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.
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 xe2x80x9creciprocalxe2x80x9d 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 xe2x80x9coptical sectioningxe2x80x9d.
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 (xcex8) 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 xe2x80x9cConfocal microscope with large field and working distancexe2x80x9d, Applied Optics, Vol.38, No.22, pp.4870; and by Stelzer et al. in xe2x80x9cA new tool for the observation of embryos and other large specimens: confocal theta fluorescence microscopyxe2x80x9d, 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 xe2x80x9cmain lobexe2x80x9d 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 xcex8=90xc2x0 in xe2x80x9cFundamental reduction of the observation volume in far-field light microscopy by detection orthogonal to the illumination axis: confocal theta microscopyxe2x80x9d, 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 (xcex8=90xc2x0). 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 90xc2x0. 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 xe2x80x9cConfocal microscope with large field and working distancexe2x80x9d, 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 xcex8. 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 throughout the scanning. U.S. Pat. No. 6,064,518 describes a confocal theta microscope that uses one objective lens for focusing the illumination beam onto an object and the same objective for collecting the observation beam reflected from the object. This system employs a beam deflection unit for directing the illumination beam from the objective lens onto an object and for passing the observation beam reflected from the object to the same objective lens. The patent points out that the scanning may be obtained by either moving the beam deflection unit such that the illumination and observation beams scan the object, or by moving the object itself via a translation stage. However, no specific scanning mechanism is disclosed. The design of this system is such that it does not lend itself to miniaturization and fast scanning as required for in vivo imaging applications. Moreover, it provides inadequate working distance for in vivo imaging of live specimens since the object must be placed between the objective and the deflection unit.
One drawback to translating the object to be scanned is that in many applications it is difficult, if not entirely impossible, to maneuver the object such that high speed and precision scanning is attained. This problem can be particularly acute in imaging objects in scattering media, such as in vivo imaging of live tissue in biological and medical applications. Moving the two objective lenses through separate mechanisms as described in U.S. Pat. No. 5,973,828, on the other hand, requires that the translations of the illumination and observation lenses be coordinated and synchronized such that the main lobes of the illumination and observation point-spread functions intersect optimally at all target points on the path to be scanned. Such coordination can be quite cumbersome to implement, rendering fast and high precision scanning difficult to achieve. Although the possibility of scanning the illumination and observation beams is proposed in U.S. Pat. No. 6,064,518, the particular design of the confocal theta microscope disclosed in this patent does not lend itself to fast and maneuverable scanning using this scheme.
Furthermore, as described in the above prior art, confocal theta microscopes use various mechanical pinholes to provide a point light source and a point detector. The disadvantage with using mechanical pinholes is the lack of flexibility and ruggedness. The optical systems incorporating mechanical pinholes are also difficult to align and miniaturize. In addition, slight misalignment of a mechanical pinhole or any other optical element can result in asymmetric intensity distribution of the light emerging from the pinhole, causing aberrations.
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. U.S. Pat. Nos. 5,120,953, 5,161,053, 5,742,419 and 5,907,425, for instance, disclose conventional reciprocal confocal scanning microscopes using a single optical fiber to transmit light. The end of the fiber provides a point illumination source and a point detector. The scanning in these systems is achieved by maneuvering the fiber end. Confocal theta microscopy is not employed in these systems, however.
Hence, what is needed in the art is a confocal scanning microscope that attains enhanced axial resolution, a long working distance and a large field of view, fast and high precision scanning, without involving complicated coordination of scanning actions. The desired confocal scanning microscope should also achieve high sensitivity and large dynamic range when imaging in a scattering medium, so as to produce high image contrast. Additionally, the desired confocal scanning microscope should have an integrated and scalable structure, rendering it a modular and versatile device.
