fluorescent layer. 3 In this Letter we show the effect of an increased detection aperture on the resolution through the use of 4Pi(B) confocal microscopy. First, we present for the experiments with scattered light. We produced a pointlike light source by focusing a laser beam into a pinhole. The light leaving the pinhole was collimated by a lens (L1) and then split into two beams by a beam splitter (BS2). One beam was focused into the sample by a microscope objective (OBJ1), and the other was blocked with a polarizer oriented perpendicular to the polarization of the laser light. A second objective (OBJ2) was placed on the other side of the sample and focused into the focal point of the first objective. The light coming from the sample was collected on both sides through the objective lenses. The two detection beams were deflected from the illumination path by a beam splitter (BS1) and focused into the detection pinhole by a lens (L2). Another crossed polarizer placed in front of the photomultiplier tube prevented light with the original polarization from being detected.Coherent detection of the light requires spatial and temporal coherence of the detected light. Temporal coherenc...

In a 4Pi-confocal microscope the specimen is illuminated and observed coherently from above and below such that the numerical aperture is increased [S. W. Hell, European Patent Application 91121368.4 (filed 1990, published 1992), S. W. Hell and E. H. K. Stelzer, J. Opt. Soc. Am. A 9, 2159 (1992)]. The point spread functions of 4Pi-confocal and confocal microscopes were measured. Our measurements prove a three- to seven-fold increase of axial resolution, thus opening the prospect for a powerful three-dimensional imaging technique with an axial resolution down to 75 nm.

No abstract

No abstract