August 15, 2013 / Vol. 38, No.

16 / OPTICS LETTERS

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Miniature varifocal objective lens for endomicroscopy

Dimitre G. Ouzounov,1,* David R. Rivera,1 Watt W. Webb,1 Julie Bentley,2 and Chris Xu1
1

School of Applied and Engineering Physics, Cornell University, Ithaca, New York 14853, USA
2

The Institute of Optics, University of Rochester, Rochester, New York 14627, USA *Corresponding author: dgo4@cornell.edu Received May 23, 2013; revised July 8, 2013; accepted July 9, 2013; posted July 9, 2013 (Doc. ID 190790); published August 13, 2013

A miniature catadioptric lens for endoscopic imaging based on the principle of wavelength division multiplexing is presented. We demonstrate change of the magnification and the field of view (FOV) of the lens without any mechanical adjustment of the optical elements. The lens provides magnifications of 1.5 at 406750 nm and 0.2 at 800 nm. The lens is used to demonstrate large-FOV (1.3 mm) reflectance imaging and high-resolution (0.57 m) multiphoton fluorescence imaging of unstained mouse tissues. 2013 Optical Society of America OCIS codes: (120.3890) Medical optics instrumentation; (170.3880) Medical and biological imaging; (180.4315) Nonlinear microscopy. http://dx.doi.org/10.1364/OL.38.003103

A challenge faced in the development of clinically useful endoscopes is the desire to provide a large field of view (FOV) and high spatial resolution within a miniature probe. A large FOV is useful so that a clinician can quickly survey a large tissue area, whereas high spatial resolution is necessary to resolve cellular details for accurate medical diagnosis. Because these two requirements cannot be achieved simultaneously by a miniature objective lens, optical zoom is an essential functionality for practical endoscopes. To date, many clinical biopsy procedures are guided by optical endoscopes. Although conventional wide-field endoscopes have large FOVs, they provide only a gross inspection of tissue morphology, which is often inadequate for reliable diagnostics. Over the past decade, a number of new high-spatial-resolution imaging modalities (e.g., optical coherence tomography [1], multiphoton microscopy [2,3], and confocal microscopy [4]) have been used for endoscopic imaging [513]. Although each imaging modality derives its contrast from different physical phenomena, the endoscopic probes have similar architectures [14], including optical fiber delivery of excitation light and collection of relevant signal, a scanning mechanism (e.g., fiber scanner, microelectromechanical systems), and a miniature objective lens. A variety of objective lenses have been used: simple monolithic gradient-index (GRIN) lenses [1519], compound GRIN lenses, [12,20,21], compound spherical [10,22] or aspherical lenses [11,23], micromachined lenses [24], and glass or plastic lenses [25,26]. These objective lenses perform successfully in terms of resolution, size, and cost, but achieving optical zoom capability in miniature endoscopes has been elusive. Currently, pathologists study ex vivo biopsy samples with at least two optical magnifications (e.g., a 4 objective for viewing the tissue architecture and a 20 or 40 objective for resolving cellular details). This variable magnification and FOV is invaluable in helping obtain an accurate diagnosis of a patients health state. While optical zoom capability is easily implemented in a conventional light microscope by switching among multiple objective lenses, this mechanical approach is difficult to implement in a miniature endoscope due to size limitations. For practical implementation, both large FOV imaging and high-resolution imaging must be obtained
0146-9592/13/163103-04$15.00/0