Accordingly it is a principal object of the present invention to provide an angled-dual-axis confocal scanning microscope for imaging in tissue or a scattering medium that:
a) uses low NA and therefore inexpensive objective lenses;
b) attains improved axial resolution;
c) provides a larger field of view;
d) affords a longer working distance;
e) achieves higher sensitivity and larger dynamic range of detection;
f) is fiber-coupled;
g) has higher power efficiency;
h) performs vertical cross-section scanning; and
i) has small, compact, integrated, and simple construction.
It is a further object of the present invention to provide an assembly of fiber-based angled-dual-axis confocal scanning systems that advantageously combine the angled-dual-axis confocal scanning microscope of the present invention and fiber-optic components.
These and other objects and advantages will become apparent from the following description and accompanying drawings.
This invention provides an angled-dual-axis confocal scanning microscope, comprising an angled-dual-axis confocal scanning head and a vertical scanning unit. The angled-dual-axis confocal scanning head further comprises a first end of a first single-mode optical fiber serving as a point light source, an angled-dual-axis focusing means, a scanning means, and a first end of a second single-mode optical fiber serving as a point light detector.
From the first end of the first optical fiber an illumination beam emerges. The angled-dual-axis focusing means serves to focus the illumination beam to a diffraction-limited illumination focal volume along an illumination axis within an object. The angled-dual-axis focusing means further receives an observation beam emanated from an observation focal volume along an observation axis within the object, and focuses the observation beam to the first end of the second optical fiber. The angled-dual-axis focusing means is so designed that the illumination axis and the observation axis intersect at an angle xcex8 within the object, such that the illumination and observation focal volumes intersect optimally at a confocal overlapping volume. The scanning means, in the form of a single scanning element disposed between the angled-dual-axis focusing means and the object, is positioned such that it receives the illumination beam from the angled-dual-axis focusing means and directs the illumination beam to the object; and that it collects the observation beam emanated from the object and passes the observation beam to the angled-dual-axis focusing means. The scanning means is further capable of pivoting the illumination and observation beams jointly in such a way that the illumination and observation axes remain intersecting optimally at a fixed angle xcex8 and that the confocal overlapping volume moves along an arc-line within the object, thereby producing an arc-line scan. The vertical scanning unit comprises a vertical translation means and a compensation means. The vertical translation means is mechanically coupled to the angled-dual-axis confocal scanning head, such that it causes the angled-dual-axis confocal scanning head to move towards or away from the object, whereby a succession of arc-line scans that progressively deepen into the object is produced, providing a two-dimensional vertical cross-section scan of the object. The compensation means keeps the optical path lengths of the illumination and observation beams substantially unchanged, thereby ensuring the optimal intersection of the illumination and observation focal volumes in the course of vertical scanning. Altogether, the angled-dual-axis confocal scanning microscope of the present invention is designed such that it is capable of performing vertical cross-section scanning in a line-by-line fashion with enhanced axial (i.e., vertical) resolution and greater speed, while maintaining a workable working distance and a large field of view. Additionally, the object may be moved incrementally in a direction perpendicular to the vertical cross-section scan plane as each vertical cross-section scan is completed, resulting in a plurality of vertical cross-section images that can be assembled into a three-dimensional image of a region within the object.
It is to be understood that the term xe2x80x9cemanatingxe2x80x9d as used in this specification is to be construed in a broad sense as covering any light transmitted back from the object, including reflected light, scattered light, and fluorescent light. It should be also understood that when describing the intersection of the illumination and observation beams in this specification, the term xe2x80x9coptimalxe2x80x9d means that the illumination and observation focal volumes (i.e., the main lobes of the illumination beam""s point-spread function and the observation beam""s point-spread function) intersect in such a way that their respective centers substantially coincide and the resulting overlapping volume has comparable transverse and axial extents. This optimal overlapping volume is termed xe2x80x9cconfocal overlapping volumexe2x80x9d in this specification.
