Radiant energy – Photocells; circuits and apparatus – Photocell controls its own optical systems
Reexamination Certificate
2000-07-28
2002-07-23
Kim, Robert H. (Department: 2882)
Radiant energy
Photocells; circuits and apparatus
Photocell controls its own optical systems
C250S235000, C359S215100, C359S385000
Reexamination Certificate
active
06423956
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to the field of confocal microscopes, and in particular, to a new class of fiber-coupled, angled-dual-axis confocal scanning microscopes with integrated structure, enhanced resolution, low noise, and vertical cross-section scanning.
BACKGROUND ART
The advent of fiber optics and laser technology has brought a renewed life to many areas of conventional optics. Confocal microscopes, for example, have enjoyed higher resolution, more integrated structure, and enhanced imaging capability. Consequently, confocal microscopes have become increasingly powerful tools in a variety of applications, including biological and medical imaging, optical data storage and industrial applications.
The original idea of confocal microscopy traces back to the work of Marvin Minsky. Described in his seminal U.S. Pat. No. 3,013,467 is a system in which an illumination beam passes through a pinhole, traverses a beamsplitter, and is focused by an objective to a focal volume within an object. An observation beam that emanates from the focal volume is in turn converged by the same objective lens, reflected by its second encounter with the beamsplitter, and passes through a second pinhole to an optical detector. The geometry of this confocal arrangement is such that only the light beam originating from the focal volume is able to pass through the second pinhole and reach the optical detector, thus effectively discriminating all other out-of-focus signals.
Contemporary confocal microscopes tend to adopt one of two basic confocal geometries. In the transmission arrangement using two objectives, one objective focuses an illumination beam from a point source onto a focal volume within an object and another objective collects an observation beam that emanates from the focal volume. Whereas in the so-called “reciprocal” reflection arrangement, a single objective plays a dual role of focusing light on the object and collecting the light emanated from the object. In either case, the confocal arrangement enables the confocal microscope to attain a higher resolution and sharper definition than a conventional microscope, because out-of-focus signals are rejected. This unique ability has made confocal microscopes particularly useful tools in the examination of biological specimens, since they can view a specific layer within a sample and avoid seeing other layers, the so-called “optical sectioning”.
In order to image a thin layer about a few micrometers thick within a sample, however, the numerical aperture (NA) of the objective lenses must be large, so as to provide adequate resolution particularly in the axial direction. This generally results in a short working distance, which is undesirable in practice. Moreover, when imaging within tissue or scattering media, the signal is typically dominated by scattering from points far away from the focus of the large NA objective, thus resulting in noisy (low contrast) images.
A great deal of ingenuity has accordingly been devoted to improving the axial resolution of confocal microscopes without using high NA lenses. A particularly interesting approach is to spatially arrange two separate illumination and observation objective lenses, or illumination and observation beam paths, in such a way that the illumination beam and the observation beam intersect at an angle theta (&thgr;) at the focal points, so that the overall point-spread function for the microscope, i.e., the overlapping volume of the illumination and observation point-spread functions results in a substantial reduction in the axial direction. A confocal microscope with such an angled, dual-axis design is termed a confocal theta microscope, or an angled-dual-axis confocal microscope, hereinafter. Its underlying theory is stated below for the purpose of elucidating the principle of this invention. A more detailed theory of confocal theta microscopy can be found in U.S. Pat. No. 5,973,828; by Webb et al. in “Confocal microscope with large field and working distance”, Applied Optics, Vol.38, No.22, pp.4870; and by Stelzer et al. in “A new tool for the observation of embryos and other large specimens: confocal theta fluorescence microscopy”, Journal of Microscopy, Vol.179, Part 1, pp. 1; all incorporated by reference.
The region of the point-spread function of a microscope's objective that is of most interest is the region in which the point-spread function reaches its maximum value. This region is referred to as the “main lobe” of the point-spread function in the art. It is typically characterized in three dimensions by an ellipsoid, which extends considerably further in the axial direction than in the transverse direction. This characteristic shape is the reason that the axial resolution is inherently poorer than the transverse resolution in a conventional confocal microscope. When the main lobes of the illumination and observation point-spread functions are arranged to intersect at an angle in a confocal theta microscope, however, a predominantly transverse and therefore narrow section from one main lobe is made to multiply (i.e., zero out) a predominantly axial and therefore long section from the other main lobe. This optimal multiplication of the two point-spread functions reduces the length of the axial section of the overall point-spread function, thereby enhancing the overall axial resolution. The shape of the overall point-spread function can be further adjusted by varying the angle at which the main lobes of the illumination and observation point-spread functions intersect.
The past few years have seen a few confocal theta microscopes with similar designs in the art. For example, Stelzer et al. describe the theory of confocal theta microscopy with two and three objective high NA lenses and an angle of &thgr;=90° in “Fundamental reduction of the observation volume in far-field light microscopy by detection orthogonal to the illumination axis: confocal theta microscopy”, Optics Communications 111, pp.536. German Patent DE-OS 43 26 473 A1 demonstrates a confocal theta microscope in which the axes of two high NA objective lenses are oriented at a right angle (&thgr;=90°). It also discloses a confocal theta microscope with three high NA objective lenses, in which the axes of two objectives are perpendicular to each other, while the axis of the third objective lies on the axis of one of the other two objectives. The patent does not disclose how scanning is carried out in these confocal theta microscopes. Although scanning might be performed by translating the object to be examined in such systems, the designs of these confocal theta microscopes are such that they do not appear to readily accommodate scanning that probes into the interior of the object. U.S. Pat. No. 5,969,854 discloses a confocal theta microscope with the axes of two high NA objective lenses positioned at an angle approximately 90°. This system incorporates a scanning mechanism that translates the object for imaging purposes.
Webb et al. describe a confocal scanning microscope with angled objective lenses that have relatively low NA in “Confocal microscope with large field and working distance”, Applied Optics, Vol.38, No.22, pp.4870. The design of this microscope attains usable resolutions for biological applications in both transverse and axial directions, while achieving a large field of view and a long working distance. The scanning in this case is achieved by moving a stage on which the object is mounted. U.S. Pat. No. 5,973,828 discloses a confocal theta scanning microscope in which the axes of two objective lenses intersect at a variable angle &thgr;. Two-dimensional scanning is achieved by steering the illumination and observation beams in the back focal plane of the objective lenses to provide scanning in one direction, and by separately moving and coordinating the illumination and observation lenses to bring about overlap of the focal volumes during scanning in the other direction. It is disclosed that without such coordination the overlap cannot be maintained throu
Garrett Mark H.
Kino Gordon S.
Mandella Michael J.
Kao Chih-Cheng Glen
Kim Robert H.
Optical Biopsy Technologies
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