with the same endoscope apparatus without mechanical adjustments of the distal endoscope parts. Chen et al. reported [27] a small dual-zone lens, but nearly half of the excitation power incident on the sample does not produce useful information. An optical endoscope that provides high-spatial-resolution imaging with optical zoom capability has never before been demonstrated. Traditional varifocal/zoom lenses, including catadioptric lenses [2831], achieve their functionality by moving one or more of their components. In this Letter, we present a miniature catadioptric varifocal lens based on the idea of separating the optical paths of excitation light with different wavelengths. The lens provides two magnifications and FOVs, and the zoom operation is achieved by changing the wavelength of the excitation light without any mechanical adjustments of the lens components. The objective lens consists of 3 elements, has a 3 mm outside diameter (OD) and is 8 mm in length [Fig. 1(a)]. The variation of magnification and FOV is enabled by the dichroic coatings deposited at the central part of the proximal surface of element 3 and at the peripheral region of the proximal surface of element 2 [Fig. 1(a)]. The central part of the proximal surface of element 2 is uncoated. Starting from the left in Fig. 1(a), the excitation light emerges from the delivery/scanning fiber and is directed by element 1 onto the dichroic coating at the central part of the proximal surface of element 3. Depending on the incident wavelength (i ), the excitation light is either reflected (e.g., i 800 nm) to the patterned dichroic coating on the proximal surface of element 2 and then focused to the sample with high numerical aperture (NA), or transmitted (e.g., i 406 nm) and focused to the sample with low NA. Therefore, the change of magnification and FOV is achieved simply by changing the wavelength of the excitation light without any mechanical adjustment. The high-resolution multiphoton imaging mode is designed (Table 1) to operate between 800 and 900 nm. At i 800 nm, the calculated full width at halfmaximum (FWHM) of the lateral point spread function (PSF) is 0.7 m [Fig. 3(a)], and the Strehl ratio is approximately 1 over the central 160 m FOV, indicating diffraction-limited optical performance. The
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Fig. 2. Lens characterization and imaging setup.

Fig. 1. (a) Lens design layout, drawn to scale. (b) Transmission spectrum of the patterned dichroic coating deposited on the proximal surface of element 2 (shown in the inset). The patterned structure of the coating is clearly seen. (c) Transmission spectrum of the dichroic coating deposited on the proximal surface of element 3 (shown in the inset). (d) 3D lens layout, indicating the positions of the plastic fibers for signal collection. (e) Photograph of the objective lens assembly.

low-magnification imaging mode (Table 1) operates between 350 and 730 nm. At i 406 nm, the lateral resolution (FWHM) is 4.5 m, and the FOV is 1.3 mm. We chose 406 nm for one-photon reflectance imaging since it is one of the preferred excitation wavelengths for narrowband imaging [32]. In both imaging modes, the focal planes of the miniature zoom lens have small curvatures. Such a curved image plane allows significantly better aberration correction than a planar image plane and is inconsequential for in vivo tissue imaging. The radius of curvature of the image surface is 1.09 and 2.01 mm for high-magnification and low-magnification modes, respectively. For in vivo imaging, this is not a problem because tissue features are not flat. Since the plastic optical fibers (POFs) collect through nonimaging pathways, the curvature of the imaging surface will have little effect on signal collection. We performed tolerance analysis by calculating the root-mean-squared (rms) wavefront error, that is, deviations from an ideal spherical wavefront. The nominal error of the lens design is 0.007. We performed Monte Carlo simulations with tolerances listed in Table 2; for 5000 runs, more than 90% of
Table 1. Design Specifications for Miniature Zoom Objective Parameters High-Mag Mode Low-Mag Mode

Sample space Water Air Magnification 0.2 1.47 NA 0.55 (at 800 nm) 0.075 (at 405 nm) FOV 160 m 1.3 mm One-photon resolution 0.7 m 4.5 m Wavelength 800900 nm 350730 nm