In an angled-dual-axis confocal scanning head of the present invention, the angled-dual-axis focusing means generally comprises an assembly of beam focusing, collimating, and deflecting elements. Such elements can be selected from the group of refractive lenses, diffractive lenses, GRIN lenses, focusing gratings, micro-lenses, holographic optical elements, binary lenses, curved mirrors, flat mirrors, prisms and the like. A crucial feature of the angled-dual-axis focusing means is that it provides an illumination axis and an observation axis that intersect optimally at an angle xcex8. The scanning means typically comprises an element selected from the group consisting of mirrors, reflectors, acousto-optic deflectors, electro-optic deflectors, mechanical scanning mechanisms, and Micro-Electro-Mechanical-Systems (MEMS) scanning micro-mirrors. A key feature is that the scanning means is in the form of a single element, as opposed to two or more separate scanning elements in many prior art confocal scanning systems. A preferred choice for the scanning means is a flat pivoting mirror, particularly a silicon micro-machined scanning mirror for its compact and light-weight construction. Moreover, the optical fibers can be single-mode fibers, multi-mode fibers, birefrigent fibers, polarization maintaining fibers and the like. Single-mode fibers are preferable in the present invention, for the ends of single-mode fibers provide a nearly point-like light source and detector.
A unique feature of the angled-dual-axis confocal scanning head of the present invention is that the scanning means is placed between the angled-dual-axis focusing means and the object to be examined. This enables the best on-axis illumination and observation point-spread functions to be utilized throughout the entire angular range of an arc-line scan, thereby providing greater resolution over a larger transverse field of view, while maintaining diffraction-limited performance. Such an arrangement is made possible by taking advantage of the longer working distance rendered by using relatively lower NA focusing elements or lenses in the angled-dual-axis focusing means.
Another important advantage of the angled-dual-axis arrangement of the present invention is that 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 where the beams overlap. This substantially reduces scattered photon noise in the observation beam, thus enhancing the sensitivity and dynamic range of detection. This is in contrast to the direct coupling of scattered photon noise between the illumination and observation beams in a transmission or reciprocal confocal microscope, due to the collinear arrangement between the beams. Moreover, by using low NA focusing elements (or lenses) in an angled-dual-axis confocal scanning system of the present invention, the illumination and observation beams do not become overlapping until very close to the focus. Such an arrangement further prevents scattered light in the illumination beam from directly xe2x80x9cjumpingxe2x80x9d to the observation beam, hence further filtering out scattered photon noise in the observation beam. Altogether, the angled-dual-axis confocal system of the present invention has much lower noise and is capable of providing much higher contrast when imaging in a scattering medium than the prior art confocal systems employing high NA lenses, rendering it highly suitable for imaging within biological specimens.
A further advantage of the present invention is that the entire angled-dual-axis confocal scanning head can be mounted on a silicon substrate etched with precision V-grooves where various optical elements are hosted. Such an integrated device offers a high degree of integrity, maneuverability, scalability, and versatility, while maintaining a workable working distance and a large field of view. In particular, a micro-optic version of an integrated, angled-dual-axis confocal scanning head of the present invention can be very useful in biological and medical imaging applications, e.g., endoscopes and hand-held optical biopsy instruments.
The present invention further provides a first angled-dual-axis confocal scanning system, comprising an angled-dual-axis confocal scanning microscope of the present invention, a light source, and an optical detector. The light source is optically coupled to the second end of the first optical fiber of the angled-dual-axis confocal scanning microscope, providing an illumination beam; and the optical detector is optically coupled to a second end of the second optical fiber of the angled-dual-axis confocal scanning microscope, receiving an observation beam collected from an object. The light source can be a continuous wave (CW) or a pulsed source such as a fiber laser, a semiconductor optical amplifier, an optical fiber amplifier, a semiconductor laser, a diode pumped solid state laser, or other suitable fiber-coupled light source known in the art. The optical detector can be a PIN diode, an avalanche photo diode (APD), or a photomultiplier tube. Such an angled-dual-axis confocal scanning system provides a simple and versatile imaging tool with high resolution and fast scanning capability.
