Optical: systems and elements – Deflection using a moving element – Using a periodically moving element
Reexamination Certificate
2002-05-10
2003-02-18
Phan, James (Department: 2872)
Optical: systems and elements
Deflection using a moving element
Using a periodically moving element
C359S212100, C359S201100, C359S202100, C359S205100, C359S208100, C359S368000, C359S372000, C359S900000, C359S225100, C359S226200, C385S083000, C385S088000
Reexamination Certificate
active
06522444
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to the field of confocal microscopes. In particular, it is related to a.novel class of integrated, miniaturized, fiber-coupled, high-precision confocal scanning endoscopes that are particularly suitable for biomedical applications.
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.
In recent years, a great deal of ingenuity has accordingly been devoted to improving the axial resolution of confocal microscopes. A particularly effective 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 described below for the purpose of elucidating the principle of the present invention. A more detailed theory of the 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 confocal 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 synergistic 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.
This enhancement of axial resolution is even more dramatic when this technique is used with relatively low numerical aperture (NA) lenses. 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.
In addition to achieving higher resolution, an angled-dual-axis confocal microscope described above renders a number of additional important advantages. For instance, since the observation beam is positioned at an angle relative to the illumination beam, scattered light along the illumination beam does not easily get passed into the observation beam, except in the region where the beams overlap. This substantially reduces scattered photon.noise in the observation beam, thus enhancing the sensitivity and dynamic range of detection. Moreover, the illumination and observation beams do not become overlapping until very close to the focus—this effect is particularly prominent when using low NA focusing elements (or lenses) for focusing the illumination and observation beams. Therefore, such an arrangement prevents scattered light in the illumination beam from directly “jumping” to the corresponding observation beam, thereby further filtering out scattered photon noise in the observation beam. As such, an angled-dual-axis confocal microscope has much lower noise and is capable of providing much higher contrast when imaging in a scattering medium, rendering it highly suitable for imaging within biological specimens.
Furthermore, in recent years optical fibers have been used in confocal systems to transmit light more flexibly. A single-mode fiber is typically used so that an end of the fiber is also conveniently utilized as a pinhole for both light emission and detection. This arrangement is not susceptible to the alignment problems the mechanical pinholes in the prior art systems tend to suffer. Moreover, the use of optical fibers enables the microscopes to be more flexible and compact in structure, along with greater maneuverability in scanning.
The aforementioned angled-dual-axis confocal arrangement can be further utilized to perform two-photon (and multi-photon) fluorescence microscopy, so as to exploit its high resolution and low noise capabilities. Two-photon (and multi-photon) fluorescence microscopy has been described and performed in the art, as exemplified by Lakowicz et al. in “Two-color Two-Photon Excitation of Fluorescence”, Photochemistry and Photobiology, 64(4), (1996) pp.632-635; by Beaurepaire et al. in “Combined scanning optical coherence and two-photon-excited fluorescence microscopy”, Optics Letters, Vol.24, No.14, (1999) pp. 969-971; and by Lindek et al. in “Resolution improvement in nonconfocal theta microscopy”, Optics Letters, Vol.24, No.21, (1999) pp.1505-1507. In such an arrangement, two illumination beams are directed to intersect optimally, such that each of the two observation beams thus produced is in an optimal confocal arrangement with its corresponding illumination beam. Whereas traditional single-photon fluorescence laser microscopy requires only a single photon &lgr;
3
for excitation, two-photon fluorescence microscopy requires simultaneous absorption of two photons &lgr;
1
and &lgr;
2
for excitation. In terms of energy, hc/&lgr;
3
=hc/&lgr;
1
+hc/&lgr;
2
. Thus, &lgr;
1
and &lgr;
2
are both longer in wavelength than &lgr;
3
. However, it is important to note that &lgr;
2
need not necessarily equal &lgr;
1
. Indeed, any combination of wavelengths can be used, so long as the net energy requirements for exciting a particular type of fluorophores are satisfied. Accordingly, two-photon (or multi-photon) fluorescence microscopy has been used in the art for imaging various types of fluorophores (or fluorophore indicators attached to proteins and biological cells) that are of particular interest to biomedical applications.
The past few years have seen a number of confocal theta microscopes in the art for performing scanning reflectance and fluorescence microscopy, as exemplified by German Patent DE-OS 43 26 473 A1; by Webb et al. in “Confocal microscope with large field and working distance”, Applied Optics, Vol.38, No.22, pp.4870; by U.S. Pat. No. 5,973,828 of; by U.S. Pat. No. 6,064,518 of; by U.S. Pat. No. 5,034,613 of Denk et al.; and by U.S. Pat. No. 6,020,591 of Harter et al. None of these prior art confocal systems, however, perform the scanning microscopy in an angled-dual-axis confocal arrangement that is easily scalable to a small size instrument. Moreover, the designs of these prior art confocal systems are such that they do not lend these systems to be miniaturized confocal scanning endoscopes, suitable for biomedical imaging and other applications where relatively long working distance, large field of view, high resolution, fast scanning, and highly compact and maneuverable imaging
Garrett Mark H.
Kino Gordon S.
Mandella Michael J.
Lumen Intellectual Property Services Inc.
Optical Biopsy Technologies, Inc.
Phan James
LandOfFree
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