Optical: systems and elements – Deflection using a moving element – Using a periodically moving element
Reexamination Certificate
2000-11-01
2002-04-09
Robinson, Mark A (Department: 2872)
Optical: systems and elements
Deflection using a moving element
Using a periodically moving element
C359S214100, C359S372000, C359S385000
Reexamination Certificate
active
06369928
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to the field of confocal microscopes and two-photon fluorescence microscopy. In particular, it is related to an assembly of fiber-coupled, angled-dual-illumination-axis confocal scanning microscopes with integrated structure, enhanced resolution, higher sensitivity, and versatile imaging capabilities.
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 using high numerical aperture (NA) lenses. 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 “Fundamental reduction of the observation volume in far-field light microscopy by detection orthogonal to the illumination axis: confocal theta microscopy” of Stelzer et al., Optics Communications 111 (1994), pp.536-547; U.S. Pat. No. 5,973,828; “Confocal microscope with large field and working distance” of Webb et al., Applied Optics, Vol.38, No.22, pp.4870; and “A new tool for the observation of embryos and other large specimens: confocal theta fluorescence microscopy” of Stelzer et al., Journal of Microscopy, Vol.179, Part 1, pp. 1; all incorporated by reference. It should be noted that high NA objectives are used in these prior art systems to achieve high resolution.
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 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.
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, by using low NA focusing elements (or lenses) for focusing the illumination and observation beams, the illumination and observation beams do not become overlapping until very close to the focus. 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 using relatively low NA lenses 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.
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. 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 the particular types of fluorophores being used are satisfied. An inherent advantage of two-photon fluorescence is that the two-photon absorption occurs only within a confined region where the two incident beams overlap, hence eliminating unwanted, spurious fluorescence and scattered light. Moreover, because two-photon excitation depends on the square of the excitation power, the excited volume is restricted to the focal point, providing an equivalent of confocal conditions. Additional advantages provided by two-photon (and multi-photon) excitation include longer penetration depth within a specimen (since longer wavelengths are employed, thus reducing scattering losses), reduced photobleaching and phototoxicity, and reduced background noise.
Accordingly, two-photon excitation has been of considerable interest for microscopy, fluorescence spectroscopy, and for single-molecule detection. For instance, two-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. It has also been used as an alternative way of attaining enhanced resolution and greater flexibility in imaging. The prior art effort in utilizing two-photon microscopy is exemplified by U.S. Pat. No. 5,034,613 of Denk et al.; U.S. Pat. No. 6,020,591 of Harter et al.; “Two-color Two-Photon Excitation of Fluorescence” by Lakowicz et al. in Photochemistry and Photobiology, 64(4), (1996) pp.632-635; “Combined scanning optical coherence and two-photon-excited fluorescence microscopy” by Beaurepaire et al. in Optics Letters, Vol.24, No.14, (1999) pp. 969-971; and “Resolution improvement in nonconfocal theta microscopy” by Lindek et al. in Optics Letters, Vol.24, No.21, (1999) pp.1505-1507. None of these prior art systems, however, exploit advantages gained by using relatively low NA lenses and hence allow themselves to be miniaturized or have sufficiently long working distances needed for in-vivo biological applications. Moreover, the scanning me
Garrett Mark H.
Kino Gordon S.
Mandella Michael J.
Lumen Intellectual Property Services Inc.
Optical Biopsy Technologies, Inc.
Robinson Mark A
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