the trials had an rms wavefront error less than 0.05, which is below the accepted practical limit (0.07) for a diffraction-limited optical system. The dichroic coatings on elements 2 and 3 [Figs. 1(a) and 1(b)] that enable the varifocal operation lead to nonreciprocal propagation of the excitation and the fluorescence light. Therefore, epicollection of the two-photon excited fluorescence signal through the excitation pathway is inefficient. As a result, we use 10 large-core (500 m OD) POFs (ESKA acrylic, Mitsubishi) located just behind element 2 [Fig. 1(d)] to collect the fluorescence signal. The same POFs collect the scattered light at low-magnification mode. Different locations of the imaging planes result in different collection efficiency, which is smaller for the low-magnification mode due to the longer working distance. However, at low magnification the signal level is much higher due to the linear contrast mechanism. The optical elements were manufactured [Fig. 1(e)] and assembled by Optics Technology Inc., Pittsford, New York. We tested the performance of this miniature objective by imaging a US Air Force (USAF) test target in transmission. The experimental setup is shown in Fig. 2. Characterization and imaging in high-resolution mode is done with a mode-locked Ti:sapphire laser (Tsunami, Spectra Physics) operating at 800 nm. For low-magnification, large-FOV imaging, we use a fiber-coupled continuous wave semiconductor laser operating at 406 nm (LP406SF20, Thorlabs). The laser beam is raster-scanned by a pair of galvo mirrors and is focused to the object plane of the zoom endoscope lens by a low-NA objective (Olympus, 0.1 NA, 4). We analyzed the intensity line profile across the indicated feature [Fig. 3(a)] to quantify the lateral resolution for both modes. The intensity profile at the edge of the feature is the edge-response function, and its derivative is the line-spread function, which corresponds to the cross section of the PSF. The lateral resolution of the high-magnification mode (FWHM) is 0.75 m [Fig. 3(a)], which corresponds to a two-photon resolution (FWHM) of 0.5 m. The side lobes of the PSF are caused by the obstruction at the center of the back aperture, which is a well-known limitation for a reflective objective lens. However, these side lobes will be significantly suppressed in two-photon excitation because of

August 15, 2013 / Vol. 38, No. 16 / OPTICS LETTERS Table 2. Lens Prescription and Tolerances for Design in Fig. 1a Radius Value, (mm) infinity 2.218 3.022 1.6 1.6 infinity 1.092 Radius Tolerance (fr/mm) 5 fringe 0.05 0.01 0.01 0.01 5 fringe Thickness Value (mm) 1 1 3 1.567 2.269 0.1 Thickness Tolerance (mm) 0.1 0.15 0.25 0.01 0 0.0 0.1 Index Tolerance 0.001 0.001 0.001 0.02 Wedge/ Roll TIRb (mm) 0.05 0.05 0.004 0.004 Irregularity Tolerance (fringes) 0.25 0.25 0.1 0.1 0.25 0.25

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Elements Fiber tip Element 1R1 Element 1R2 Element 2R1 Element 2R2 Element 3R1 Element 3R2 Image
a b

Glass air silica air silica NLAF2 seawater seawater

Image plane values are for high-magnification mode. Total indicator reading.

the quadratic dependence on excitation intensity. The FOV of the high-magnification mode is 160 m [Fig. 3(b)]. The output beam of the miniature zoom lens operating in high-magnification mode has an annular profile, which reduces the axial resolution [33,34]. We characterized the two-photon axial resolution of the high-magnification mode by stepping a 500 nm Rhodamine B thin film through its focus. The measured FWHM of the thin film response is 10.5 m [Fig. 3(d)], which is sufficient for resolving cellular layers in biological tissues. Using the USAF test target, the one-photon lateral resolution of the low-magnification mode was measured to be 4.5 m. The low-magnification imaging mode achieved a large FOV of 1.3 mm [Fig. 3(c)]. The transmission of the varifocal lens is 40% and 70%, respectively, for the high- and low-magnification modes. In this prototype, the only coatings deposited were the patterned dichroic coatings on the proximal surfaces of elements 2 and 3 (Fig. 1) needed for varifocal operation. Antireflection coatings on the other optical

Fig. 3. (a) Calculated (red solid line) and measured (blue dashed line) lateral PSF for the high-resolution imaging mode. Inset: Group 9 of USAF resolution target imaged using the highresolution imaging mode at 800 nm. Inset: Intensity line profile across the indicated feature and its derivative. (b) USAF resolution target imaged using the high-magnification mode (i 800 nm). (c) USAF resolution target imaged in transmission using the low-magnification mode (i 406 nm). (d) Axial scan of a thin Rhodamine B film showing the two-photon axial resolution (FWHM) of 10.5 m.

