1. Field of Invention
The field of the currently claimed embodiments of this invention relates to motion-compensated confocal microscopes.
2. Discussion of Related Art
Confocal microscopy is a well-established 3-D imaging technique with high lateral and axial resolution [1]. The concept of using fiber-optic-component based confocal microscopy has been demonstrated to show high stability, ease of use, and flexibility [2-4]. Flexible coherent fiber bundles—consisting of tens of thousands of fiber channels—have been widely implemented for use in endoscopic confocal reflectance microscopy [5,6], two-photon laser scanning [7], and optical coherence tomography [8-10]. This design allows for a scan-less probe and probe miniaturization. It also has the advantage of separation of the scanning end and sample end and miniaturization. To improve imaging quality in vivo, a lens system must be customized and fitted to the fiber bundle. Confocal microscopy, based on a pair of GRIN lenses or objective lenses attached to a fiber bundle probe, has been studied [5, 11]. However, in vivo imaging of live samples can be significantly degraded due to the motions of live samples such as breathing, heart-beating, blood-flowing, and other physiological activities. Such motions result in intra- and inter-frame distortions, or even loss of the whole image frame [12]. For example, during the imaging of an embryo of a fruit fly during stem cell study, the accumulated muscle motion effect of the embryo can cause the imaging area to be completely out of the view. Thus, motion compensation is critical to obtaining reasonable confocal imaging in vivo—especially when video imaging is required. Therefore, there remains a need for improved motion-compensated confocal microscopes.
A motion-compensated confocal microscope according to an embodiment of the current invention includes a laser scanning system, a fiber-optic component having a proximal end and a distal end such that the fiber-optic component is optically coupled to the laser scanning system to receive illumination light at the proximal end and to emit at least a portion of the illumination light at the distal end, and a detection system configured to receive and detect light returned from a specimen being observed and to output an image signal. The light returned from the specimen is received by the distal end of the fiber-optic component and transmitted back and out the proximal end of the fiber-optic component. The motion-compensated confocal microscope also includes a motion compensation system connected to at least one of the distal end of the fiber-optic component or to the specimen to move at least one of the distal end of the fiber-optic component or the specimen to compensate for relative motion between the distal end of the fiber-optic component and a portion of the specimen being observed.
Further objectives and advantages will become apparent from a consideration of the description, drawings, and examples.
Some embodiments of the current invention are discussed in detail below. In describing embodiments, specific terminology is employed for the sake of clarity. However, the invention is not intended to be limited to the specific terminology so selected. A person skilled in the relevant art will recognize that other equivalent components can be employed and other methods developed without departing from the broad concepts of the current invention. All references cited anywhere in this specification, including the Background and Detailed Description sections, are incorporated by reference as if each had been individually incorporated.
Some embodiments of the current invention provide a motion compensated fiber-optic confocal microscope system. Some examples demonstrate employing a Fourier domain common-path optical coherence tomography (CP-OCT) distance sensor and a high-speed linear motor at the distal end of the fiber optic confocal microscope imaging probe according to an embodiment of the current invention. The fiber-optic confocal microscope in this example is based on a 460 micron diameter fiber bundle terminated with a gradient index (GRIN) lens. Using the peak detection of 1-D A-scan data of CP-OCT, the distance deviation from the focal plane was monitored in real-time. When the distance deviation surpasses a pre-determined value, the linear motor moves the confocal microscope probe to maintain the deviation within a predetermined value. The motion compensation was achieved for a confocal microscope imaging rate of 1 Hz, with an average distance error of 2 microns in the examples.
A system according to some embodiments of the current invention can correct intra-frame and inter-frame distortion caused by biological activities of live samples, for example, such as breathing, heart beating, and blood flowing during in vivo confocal microscopy imaging to improve the imaging quality of confocal microscopes.
For imaging probes with a small field of view held perpendicular to the samples, image distortion is mainly caused by the axial motion of the sample which moves the sample's imaging surface away from the focal plane. Common-path optical coherence tomography (CP-OCT) has recently been demonstrated with its ability to precisely sense distance and has compact features for instrument integration [13-15].
The motion-compensated confocal microscope 100 further includes a motion compensation system 112 connected to at least one of the distal end 108 of the fiber-optic component 104 or to the specimen to move at least one of the distal end 108 of the fiber-optic component 104 or the specimen to compensate for relative motion between the distal end 108 of said fiber-optic component 104 and a portion of the specimen being observed.
In an embodiment of the current invention, the motion compensation system 112 can include a distance detector 114 arranged to detect a relative distance between the distal end 108 of the fiber-optic component 104 and the portion of the specimen being observed. The distance detector 114 can be a common-path Fourier domain optical coherence tomography system in an embodiment that includes an optical fiber probe 116 having an end fixed at a substantially constant position relative to the distal end 108 of the fiber-optic component 104.
In an embodiment of the current invention, the motion compensation system 112 can also include a moveable stage 118 attached to the distal end 108 of the fiber-optic component 104. Although not shown in
In an embodiment of the current invention, the fiber-optic component 104 can be, or can include, an optical fiber bundle. The fiber-optic component 104 can further include a gradient refractive index (GRIN) lens at the distal end 108 of the fiber-optic component 104 according to some embodiments of the current invention. In some embodiments, two or more GRIN lenses can be used. In another embodiment of the current invention, the fiber-optic component 104 can further include an imaging system at the distal end 108 of the fiber-optic component 104.
In some embodiments of the current invention, the laser scanning system 102 can further include a light scanning unit 120 configured to scan a laser beam of light across the proximal end 106 of the fiber-optic component 104 to thereby scan illumination and detection across a portion of the specimen. In an embodiment of the current invention, the laser scanning unit 120 can include a Galvanic mirror system, for example.