It is known in the art that many biological tissues, such as tendons, muscle, nerve, bone, cartilage and teeth, exhibit birefrigence due to their linear or fibrous structure. Birefrigence causes the polarization state of light to be altered (e.g., rotated) in a prescribed manner upon refection. Skin is another birefrigent medium. Collagen contained in skin is a weakly birefrigent material. At temperatures between 56-65xc2x0 C., collagen denatures and loses its birefrigence. Thus, by detecting induced changes in the polarization state of light reflected from a skin sample, an image representing the regions of skin where thermal injury occurs can be identified. The angled-dual-axis confocal scanning system described above can be modified to image such a birefrigent-scattering (or other polarization-altering) medium. A polarized light source is optically coupled to a second end of the first optical fiber of the angled-dual-axis confocal scanning microscope, providing a polarized illumination beam. The birefrigent (or other polarization-altering) xe2x80x9cscatterersxe2x80x9d emanate an observation beam whose polarization is altered (e.g., rotated) relative to the polarization of the illumination beam. Such a rotated polarization can be represented in two orthogonal polarization components. A polarizing beamsplitter is then optically coupled to a second end of the second optical fiber of the angled-dual-axis confocal scanning microscope, serving to route the two orthogonal polarization components of the observation beam to two separate optical detectors. An image representing the birefrigent (or other polarization-altering) xe2x80x9cscatterersxe2x80x9d can be accordingly constructed.
The present invention also provides an angled-dual-axis confocal scanning module, comprising an angled-dual-axis confocal scanning microscope of the present invention optically coupled to a non-reciprocal three-port optical circulator. The third and first ports of the optical circulator are optically coupled to the second ends of the first and second optical fibers of the angled-dual-axis confocal scanning microscope, respectively; and the second port of the optical circulator serves as a bi-directional input/output port. The configuration of the angled-dual-axis confocal scanning module is such that an illumination beam transmitted to the second port is in turn passed into the third port of the optical circulator and then coupled to the second end of the first optical fiber of the angled-dual-axis confocal scanning microscope in nearly its entirety; and an observation beam collected by the angled-dual-axis confocal scanning microscope is delivered to the first port and then routed to the second port of the optical circulator, to be further utilized or detected in nearly its entirety. As such, the angled-dual-axis confocal scanning module of the present invention provides a modular angled-dual-axis confocal scanning device with a single input/output port, and can be readily adapted in a variety of applications, as the following embodiments demonstrate.
For example, by coupling the angled-dual-axis confocal scanning module of the present invention to a first output aperture of a self-detecting laser source having two output apertures, an illumination beam is transmitted from the first output aperture of the laser to the angled-dual-axis confocal scanning module, and an observation beam collected by the module is in turn back coupled to the laser via the same output aperture. The feedback of the observation beam emanated from an object alters the light intensity as well as the modes supported by the laser cavity, and the resulting changes or perturbations can be detected by coupling an optical detector to a second output aperture of the laser. The presence of the non-reciprocal optical circulator in the angled-dual-axis confocal scanning module allows nearly 100% of the observation beam to be back coupled to the laser, hence maximizing the signal-to-noise ratio in detection. The use of a self-detecting laser as an integrated light source and detector further simplifies the structure of this angled-dual-axis confocal scanning system. Moreover, a frequency shifter (or a phase modulator) can be optically coupled to this angled-dual-axis confocal scanning system, arranged such that the frequency of the observation beam is shifted. The feedback of the frequency-shifted (or phase-modulated) observation beam to the laser results in the laser""s output beam being modulated at a beat frequency, thereby allowing for more sensitive heterodyne detection. The system thus described constitutes the second angled-dual-axis confocal scanning system of the present invention.