surfaces will result in significantly higher power transmission. To demonstrate and test the imaging capabilities of the dual magnification objective, we acquired ex vivo images from various unstained mouse tissues. The two-photon fluorescence and one-photon reflected/scattered light emitted from these tissues was epicollected using the 10 large-core POFs and detected by an ultra bialkali (R7600U-200, Hamamatsu) photomultiplier tube (PMT). The PMT output current was amplified and converted to voltage (C7319, Hamamatsu). Prior to imaging, the excised tissue samples (i.e., a segment of colon, a single lung lobe, and a whole kidney obtained from an adult CD-1 mouse) were embedded in agarose gel and mounted on microscope slides. The slide was mounted on a three-dimensional (3D) translation stage (MP-285, Sutter). Low-magnification images were acquired first. To acquire high-magnification images, the site of interest was placed at the center of the FOV, and then the sample was moved axially to the image plane of the high-magnification mode. Image acquisition is performed with the software ScanImage. Figure 4(a) shows a high-resolution two-photon image of unstained mouse colon tissue (inner epithelial lining of the colon). Typical features (e.g., enterocytes, goblet cells, and crypts) are visible. A corresponding low-magnification, reflection/scattering image of mouse colon is shown in Fig. 4(b). Figure 4(c) shows a high-magnification multiphoton image of unstained mouse lung, where characteristic features including alveolar walls and lumens are clearly distinguishable. The corresponding low-magnification image is shown in Fig. 4(d). All images were acquired at a rate of 2 framess. The average power at the sample during multiphoton imaging was 25 mW. In Fig. 5(a), we show a low-magnification image of unstained mouse kidney. High-magnification two-photon fluorescence and second harmonic generation (SHG) images of unstained mouse kidney at the surface and 80 m below the surface are shown in Fig. 5(b) and Fig. 5(c), respectively. In Fig. 5(b), the fibrous components of the kidney capsule are visible. Figure 5(c) displays an optical cross section of the proximal convoluted tubules, which are separated by renal interstitium (i.e., dark, nonfluorescent spaces containing sparse amount of connective tissue). In conclusion, we have designed, built, and characterized a miniature endoscopic objective lens that provides

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We thank members of the Xu and Webb groups for valuable discussions. The project was supported by the NIH Grants R01-CA133148 and R01-EB006736.
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Fig. 4. High-magnification two-photon intrinsic fluorescence images of unstained ex vivo mouse (a) colon and (c) lung. Lung image is unaveraged; colon image is averaged over three frames. In (a), enterocytes (e), goblet cells (g), and crypts (c) are visible. In (c), alveolar walls (w) and lumens (L) are distinguishable. (b) Low-magnification reflection/scattering image of unstained mouse colon (b) and lung (d). All images are acquired at two frames per second. Average power at the sample during multiphoton imaging was 25 mW. The white circles in (b) and (d) indicate the approximate location of the sites from which the multiphoton images shown in (a) and (c) are obtained.

Fig. 5. (a) Low-magnification reflection/scattering image of unstained mouse kidney. High-magnification two-photon intrinsic fluorescence and SHG image of unstained ex vivo mouse kidney (b) at the surface and (c) approximately 80 m below the surface. In (b), the collagen fibers of the kidney capsule are well distinguishable. In (c), cuboidal epithelium (CE) and the renal interstitium (RI) are visible. The white circle in (a) indicates the approximate location of the site from which the multiphoton images shown in (b) and (c) is obtained.

dual magnification and FOV without any mechanical adjustments of its elements. The operation is based on separating the optical path for excitation light with different wavelengths. We fully characterized the two modes of operation by imaging a USAF resolution target in transmission. We demonstrated the feasibility to employ this lens for dual magnification and FOV endoscopic imaging by acquiring multiphoton and reflectance images of unstained ex vivo mouse tissues. Future work will include the integration of the objective into a fully functional endomicroscope probe and demonstrating its imaging capabilities.