Steffen Lindek

Confocal microscopy with an increased detection aperture: type-B 4Pi confocal microscopy

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.

Measurement of the 4Pi‐confocal point spread function proves 75 nm axial resolution

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.

Nonlinear absorption extends confocal fluorescence microscopy into the ultra-violet regime and confines the illumination volume

It is shown that two-photon absorption confines the illumination volume and present quantitative evidence that an additional confocal arrangement of the detector further improves the resolution by 48%. The axial resolution in a confocal fluorescence microscope using two-photon absorption with infra-red light is comparable to that achievable with ultra-violet light half the wavelength. An important advantage of two-photon microscopy over single-photon microscopy is that absorption is almost confined to the observed volume. This means no photodamage is caused outside the observed volume.

Enhancing the Axial Resolution in Far-field Light Microscopy: Two-photon 4Pi Confocal Fluorescence Microscopy

Abstract We demonstrate theoretically and experimentally a fourfold increase in axial point resolution in far-field light microscopy. The resolution enhancement is achieved by coherently illuminating the specimen with two opposing objective lenses (4Pi confocal microscopy) and applying two-photon excitation. The point spread function and the axial resolution of this set-up are calculated in optical units. The axial resolution is measured and compared with predictions, both for the confocal and for the 4Pi confocal set-up, in single as well as in two-photon excitation mode.

Confocal theta fluorescence microscopy with annular apertures

In a confocal theta fluorescence microscope, two objective lenses with circular apertures are used, one to illuminate the sample and the other to detect the emitted light at an angle to the illumination axis. We show that annular illumination and detection apertures lead to a reduction in the extent of the point-spread function. A spatial resolution improved by more than 50% can be achieved with a central obstruction blocking the inner 80% of the diameter. For the limit of a very narrow annular aperture and a numerical aperture of 0.75, the volume at half-maximum of the point-spread function is reduced from 15to5 aL. Amixed setup with anannular illumination aperture and a circular detection aperture is also considered.

Optical transfer functions for confocal theta fluorescence microscopy

Optical transfer functions are presented for the confocal theta fluorescence microscope, which uses an orthogonally placed objective lens to detect the fluorescence emission. Therefore the transfer functions do not exhibit cylindrical symmetry about the illumination axis. We show that in an ideal noise-free system, confocal theta microscopy increases the cutoff spatial frequency along the illumination axis by a factor of 3.5 to 3.7 for a numerical aperture of 0.75. In a system with 10% noise the improvement by confocal theta microscopy is even greater, between a factor of 4.1 and 4.4. This explains the improved three-dimensional imaging properties of theta microscopy.

Two New High-Resolution Confocal Fluorescence Microscopies (4Pi, Theta) with One- and Two-Photon Excitation

Improving spatial resolution has been one of the main goals of research since the early beginnings of light microscopy. As a microscope objective lens cannot cover more than 35% of the full solid angle of 4rc steradians, the intensity distribution of a focused spot is elongated along the optical axis and the axial resolution in a conventional microscope is generally many times poorer than the lateral resolution. Much effort has been exerted to reduce the axial extent of this light distribution. An important step toward an improved axial resolution was the development of confocal arrangements (Minsky, 1961; Brakenhoff et al., 1979; Wilson et al., 1980; Wijnaendts van Resandt et al., 1985; Carlsson et al., 1985) that permit the investigation of thick samples along their optical axis. However, the axial resolution in a confocal microscope is still poorer than the lateral resolution (Wilson and Sheppard, 1984, pp. 70–72). Further improvement was limited by the fact that, according to diffraction theory, an improvement is only feasible by decreasing the wavelength or by increasing the numerical aperture (NA) of the objective lens.

Single-lens theta microscopy: Resolution, efficiency and working distance

In single-lens theta microscopy (SLTM), a single objective lens in combination with a mirror unit is used to achieve a theta configuration, which leads to axial and volume resolution improvements. In this type of microscope, the usable numerical aperture (NA) is not limited by the physical size of the objective lenses, as it is in two-lens theta microscopy. Although the outer parts of larger apertures are not entirely used, an increase in volume resolution by a factor of 2.9 can be achieved by increasing the NA from 0.75 to 1.1 in an orthogonal system. In addition, the angle between the faces of the mirror unit can be decreased in order to accommodate even larger NA and thereby to improve the spatial resolution further. The trade-off between resolution and collection efficiency improvements and the usable working distance in SLTM is also quantitatively discussed.

Confocal theta microscopy and 4Pi-confocal theta microscopy

Confocal theta microscopy is a novel method by which the axial resolution in confocal fluorescence microscopy can be substantially improved. The basic idea is to observe the sample with two or more objective lenses and to detect the emission light at an angle theta to the illumination axis. The observation volume is considerably decreased when the detection axis is rotated by 90 degree(s) relative to the illumination axis. This leads to an almost spherical observation volume, which is three times smaller than in a comparable confocal fluorescence microscope. Confocal theta microscopy can be combined with 4Pi-confocal microscopy and is the first viable method proposed for fully exploiting the resolution increase achievable with 4Pi-techniques. The axial side lobes of the 4Pi-point spread function are suppressed, and the observation volume is reduced by a factor of 4. The resolution properties of confocal theta fluorescence microscopies are investigated for single- and two-photon absorption. Evaluations of confocal theta point spread functions are presented and the resolution improvement achieved by theta observation is discussed.