If the self-detection laser source is equipped with only one output aperture, the angled-dual-axis confocal scanning module of the present invention can be optically coupled to the laser via a beam-splitting means, such as a 90/10 fiber-optic coupler or other low-coupling tap coupler. The beam-splitting means serves to divert a portion of the laser""s output beam, which carries the perturbations due to the back coupling of the observation beam, to a detection path to which an optical detector may be coupled. Such a system constitutes the third angled-dual-axis confocal scanning system of the present invention.
The self-detecting characteristics of lasers have been advantageously exploited in the art to provide an integrated light source and detector, which also demonstrates the inherent high sensitivity of this method of optical detection. A great deal of effort has also been devoted to eliminate such sensitive feedback effects (e.g., optical isolators with non-reciprocal optical elements such as Faraday rotators are designed to eliminate or block the back-coupling of light). In the present invention, the self-detecting laser can be a fiber laser, a semiconductor laser, or a diode pumped solid state laser. A fiber-based laser system, such as the fiber laser disclosed by the inventors of this application in U.S. Pat. No. 5,887,009, may be used to take advantage of the inherent flexibility of laser cavity parameters. A semiconductor laser may also be desirable as a low cost device.
The angled-dual-axis confocal scanning module of the present invention can also be optically coupled to a light source via a second non-reciprocal, three-port optical circulator. In this embodiment, an output aperture of the light source is optically coupled to a first port of the second optical circulator and a second port of the second optical circulator is in turn optically coupled to the input/output port of the angled-dual-axis confocal scanning module, such that an illumination beam is passed from the light source into the angled-dual-axis confocal scanning module in nearly its entirety. The optical coupling between the second optical circulator and the angled-dual-axis confocal scanning module is preferably provided by a single optical fiber, though other optical coupling means can also be implemented. An observation beam collected by the angled-dual-axis confocal scanning module is then routed to a third port of the second optical circulator, which further leads to a detection path, preferably in the form of a detection optical fiber. An optical detector may be optically coupled to the detection optical fiber. In this angled-dual-axis confocal scanning system, the light source may be any suitable laser or non-laser source, which operates in either continuous or pulsed mode. In fact, a skilled artisan may implement any light source suitable for a given application. Moreover, the non-reciprocal nature of the second optical circulator allows nearly 100% of the observation beam to be used for detection, hence maximizing the signal-to-noise ratio. The system thus described constitutes the fourth angled-dual-axis confocal scanning system of the present invention.
The fourth angled-dual-axis confocal scanning system described above can be further modified into an interferometer configuration, such that the observation beam is combined with a portion of the output beam from the light source to create coherent interference. This can be achieved by inserting a beam-splitting means, such as a fiber-optic coupler or a beamsplitter, between the light source and the second optical circulator. In such an arrangement, the beamsplitting means diverts a portion of the output beam emitted from the light source to the first port of the second optical circulator, which is in turn routed to the angled-dual-axis confocal scanning module, providing an illumination beam. The remainder of the output beam from the light source is diverted to a reference path, preferably in the form of a reference optical fiber, providing a reference beam. The third port of the second optical circulator then routes an observation beam collected by the angled-dual-axis confocal scanning module to a detection path, preferably in the form of a detection optical fiber. The reference and detection optical fibers may be coupled by a 50/50 fiber-optic coupler to mix the observation and reference beams, and produce two outputs with a xcfx80 phase difference for use in a balanced detection scheme. In this way, an interferometer is created and the length of the reference optical fiber can be adjusted to achieve coherent interference between the observation and reference beams.