In some embodiments of the current invention, the motion compensation system 112 can perform motion compensation in real time such that the motion compensation is performed on a frame-by-frame basis as the laser scanning unit 120 completes each scan.
The following examples are provided to help explain some concepts of the current invention. The broad concepts of the current invention are not limited to these specific examples.
GRIN Lens Terminated Fiber Bundle Probe
A fiber bundle probe terminated with GRIN lenses was assembled by gluing two GRIN lenses together at the distal end of a fiber bundle (Fujikura FIGH-10-500N, with an imaging diameter of 460 μm and 10K fiber cores) using UV curing adhesive. We used the GRIN lenses (NT64-525, 0.25 pitch, N.A.=0.55 and NT64-526, 0.23 pitch, N.A.=0.55) from Edmund Optics. The length of the 0.25 pitch lens was 4.34 mm; the length of the 0.23 pitch lens was 3.96 mm. When assembled, Zemax [16] simulation showed a working distance of 200 microns with a 1× image magnification. However, our experiment showed a working distance of ˜140 microns with a 1× image magnification for the probe. This was due to the forming of a small gap between the two GRIN lenses during the assembling process.
We built an axial motion-compensated confocal microscope system according to an embodiment of the current invention by combining a fiber-bundle-based confocal microscope with a CP-OCT distance sensor. The schematic of the whole system is shown in
A CP-OCT distance-sensing system was operated separately with the confocal scanning system. The light from a SUPERLUM Broadband Light Source (center wavelength: 878.6 nm, bandwidth: 180 nm) was coupled into a single-mode fiber by a 50/50 broadband coupler. The single-mode fiber probe was cleaved in a right angle to provide reflection at the fiber end. The Fresnel reflection at the fiber tip served as reference light. A needle tube was used to protect the single-mode fiber reference surface by leaving a distance offset between the fiber inside the tube and the tube tip. The back-reflected/scattered light from the reference and the sample was directly coupled into the fiber and routed by the coupler to a customized spectrometer.
The fiber bundle scanning probe and the single-mode fiber probe were glued together at the probe stage, which was connected to the shaft of a high-speed linear motor (LEGS-L01S-11, Piezo LEGS). We used Workstation (DELL, Precision T7500) to obtain the distance information from the CP-OCT signal and deliver commands to the linear motor through a motor driver.
The LEGS-L01S-11 has a 35 mm travel range, 20 mm/s maximum speed, less than 1 nm resolution depending on different control modes, and a 10N maximum driving force. The CP-OCT system has an axial resolution of 3.6 micron in air and 2.8 micron in water. Using the peak detection [17], we achieved a position accuracy of 1.6 micron. The reference signal is obtained from a partial reflector near the distal end of the fiber-optic probe. Any distance can be measured from the reference plane by analyzing the CP-OCT spectral signal where the absolute value of the optical distance can be simply calculated from d=λ2/2nδλ where δλ, is the spectral modulation period detected by the OCT spectrometer and n is the refractive index. To validate its accuracy, we measured the d deduced from the OCT corresponding to the change in displacement of nerve tissue placed on top of a precision translation stage. We found a distance error of ±1.6 μm. As long as the OCT peak is above 10 dB above the noise floor, the distance accuracy remains relatively constant and for most surfaces provides more than 30 dB peaks. The system control flowchart is shown in
The fiber bundle has 10K cores and the imaging plain was over-sampled 200 pixels by 200 pixels (460 micron by 460 micron) to follow the Nyquist Sampling theorem. To obtain good image quality, the data card was set at a sampling rate of 40 K/s, which sets the imaging frame rate to ˜1 fps. NBS 1963A Resolution Target was used as test sample. By choosing a pinhole size of ˜50 micron in front of the detector, we effectively suppressed background and non-signal rays while maintaining a relatively high sensitivity. The axial resolution of the confocal system was ˜40 microns, this was measured using a mirror as a target and moving the target along the axial direction of the confocal microscope. The peak signal to noise ratio measured using the mirror target was 22 dB. A typical SNR for the airforce target was 20 dB. When we moved the glass sample 50 microns away from the focal plane, the target ‘number 6’ completely disappeared, as shown in
We placed the sample on a Newport XYZ 3D translation stage to simulate target movement. During image acquisition, the stage was driven back and forth along the axial direction of the probe. Without the motion compensation, some part of the frame comes into focus while some part of the frame is out of focus (intra-frame distortion). The consequence of the target movement is that some part of the image will be annulled by the depth discrimination of the confocal microscopy. With the motion compensation, the whole frame remains in focus, as is shown in
To show the influence of the motion compensation on inter-frame distortion, two sets of images were recorded over 50 seconds as is shown in
In this example, our results indicate that the system can compensate motion amplitude up to 60 microns at the rate of 840 Hz while maintaining a sample focus error within 5 microns. However, the concepts of the current invention are not limited to this example.
The embodiments illustrated and discussed in this specification are intended only to teach those skilled in the art how to make and use the invention. In describing embodiments of the invention, specific terminology is employed for the sake of clarity. However, the invention is not intended to be limited to the specific terminology so selected. The above-described embodiments of the invention may be modified or varied, without departing from the invention, as appreciated by those skilled in the art in light of the above teachings. It is therefore to be understood that, within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described.
This application claims priority to U.S. Provisional Application No. 61/482,300 filed May 4, 2011, the entire contents of which are hereby incorporated by reference.
This invention was made with Government support of Grant No. R21 1R21NS063131-01A1, awarded by the Department of health and Human Services, The National Institutes of Health (NIH); and Grant No. IIP-0822695, awarded by NSF. The U.S. Government has certain rights in this invention.
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/US2012/036643 | 5/4/2012 | WO | 00 | 10/30/2013 |
Number | Date | Country | |
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61482300 | May 2011 | US |