The system described above, hence the fifth angled-dual-axis confocal scanning system of the present invention, may further include a frequency shifter (or a phase modulator), arranged such that the frequency of either the reference or the observation beam is shifted, so as to generate coherent heterodyne interference between the observation and reference beams. Heterodyne balanced detection technique, well-known in the art of optical coherence tomography (OCT), can be accordingly utilized. An adjustable optical delay device can also be implemented in such a way to maintain coherent interference between the reference and observation beams. If the light source has a short coherence length, then the delay can be adjusted such that only single-scattered light in the observation beam is coherent with the reference beam at the 50/50 fiber-optic coupler and multiple-scattered light, which traverses over a larger optical path length in the observation beam, does not contribute to the coherent interference, therefore providing further filtering of multiple-scattered light. To enhance the signal-to-noise ratio in detection, an optical amplifier, such as a two-port fiber amplifier or semiconductor optical amplifier (SOA), can be coupled to the detection optical fiber, such that the observation beam is amplified. An amplified observation beam also allows faster scanning rates and consequently higher pixel rates without appreciable loss in signal-to-noise ratio, because a shorter integration time per pixel of an image is required in data collection.
The light source in the fifth angled-dual-axis confocal scanning system of the present invention can be an optical fiber amplifier, a semiconductor optical amplifier, a fiber laser, a semiconductor laser, a diode-pumped solid state laser, or a continuous wave or pulsed broadband OCT source having a short coherence length, as is well known in the art. If polarized light is provided by the light source, the beam-splitting means should be a polarizing beamsplitter, such as a polarizing beamsplitter evanescent wave optical fiber coupler, and the various optical fibers in the system should be polarization maintaining (PM) fibers. In this case, the observation and reference beams can be brought into the same polarization by rotation of either the reference or detection optical fiber. Alternatively, a polarization rotation means, such as a Faraday rotator, can be coupled to either the reference or detection optical fiber, such that the reference and observation beams have substantially the same polarization when combined. Furthermore, the 50/50 fiber-optic coupler can be a polarization maintaining fiber coupler to optimally mix the polarized observation and reference beams.
A distinct advantage of the angled-dual-axis confocal scanning microscope of the present invention is that the scanning is achieved by pivoting both the illumination and observation beams, as opposed to moving either the object or the microscope""s objective lenses in the prior art confocal theta scanning microscopes, which adversely limits the speed and maneuverability of scanning. Moreover, a single-element scanning means, such as a micro-machined scanning mirror, is used to pivot the illumination and observation beams jointly, in contrast to the prior art systems where the two beams are scanned individually by way of moving the microscope""s objectives lenses separately, which requires precise synchronization and coordination in maneuvering the lenses. In addition, by disposing the scanning means between the angled-dual-axis focusing means and the object, fast and high-precision scanning at high resolution is obtained over a large field of view. Such an arrangement takes advantage of the long working distance rendered by using low NA focusing elements (or lenses). Another important advantage gained by using low NA focusing elements is that the illumination and observation beams do not become overlapping until sufficiently close to the focus. This prevents scattered light in one beam from directly xe2x80x9cjumpingxe2x80x9d to another beam, hence eliminating scattered photon noise in the observation beam. Furthermore, low NA lenses can be easily designed for aberration correction, thus allowing diffraction-limited performance at relatively low cost. In the present invention, diffraction-limited focusing is only required xe2x80x9con-axisxe2x80x9d, hence further simplifying the lens requirements. The angled-dual-axis confocal scanning microscope of the present invention further advantageously exploits the flexibility, scalability and integrity afforded by optical fibers and silicon micro-machining techniques, rendering it a highly versatile and modular device. Accordingly, the angled-dual-axis confocal scanning microscope of the present invention is particularly suited for applications in which high resolution and fast scanning are required, such as in vivo imaging of live tissue for performing optical biopsies in medical applications.
By integrating the angled-dual-axis confocal scanning microscope of the present invention with fiber-optic components and a fiber-coupled laser, the angled-dual-axis confocal scanning systems of the present invention provide a diverse assembly of fiber-based, high resolution and fast scanning systems that can be adapted in a variety of applications, such as in biological and medical imaging, and industrial applications.
The novel features of this invention, as well as the invention itself, will be best understood from the following drawings and detailed description.
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